GAG BINDING PROTEIN

Abstract

A method is provided for introducing a GAG binding site into a protein comprising the steps: identifying a region in a protein which is not essential for structure maintenance introducing at least one basic amino acid into said site and/or deleting at least one bulky and/or acidic amino acid in said site, whereby said GAG binding site has a GAG binding affinity of Kd≦10 μM, preferably ≦1 μM, still preferred ≦0.1 μM, as well as modified GAG binding proteins.

Description

[0001] This application is a divisional of U.S. application Ser. No. 11/422,169 filed on Jun. 5, 2006, which is a 371 of PCT/EP2004/013670 filed on Dec. 2, 2004. The entire contents of the above-identified applications are hereby incorporated by reference.

[0002] The present invention relates to methods and tools for the inhibition of the interaction of chemokines and their high-affinity receptors on leukocytes and methods for the therapeutic treatment of inflammatory diseases.

[0003] Chemokines, originally derived from chemoattractant cytokines, actually comprise more than 50 members and represent a family of small, inducible, and secreted proteins of low molecular weight (6-12 kDa in their monomeric form) that play a decisive role during immunosurveillance and inflammatory processes. Depending on their function in immunity and inflammation, they can be distinguished into two classes. Inflammatory chemokines are produced by many different tissue cells as well as by immigrating leukocytes in response to bacterial toxins and inflammatory cytokines like IL-1, TNF and interferons. Their main function is to recruit leukocytes for host defence and in the process of inflammation. Homing chemokines, on the other hand, are expressed constitutively in defined areas of the lymphoid tissues. They direct the traffic and homing of lymphocytes and dendritic cells within the immune system. These chemokines, as illustrated by BCA-1, SDF-1 or SLC, control the relocation and recirculation of lymphocytes in the context of maturation, differentiation, activation and ensure their correct homing within secondary lymphoid organs.

[0004] Despite the large number of representatives, chemokines show remarkably similar structural folds although the sequence homology varies between 20 to 70 percent. Chemokines consist of roughly 70-130 amino acids with four conserved cysteine residues. The cysteines form two disulphide bonds (Cys 1→Cys 3, Cys 2→Cys 4) which are responsible for their characteristic three-dimensional structure. Chemotactic cytokines consist of a short amino terminal domain (3-10 amino acids) preceding the first cysteine residue, a core made of β-strands and connecting loops found between the second and the fourth cysteine residue, as well as a carboxy-terminal α-helix of 20-60 amino acids. The protein core has a well ordered structure whereas the N- and C-terminal parts are disordered. As secretory proteins they are synthesised with a leader sequence of 20-25 amino acids which is cleaved off before release.

[0005] The chemokines have been subdivided into four families on the basis of the relative position of their cysteine residues in the mature protein. In the α-chemokine subfamily, the first two of the four cysteines are separated by a single amino acid (CXC), whereas in the β-chemokines the corresponding cysteine residues are adjacent to each other (CC). The α-chemokines can be further classified into those that contain the ELR sequence in the N-terminus, thereby being chemotactic for neutrophils (IL-8 for example), and those that lack the ELR motif and act on lymphocytes (I-TAC for example). Structurally the β-chemokines can be subdivided into two families: the monocyte-chemoattractant protein eotaxin family, containing the five monocyte chemoattractant proteins (MCP) and eotaxin which are approximately 65 percent identical to each other, and the remaining β-chemokines. As with the CXC-family, the N-terminal amino acids preceding the CC-residues are critical components for the biologic activity and leukocyte selectivity of the chemokines. The β-chemokines, in general, do not act on neutrophils but attract monocytes, eosinophils, basophils and lymphocytes with variable selectivity.

[0006] Only a few chemokines do not fit into the CC- or the CXC-family. Lymphotactin is so far the only chemokine which shows just two instead of the four characteristic cysteines in its primary structure, and is thus classified as γ- or C-chemokine. On the other hand, by concluding this classification, fractalkine has to be mentioned as the only representative of the δ- or CXXXC-subfamily with three amino acids separating the first two cysteines. Both of them, Lymphotaxin and fractalkine, induce chemotaxis of T-cells and natural killer cells.

[0007] Chemokines induce cell migration and activation by binding to specific cell surface, seven transmembrane-spanning (7TM) G-protein-coupled receptors on target cells. Eighteen chemokine receptors have been cloned so far including six CXC, ten CC, one CX3C and one XC receptor. Chemokine receptors are expressed on different types of leukocytes, some of them are restricted to certain cells (e.g. CXCR1 is restricted to neutrophils) whereas others are more widely expressed (e.g. CCR2 is expressed on monocytes, T cells, natural killer cells and basophils). Similar to chemokines, the receptors can be constitutively expressed on certain cells, whereas some are inducible. Some of them can even be down-regulated making the cells unresponsive to a certain chemokine but remaining responsive to another. Most receptors recognise more than one chemokine and vice versa but recognition is restricted to chemokines of the corresponding subfamily (see Table 1).

TABLE-US-00001 TABLE 1 Inflammatory Chemokine Receptor Chemotactic for Diseases CXC- IL-8 CXCR1 Neutrophils Acute respiratory distress Chemokine CXCR2 syndrome [71]; (+ELR-motif) Bacterial pneumonia [72]; Rheumathoid arthritis [73]; Inflammatory bowel disease [74]; Psoriasis [75]; Bacterial meningitis [761 CC- MCP-1 CCR2 Basophils; Monocytes; Asthma [77]; Chemokine Activated T cells; Glomerulonephritis [78]; Dentritic cells; Natural Atheroscleosis [79]; killer cells Inflammatory bowel disease [80]; Psoriasis [81]; Bacterial and viral meningitis [82, 83] RANTES CCR1 Eosinophils; Monocytes; Asthma [84]; Activated T cells; Glomerulonephritis [85] Dentritic cells CCR3 Eosinophils; Basophils; Dentritic cells CCR5 Monocytes; Activated T cells; Dentritic cells; Natural killer cells

[0008] Chemokines have two main sites of interaction with their receptors, one in the amino-terminal domain and the other within an exposed loop of the backbone that extends between the second and the third cysteine residue. Both sites are kept in close proximity by the disulphide bonds. The receptor recognises first the binding site within the loop region which appears to function as a docking domain. This interaction restricts the mobility of the chemokine thus facilitating the proper orientation of the amino-terminal domain. Studies have been performed with mutant chemokines that still bound effectively to their receptors but did not signal. These mutants were obtained by amino acid deletion or modification within the N-termini of, for example, IL-8, RANTES and MCP-1.

[0009] Multiple intracellular signalling pathways occur after receptor activation as a result of chemokine binding. Chemokines also interact with two types of nonsignalling molecules. One is the DARC receptor which is expressed on erythrocytes and on endothelial cells and which binds CC- as well as CXC-chemokines to prevent them from circulation. The second type are heparan sulphate glycosaminoglycans (GAGs) which are part of proteoglycans and which serve as co-receptors of chemokines. They capture and present chemokines on the surface of the homing tissue (e.g. endothelial cells) in order to establish a local concentration gradient. In an inflammatory response, such as in rheumatoid arthritis, leukocytes rolling on the endothelium in a selectin-mediated process are brought into contact with the chemokines presented by the proteoglycans on the cell surface. Thereby, leukocyte integrins become activated which leads to firm adherence and extravasation. The recruited leukocytes are activated by local inflammatory cytokines and may become desensitised to further chemokine signalling because of high local concentration of chemokines. For maintaining a tissue bloodstream chemokine gradient, the DARC receptor functions as a sink for surplus chemokines.

[0010] Heparan sulphate (HS) proteoglycans, which consist of a core protein with covalently attached glycosaminoglycan sidechains (GAGs), are found in most mammalian cells and tissues. While the protein part determines the localisation of the proteoglycan in the cell membrane or in the extracellular matrix, the glycosaminoglycan component mediates interactions with a variety of extracellular ligands, such as growth factors, chemokines and adhesions molecules. The biosynthesis of proteoglycans has previously been extensively reviewed. Major groups of the cell surface proteoglycans are the syndecan family of transmembrane proteins (four members in mammals) and the glypican family of proteins attached to the cell membrane by a glycosylphosphatidylinositol (GPI) tail (six members in mammals). While glypicans are expressed widely in the nervous system, in kidney and, to a lesser extent, in skeletal and smooth muscle, syndecan-1 is the major HSPG in epithelial cells, syndecan-2 predominates in fibroblasts and endothelial cells, syndecan-3 abounds in neuronal cells and syndecan-4 is widely expressed. The majority of the GAG chains added to the syndecan core proteins through a tetrasaccharide linkage region onto particular serines are HS chains. Although the amino acid sequences of the extracellular domains of specific syndecan types are not conserved among different species, contrary to the transmembrane and the cytoplasmic domains, the number and the positions of the GAG chains are highly conserved. The structure of the GAGs, however, is species-specific and is, moreover, dependent upon the nature of the HSPG-expressing tissue.

[0011] Heparan sulphate (HS) is the most abundant member of the glycosaminoglycan (GAG) family of linear polysaccharides which also includes heparin, chondroitin sulphate, dermatan sulphate and keratan sulphate. Naturally occurring HS is characterised by a linear chain of 20-100 disaccharide units composed of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA) which can be modified to include N- and O-sulphation (6-O and 3-O sulphation of the glucosamine and 2-O sulphation of the uronic acid) as well as epimerisation of β-D-gluronic acid to α-L-iduronic acid (IdoA).

[0012] Clusters of N- and O-sulphated sugar residues, separated by regions of low sulphation, are assumed to be mainly responsible for the numerous protein binding and regulatory properties of HS. In addition to the electrostatic interactions of the HS sulphate groups with basic amino acids, van der Waals and hydrophobic interactions are also thought to be involved in protein binding. Furthermore, the presence of the conformationally flexible iduronate residues seems to favour GAG binding to proteins. Other factors such as the spacing between the protein binding sites play also a critical role in protein-GAG binding interactions: For example γ-interferon dimerisation induced by HS requires GAG chains with two protein binding sequences separated by a 7 kDa region with low sulphation. Additional sequences are sometimes required for full biological activity of some ligands: in order to support FGF-2 signal transduction, HS must have both the minimum binding sequence as well as additional residues that are supposed to interact with the FGF receptor.

[0013] Heparin binding proteins often contain consensus sequences consisting of clusters of basic amino acid residues. Lysine, arginine, asparagine, histidine and glutamine are frequently involved in electrostatic contacts with the sulphate and carboxyl groups on the GAG. The spacing of the basic amino acids, sometimes determined by the proteins 3-D structure, are assumed to control the GAG binding specificity and affinity. The biological activity of the ligand can also be affected by the kinetics of HS-protein interaction. Reducing the dimension of growth factor diffusion is one of the suggested HSPG functions for which the long repetitive character of the GAG chains as well as their relatively fast on and off rates of protein binding are ideally suited. In some cases, kinetics rather than thermodynamics drives the physiological function of HS-protein binding. Most HS ligands require GAG sequences of well-defined length and structure. Heparin, which is produced by mast cells, is structurally very similar to heparan sulphate but is characterised by higher levels of post-polymerisation modifications resulting in a uniformly high degree of sulphation with a relatively small degree of structural diversity. Thus, the highly modified blocks in heparan sulphate are sometimes referred to as "heparin-like". For this reason, heparin can be used as a perfect HS analogue for protein biophysical studies as it is, in addition, available in larger quantities and therefore less expensive than HS. Different cell types have been shown to synthesise proteoglycans with different glycosaminoglycan structure which changes during pathogenesis, during development or in response to extracellular signals such as growth factors. This structural diversity of HSPGs leads to a high binding versatility emphasising the great importance of proteoglycans.

[0014] Since the demonstration that heparan sulphate proteoglycans are critical for FGF signalling, several investigations were performed showing the importance of chemokine-GAG binding for promoting chemokine activity. First, almost all chemokines studied to date appear to bind HS in vitro, suggesting that this represents a fundamental property of these proteins. Second, the finding that in vivo T lymphocytes secrete CC-chemokines as a complex with glycosaminoglycans indicates that this form of interaction is physiologically relevant. Furthermore, it is known that the association of chemokines with HS helps to stabilise concentration gradients across the endothelial surface thereby providing directional information for migrating leukocytes. HS is also thought to protect chemokines from proteolytic degradation and to induce their oligomerisation thus promoting local high concentrations in the vicinity of the G-coupled signalling receptors. The functional relevance of oligomerisation, however, remains controversial although all chemokines have a clear structural basis for multimerisation. Dimerisation through association of the β-sheets is observed for all chemokines of the CXC-family (e.g. IL-8), contrary to most members of the CC-chemokine family (e.g. RANTES), which dimerise via their N-terminal strands.

[0015] A wealth of data has been accumulated on the inhibition of the interaction of chemokines and their high-affinity receptors on leukocytes by low molecular weight compounds. However, there has been no breakthrough in the therapeutic treatment of inflammatory diseases by this approach.

[0016] Interleukin-8 (IL-8) is a key molecule involved in neutrophil attraction during chronic and acute inflammation. Several approaches have been undertaken to block the action of IL-8 so far, beginning with inhibition of IL-8 production by for example glucocorticoids, Vitamin D3, cyclosporin A, transforming growth factor β, interferons etc., all of them inhibiting IL-8 activity at the level of production of IL-8 mRNA. A further approach previously used is to inhibit the binding of IL-8 to its receptors by using specific antibodies either against the receptor on the leukocyte or against IL-8 itself in order to act as specific antagonists and therefore inhibiting the IL-8 activity.

[0017] The aim of the present invention is therefore to provide an alternative strategy for the inhibition or disturbance of the interaction of chemokines/receptors on leukocytes. Specifically the action of IL-8, RANTES or MCP-1 should be targetted by such a strategy.

[0018] Subject matter of the present invention is therefore a method to produce new GAG binding proteins as well as alternative GAG binding proteins which show a high(er) affinity to a GAG co-receptor (than the wild type). Such modified GAG binding proteins can act as competitors with wild-type GAG binding proteins and are able to inhibit or down-regulate the activity of the wild-type GAG binding protein, however without the side effects which occur with the known recombinant proteins used in the state of the art. The molecules according to the present invention do not show the above mentioned disadvantages. The present modified GAG binding proteins can be used in drugs for various therapeutical uses, in particular--in the case of chemokines--for the treatment of inflammation diseases without the known disadvantages which occur in recombinant chemokines known in the state of the art. The modification of the GAG binding site according to the present invention turned out to be a broadly applicable strategy for all proteins which activity is based on the binding event to this site, especially chemokines with a GAG site. The preferred molecules according to the present invention with a higher GAG binding affinity proved to be specifically advantageous with respect to their biological effects, especially with respect to their anti-inflammatory activity by their competition with wild type molecules for the GAG site.

[0019] Therefore, the present invention provides a method for introducing a GAG binding site into a protein characterised in that it comprises the steps: [0020] identifying a region in a protein which is not essential for structure maintenance [0021] introducing at least one basic amino acid into said site and/or deleting at least one bulky and/or acidic amino acid in said site, whereby said GAG binding site has a GAG binding affinity of K_d≦10 μM, preferably ≦1 μM, still preferred ≦0.1 μM. By introducing at least one basic amino acid and/or deleting at least one bulky and/or acidic amino acid in said region, a novel, improved "artificial" GAG binding site is introduced in said protein. This comprises the new, complete introduction of a GAG binding site into a protein which did not show a GAG binding activity before said modification. This also comprises the introduction of a GAG binding site into a protein which already showed GAG binding activity. The new GAG binding site can be introduced into a region of the protein which did not show GAG binding affinity as well as a region which did show GAG binding affinity. However, with the most preferred embodiment of the present invention, a modification of the GAG binding affinity of a given GAG binding protein is provided, said modified protein's GAG binding ability is increased compared to the wild-type protein. The present invention relates to a method of introducing a GAG binding site into a protein, a modified GAG binding protein as well as to an isolated DNA molecule, a vector, a recombinant cell, a pharmaceutical composition and the use of said modified protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows a CD spectra.

[0023] FIG. 2 shows secondary structure contents of various mutants.

[0024] FIG. 3 shows graphics of results from fluorescence anisotropy tests of various mutants.

[0025] Figure shows graphics of results from fluorescence anisotropy tests of two mutants.

[0026] Figure shows the graphic of results from isothermal fluorescence titrations.

[0027] Figure shows the graphic of results from unfolding experiments of various mutants.

[0028] Figure shows chemotaxis index of IL-8 mutants.

[0029] Figure shows the results of the RANTES chemotaxis assay.

[0030] The term "introducing at least one basic amino acid" relates to the introduction of additional amino acids as well as the substitution of amino acids. The main purpose is to increase the relative amount of basic amino acids, preferably Arg, Lys, His, Asn and/or Gln, compared to the total amount of amino acids in said site, whereby the resulting GAG binding site should preferably comprise at least 3 basic amino acids, still preferred 4, most preferred 5 amino acids.

[0031] The GAG binding site is preferably at a solvent exposed position, e.g. at a loop. This will assure an effective modification.

[0032] Whether or not a region of a protein is essential for structure maintenance, can be tested for example by computational methods with specific programmes known to the person skilled in the art. After modification of the protein, the conformational stability is preferably tested in silico.

[0033] The term "bulky amino acid" refers to amino acids with long or sterically interfering side chains; these are in particular Trp, Ile, Leu, Phe, Tyr. Acidic amino acids are in particular Glu and Asp. Preferably, the resulting GAG binding site is free of bulky and acidic amino acids, meaning that all bulky and acidic amino acids are removed.

[0034] The GAG binding affinity is determined--for the scope of protection of the present application--over the dissociation constant K_d. One possibility is to determine the dissociation constant (K_d) values of any given protein by the structural change in ligand binding. Various techniques are well known to the person skilled in the art, e.g. isothermal fluorescence titrations, isothermal titration calorimetry, surface plasmon resonance, gel mobility assay, and indirectly by competition experiments with radioactively labelled GAG ligands. A further possibility is to predict binding regions by calculation with computational methods also known to the person skilled in the art, whereby several programmes may be used.

[0035] A protocol for introducing a GAG binding site into a protein is for example as follows: [0036] Identify a region of the protein which is not essential for overall structural maintenance and which might be suitable for GAG binding [0037] Design a new GAG binding site by introducing (replacement or insertion) basic Arg, Lys, His, Asp and Gln residues at any position or by deleting amino acids which interfere with GAG binding [0038] Check the conformational stability of the resulting mutant protein in silico [0039] Clone the wild-type protein cDNA (alternatively: purchase the cDNA) [0040] Use this as template for PCR-assisted mutagenesis to introduce the above mentioned changes into the amino acid sequence [0041] Subclone the mutant gene into a suitable expression system (prokaryotic or eukaryotic dependent upon biologically required post-translational modifications) [0042] Expression, purification and characterisation of the mutant protein in vitro [0043] Criterion for an introduced GAG binding affinity: K_d^GAG(mutant)<10 μM.

[0044] Examples of said engineered proteins with new GAG binding sites are for example the Fc part of IgG as well as the complement factors C3 and C4 modified as follows:

TABLE-US-00002 Fc: (439)KSLSLS(444) -> KSKKLS (SEQ ID NOS 1 & 2) C3: (1297)WIASHT(1302) -> WKAKHK (SEQ ID NOS 3 & 4) C4: (1)MLDAERLK(8) -> MKKAKRLK (SEQ ID NOS 5 & 6)

[0045] A further aspect of the present invention is a protein obtainable by the inventive method as described above. The inventive protein therefore comprises a--compared to the wild-type protein--new GAG binding site as defined above and will therefore act as competitor with natural GAG binding proteins, in particular since the GAG binding affinity of the inventive protein is very high, e.g. K_d≦10 μM.

[0046] A further aspect of the present invention is a modified GAG binding protein, whereby a GAG binding region in said protein is modified by substitution, insertion, and/or deletion of at least one amino acid in order to increase the relative amount of basic amino acids in said GAG binding region, and/or reduce the amount of bulky and/or acidic amino acids in said GAG binding region, preferably at a solvent exposed position, and in that the GAG binding affinity of said protein is increased compared to the GAG binding affinity of a respective wild-type protein.

[0047] It has been surprisingly shown that by increasing the relative amount of basic amino acids, in particular Arg, Lys, His, Asn and Gln, in the GAG binding region, the modified GAG binding protein shows increased GAG binding affinity compared to the wild-type proteins, in particular when the relative amount of basic amino acids is increased at a solvent exposed position, since a positively charged area on the protein surface has shown to enhance the binding affinity. Preferably, at least 3, still preferred 4, most preferred 5, basic amino acids are present in the GAG binding region.

[0048] The term "GAG binding protein" relates to any protein which binds to a GAG co-receptor. Whether or not a protein binds to a GAG co-receptor can be tested with the help of known protocols as mentioned above. Hileman et al. (BioEssays 20 (1998), 156-167) disclose consensus sites in glycosaminoglycan binding proteins. The information disclosed in this article is also useful as starting information for the present invention. The term "protein" makes clear that the molecules provided by the present invention are at least 80 amino acids in length. This is required for making them suitable candidates for the present anti-inflammation strategy. Smaller molecules interacting with a GAG binding site and being physiologically or pathologically relevant due to such an interaction are not known and therefore not relevant for the present invention. Preferably, the molecules according to the present invention are composed of at least 90, at least 100, at least 120, at least 150, at least 200, at least 300, at least 400 or at least 500 amino acid residues.

[0049] In the scope of the present application the term "GAG binding region" is defined as a region which binds to GAG with a dissociation constant (K_d-value) of under 100 μM, preferably under 50 μM, still preferred under 20 μM, as determined by isothermal fluorescence titration (see examples below).

[0050] Any modifications mentioned in the present application can be carried out with known biochemical methods, for example site-directed mutagenesis. It should also be noted that molecular cloning of GAG binding sites is, of course, prior art (see e.g. WO96/34965 A, WO 92/07935 A, Jayaraman et al. (FEBS Letters 482 (2000), 154-158), WO02/20715 A, Yang et al. (J. Cell. Biochem. 56 (1994), 455-468), wherein molecular shuffling or de novo syntesis of GAG regions are described; Butcher et al., (FEBS Letters 4009 (1997), 183-187) (relates to artificial peptides, not proteins); Jinno-Oue et al, (J. Virol. 75 (2001), 12439-12445) de novo synthesis)).

[0051] The GAG binding region can be modified by substitution, insertion and/or deletion. This means that a non-basic amino acid may be substituted by a basic amino acid, a basic amino acid may be inserted into the GAG binding region or a non-basic amino acid may be deleted. Furthermore, an amino acid which interferes with GAG binding, preferably all interfering amino acids binding is deleted. Such amino acids are in particular bulky amino acids as described above as well as acidic amino acids, for example Glu and Asp. Whether or not an amino acid interferes with GAG binding may be examined with for example mathematical or computational methods. The result of any of these modifications is that the relative amount of basic amino acids in said GAG binding region is increased, whereby "relative" refers to the amount of basic amino acids in said GAG binding region compared to the number of all amino acids in said GAG binding region. Furthermore, amino acids which interfere sterically or electrostatically with GAG binding are deleted.

[0052] Whether or not an amino acid is present in a solvent exposed position, can be determined for example with the help of the known three dimensional structure of the protein or with the help of computational methods as mentioned above.

[0053] Whether or not the GAG binding affinity of said modified protein is increased compared to the GAG binding affinity of the respective wild-type protein, can be determined as mentioned above with the help of, for example, fluorescence titration experiments which determine the dissociation constants. The criterion for improved GAG binding affinity will be K_d (mutant)<K_d (wild-type), preferably by at least 100%. Specifically improved modified proteins have--compared with wild-type K_d--a GAG binding affinity which is higher by a factor of minimum 5, preferably of minimum 10, still preferred of minimum 100. The increased GAG binding affinity will therefore preferably show a K_d of under 10 μM, preferred under 1 μM, still preferred under 0.1 μM.

[0054] By increasing the GAG binding affinity the modified protein will act as a specific antagonist and will compete with the wild-type GAG binding protein for the GAG binding.

[0055] Preferably, at least one basic amino acid selected from the group consisting of Arg, Lys, and His is inserted into said GAG binding region. These amino acids are easily inserted into said GAG binding region, whereby the term "inserted" relates to an insertion as such as well as substituting any non-basic amino acid with arginine, lysine or histidine. Of course, it is possible to insert more than one basic amino acid whereby the same basic amino acid may be inserted or also a combination of two or three of the above mentioned amino acids.

[0056] Still preferred, the protein is a chemokine, preferably IL-8, RANTES or MCP-1. Chemokines are known to have a site of interaction with co-receptor GAG whereby this chemokine binding is often a condition for further receptor activation as mentioned above. Since chemokines are often found in inflammatory diseases, it is of major interest to block the chemokine receptor activation. Such chemokines are preferably IL-8, RANTES or MCP-1, which are well characterised molecules and of which the GAG binding regions are well known (see, for example, Lortat-Jacob et al., PNAS 99 (3) (2002), 1229-1234). By increasing the amount of basic amino acids in the GAG binding region of these chemokines, their binding affinity is increased and therefore the wild-type chemokines will bind less frequently or not at all, depending on the concentration of the modified protein in respect to the concentration of the wild-type protein.

[0057] According to an advantageous aspect, said GAG binding region is a C terminal α-helix. A typicial chemical monomer is organised around a triple stranded anti-parallel β-sheet overlaid by a C-terminal α-helix. It has been shown that this C-terminal α-helix in chemokines is to a major part involved in the GAG binding, so that modification in this C-terminal α-helix in order to increase the amount of basic amino acids results in a modified chemokine with an increased GAG binding affinity.

[0058] Advantageously, positions 17, 21, 70, and/or 71 in IL-8 are substituted by Arg, Lys, His, Asn and/or Gln. Here it is possible that only one of these aforementioned sites is modified. However, also more than one of these sites may be modified as well as all, whereby all modifications may be either Arg or Lys or His or Asn or Gln or a mixture of those. In IL-8 these positions have shown to highly increase the GAG binding affinity of IL-8 and therefore these positions are particularly suitable for modifications.

[0059] Preferably the increased binding affinity is an increased binding affinity to heparan sulphate and/or heparin. Heparan sulphate is the most abundant member of the GAG family of linear polysaccharides which also includes heparin. Heparin is structurally very similar to heparan sulphate characterised by higher levels of post-polymerisation modifications resulting in a uniformly high degree of sulphation with a relatively small degree of structural diversity. Therefore, the highly modified blocks in heparan sulphate are sometimes referred to as heparin-like and heparin can be used as a heparan sulphate analogue for protein biophysical studies. In any case, both, heparan sulphate and heparin are particularly suitable.

[0060] Still preferred, a further biologically active region is modified thereby inhibiting or down-regulating a further biological activity of said protein. This further biological activity is known for most GAG binding proteins, for example for chemokines. This will be the binding region to a receptor, for example to the 7TM receptor. The term "further" defines a biologically active region which is not the GAG binding region which, however, binds to other molecules, cells or receptors and/or activates them. By modifying this further biologically active region the further biological activity of this protein is inhibited or down-regulated and thereby a modified protein is provided which is a strong antagonist to the wild-type protein. This means that on the one hand the GAG binding affinity is higher than in the wild-type GAG binding protein, so that the modified protein will to a large extent bind to the GAG instead of the wild-type protein. On the other hand, the further activity of the wild-type protein which mainly occurs when the protein is bound to GAG, is inhibited or down-regulated, since the modified protein will not carry out this specific activity or carries out this activity to a lesser extent. With this modified protein an effective antagonist for wild-type GAG binding proteins is provided which does not show the side effects known from other recombinant proteins as described in the state of the art. This further biologically active region can for example be determined in vitro by receptor competition assays (using fluorescently labelled wt chemokines, calcium influx, and cell migration (performed on native leukocytes or on 7TM stably-transfected cell lines). Examples of such further biologically active regions are, in addition to further receptor binding sites (as in the growth factor family), enzymatic sites (as in hydrolases, lyases, sulfotransferases, N-deacetylases, and copolymerases), protein interaction sites (as in antithrombin III), and membrane binding domains (as in the herpes simplex virus gD protein). With this preferred embodiment of double-modified proteins therefore dominant (concerning GAG binding) negative (concerning receptor) mutants are provided which are specifically advantageous with respect to the objectives set for the present invention.

[0061] Still preferred, said further biologically active region is modified by deletion, insertion, and/or substitution, preferably with alanine, a sterically and/or electrostatically similar residue. It is, of course, possible to either delete or insert or substitute at least one amino acid in said further biologically active region. However, it is also possible to provide a combination of at least two of these modifications or all three of them. By substituting a given amino acid with alanine or a sterically/electronically similar residue--"similar" meaning similar to the amino acid being substituted--the modified protein is not or only to a lesser extent modified sterically/electrostatically. This is particularly advantageous, since other activities of the modified protein, in particular the affinity to the GAG binding region, are not changed.

[0062] Advantageously, said protein is a chemokine and said further biological activity is leukocyte activation. As mentioned above, chemokines are involved in leukocyte attraction during chronic and acute inflammation. Therefore, by inhibiting or down-regulating leukocyte activation inflammation is decreased or inhibited which makes this particular modified protein an important tool for studying, diagnosing and treating inflammatory diseases.

[0063] According to an advantageous aspect, said protein is IL-8 and said further biologically active region is located within the first 10 N-terminal amino acids. The first N-terminal amino acids are involved in leukocyte activation, whereby in particular Glu-4, Leu-5 and Arg-6 were identified to be essential for receptor binding and activation. Therefore, either these three or even all first 10 N-terminal amino acids can be substituted or deleted in order to inhibit or down-regulate the receptor binding and activation.

[0064] A further advantageous protein is an IL-8 mutant with the first 6 N-terminal amino acids deleted. As mentioned above, this mutant will not or to a lesser extent bind and activate leukocytes, so that it is particularly suitable for studying, diagnosing and treating inflammatory diseases.

[0065] Preferably, said protein is an IL-8 mutant selected from the group consisting of del6F17RE70KN71R, del6F17RE70RN71K and del6E70KN71K. These mutants have shown to be particularly advantageous, since the deletion of the first 6 N-terminal amino acids inhibits or down-regulates receptor binding and activation. Furthermore, the two phenylalanines in position 17 and 21 were found to make first contact with the receptor on its N-terminal extracellular domain to facilitate the later activation of the receptor. In order to prevent any neutrophil contact, these two amino acids 17 and 21 are exchanged, whereby they are exchanged to basic amino acids, since they are in close proximity to the GAG binding motif of the C-terminal α-helix as can be seen on a three dimensional model of a protein. By exchanging the position 17 and/or 21 to either arginine or lysine the GAG binding affinity is therefore increased.

[0066] A further aspect of the present invention is an isolated polynucleic acid molecule which codes for the inventive protein as described above. The polynucleic acid may be DNA or RNA. Thereby the modifications which lead to the inventive modified protein are carried out on DNA or RNA level. This inventive isolated polynucleic acid molecule is suitable for diagnostic methods as well as gene therapy and the production of inventive modified protein on a large scale.

[0067] Still preferred, the isolated polynucleic acid molecule hybridises to the above defined inventive polynucleic acid molecule under stringent conditions. Depending on the hybridisation conditions complementary duplexes form between the two DNA or RNA molecules, either by perfectly being matched or also comprising mismatched bases (see Sambrook et al., Molecular Cloning: A laboratory manual, 2^nd ed., Cold Spring Harbor, N.Y. 1989). Probes greater in length than about 50 nucleotides may accommodate up to 25 to 30% mismatched bases. Smaller probes will accommodate fewer mismatches. The tendency of a target and probe to form duplexes containing mismatched base pairs is controlled by the stringency of the hybridisation conditions which itself is a function of factors, such as the concentration of salt or formamide in the hybridisation buffer, the temperature of the hybridisation and the post-hybridisation wash conditions. By applying well-known principles that occur in the formation of hybrid duplexes conditions having the desired stringency can be achieved by one skilled in the art by selecting from among a variety of hybridisation buffers, temperatures and wash conditions. Thus, conditions can be selected that permit the detection of either perfectly matched or partially mismatched hybrid duplexes. The melting temperature (Tm) of a duplex is useful for selecting appropriate hybridisation conditions. Stringent hybridisation conditions for polynucleotide molecules over 200 nucleotides in length typically involve hybridising at a temperature 15-25° C. below the melting temperature of the expected duplex. For oligonucleotide probes over 30 nucleotides which form less stable duplexes than longer probes, stringent hybridisation usually is achieved by hybridising at 5 to 10° C. below the Tm. The Tm of a nucleic acid duplex can be calculated using a formula based on the percent G+C contained in the nucleic acids and that takes chain lengths into account, such as the formula Tm=81.5-16.6 (log [Na.sup.+)])+0.41 (% G+C)-(600/N), where N=chain length.

[0068] A further aspect of the present invention relates to a vector which comprises an isolated DNA molecule according to the present invention as defined above. The vector comprises all regulatory elements necessary for efficient transfection as well as efficient expression of proteins. Such vectors are well known in the art and any suitable vector can be selected for this purpose.

[0069] A further aspect of the present application relates to a recombinant cell which is stably transfected with an inventive vector as described above. Such a recombinant cell as well as any therefrom descendant cell comprises the vector. Thereby a cell line is provided which expresses the modified protein either continuously or upon activation depending on the vector.

[0070] A further aspect of the present invention relates to a pharmaceutical composition which comprises a protein, a polynucleic acid or a vector according to the present invention as defined above and a pharmaceutically acceptable carrier. Of course, the pharmaceutical composition may further comprise additional substances which are usually present in pharmaceutical compositions, such as salts, buffers, emulgators, colouring agents, etc.

[0071] A further aspect of the present invention relates to the use of the modified protein, a polynucleic acid or a vector according to the present invention as defined above in a method for inhibiting or supressing the biological activity of the respective wild-type protein. As mentioned above, the modified protein will act as an antagonist whereby the side effects which occur with known recombinant proteins will not occur with the inventive modified protein. In the case of chemokines this will be in particular the biological activity involved in inflammatory reactions.

[0072] Therefore, a further use of the modified protein, polynucleic acid or vector according to the present invention is in a method for producing a medicament for the treatment of an inflammatory condition. In particular, if the modified protein is a chemokine, it will act as antagonist without side effects and will be particularly suitable for the treatment of an inflammatory condition. Therefore, a further aspect of the present application is also a method for the treatment of an inflammatory condition, wherein a modified protein according to the present invention, the isolated polynucleic acid molecule or vector according to the present invention or a pharmaceutical composition according to the present invention is administered to a patient.

[0073] Preferably, the inflammatory condition is selected from a group comprising rheumatoid arthritis, psoriasis, osteoarthritis, asthma, Alzheimer's disease, and multiple sclerosis. Since the activation through chemokines can be inhibited with a modified protein according to the present invention, inflammatory reactions can be inhibited or down-regulated whereby the above mentioned inflammatory conditions can be prevented or treated.

[0074] The present invention is described in further detail with the help of the following examples and figures to which the invention is, however, not limited whereby FIG. 1 is a CD spectra; FIG. 2 shows secondary structure contents of various mutants; FIGS. 3 and 4 show graphics of results from fluorescence anisotropy tests of various mutants; FIG. 5 shows the graphic of results from isothermal fluorescence titrations; FIG. 6 shows the graphic of results from unfolding experiments of various mutants, FIG. 7 shows chemotaxis index of IL-8 mutants, and FIG. 8 shows the results of the RANTES chemotaxis assay.

EXAMPLES

Example 1

Generation of Recombinant IL-8 Genes and Cloning of the Mutants

[0075] Polymerase chain reaction (PCR) technique was used to generate the desired cDNAs for the mutants by introducing the mutations using sense and antisense mutagenesis primers. A synthetic plasmid containing the cDNA for wtIL-8 was used as template, Clontech Advantage®2 Polymerase Mix applied as DNA polymerase and the PCR reaction performed using a Mastergradient Cycler of Eppendorf. The mutagenesis primers used are summarised in the table below beginning with the forward sequences (5' to 3'):

TABLE-US-00003 (SEQ ID NO: 7) CACC ATG TGT CAG TGT ATA AAG ACA TAC TCC (primer for the mutation Δ6) (SEQ ID NO: 8) CACC ATG TGT CAG TGT ATA AAG ACA TAC TCC AAA CCT AGG CAC CCC AAA AGG ATA (primer for the mutation Δ6 F17R F21R)

[0076] The reverse sequences are (5' to 3'):

TABLE-US-00004 TTA TGA ATT CCT AGC CCT CTT (SEQ ID NO: 9) (primer for the mutation E70R) TTA TGA ATT CTT AGC CCT CTT (SEQ ID NO: 10) (primer for the mutation E70K) TTA TGA CTT CTC AGC CCT CTT (SEQ ID NO: 11) (primer for the mutation N71K) TTA TGA CTT CTT AGC CCT CTT (SEQ ID NO: 12) (primer for the mutation E70K N71K) TTA TGA CTT CCT AGC CCT CTT (SEQ ID NO: 13) (primer for the mutation E70R N71K) TTA TGA CCT CTT AGC CCT CTT (SEQ ID NO: 14) (primer for the mutation E70K N71R) TTA TGA CCT CCT AGC CCT CTT (SEQ ID NO: 15) (primer for the mutation E70R N71R)

[0077] The PCR products were purified, cloned into the pCR®T7/NT-TOPO®TA (Invitrogen) vector and transformed into TOP10F competent E. coli (Invitrogen). As a next step a confirmation of the sequence was carried out by double-stranded DNA sequencing using a ABI PRISM CE1 Sequencer.

Example 2

Expression and Purification of the Recombinant Proteins

[0078] Once the sequences were confirmed, the constructs were transformed into calcium-competent BL21(DE3) E. coli for expression. Cells were grown under shaking in 1 l Lennox Broth (Sigma) containing 100 μg/ml Ampicillin at 37° C. until an OD₆₀₀ of about 0.8 was reached. Induction of protein expression was accomplished by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Four hours later the cells were harvested by centrifugation at 6000 g for 20 minutes. The cell pellet was then resuspended in a buffer containing 20 mM TRIS/HCl, 50 mM NaCl, pH 8, sonicated at 100 watts for 5×20 s and finally centrifuged again for 20 min at 10,000 g. Since the main fraction of the recombinant IL-8 proteins was found in inclusion bodies, denaturing conditions were chosen for further purification. So the cell pellet was resuspended in a buffer of 6M Gua/HCl and 50 mM MES, pH 6.5. The suspension was then stirred at 4° C. for 4 hours, followed by a dialysis step against 50 mM MES, pH 6.5. The resulting suspension was then centrifuged and filtered to be loaded on a strong cation exchange column (SP Sepharose® from Pharmacia Biotech). The elution was accomplished by a linear gradient from 0M-1M NaCl in a 50 mM MES buffer, pH 6.5 over 60 minutes. After lyophilisation of the fractions containing the desired protein, a second purification step was carried out by reversed-phase HPLC using a C18 column. In this case a non-linear gradient from 10%-90% Acetonitril was chosen to elute the desired protein. Refolding of the denatured protein was finally accomplished by the same cation exchange column under the same conditions as described above.

[0079] The protein was then checked for purity and identity by silver stain analysis in the first case and Western Blot analysis, using a specific monoclonal antibody against wtIL-8, in the second. Refolding of the proteins was also confirmed by Circular Dichroism (CD) measurements.

Example 3

Biophysical Characterisation of the Mutants

3.1 Circular Dicroism Measurements and Analysis

[0080] In order to investigate secondary structure changes of the mutant protein in the presence and absence of heparan sulphate (HS), CD spectroscopy was carried out. Measurements were recorded on a Jasco J-710 spectropolarimeter over a range of 195-250 nm, and a cell of 0.1 cm path length was used. Spectra of the protein solutions with a concentration of 5 μM were recorded with a response time of 1 s, step resolution of 0.2 nm, speed of 50 nm/min, band width of 1 nm and a sensitivity of 20 mdeg. Three scans were averaged to yield smooth spectra. The protein spectra were then background-corrected relating to the CD-signal either of the buffer itself or buffer/HS. Secondary structure analysis of the protein in the presence and absence of HS was finally accomplished using the programme SELCON.

[0081] Since a great number of amino acids were changed in a number of novel combinations, it was tried to find out the dimension of the resulting secondary structure changes by circular dichroism methods.

[0082] Different structures were obtained depending on the mutations introduced. Except for one mutant expressed (Δ6 F17R F21R E70K N71R) which didn't show any structure, all mutants exhibited measurable α-helices, β-sheets and loops. Compared to IL-8 wt only one mutant (Δ6 E70R) showed nearly similar structure whereas the others differed mainly in their α-helix which ranged from 17.2% to 45.2% out of the total structure. Nevertheless, this fact suggests that the overall structure of IL-8 wt was maintained despite many changes within the proteins sequence. This could not have been previously predicted. Having already found that heparan sulphate oligosaccharides only, and not heparin, were able to affect IL-8 wt secondary structure, attention was focused on the effects induced by unfractionated heparan sulphate. All examined mutants showed structural changes upon HS binding which can be seen as evidence of complex formation.

[0083] To demonstrate the structural changes upon introduced mutations and heparan sulphate addition, some of the data obtained are summarised in the graphs above and below.

3.2 Fluorescence Measurements

[0084] For studying concentration and ligand dependent quaternary structure changes fluorescence spectroscopy was performed. Due to its high sensitivity, requiring only nanogram quantities of protein, fluorescence technique was the method of choice for carrying out the desired investigations. Measurements were undertaken using a Perkin-Elmer (Beaconsfield, England) LS50B fluorometer.

3.3 Fluorescence Anisotropy

[0085] By recording the concentration dependent fluorescence anisotropy of the chemokine resulting from the extrinsic chromophore bisANS it was aimed to find out the dimerisation constant of the mutants. Measurements were performed in PBS starting with high concentrations (up to 4 μM protein) followed by stepwise dilution. For each data point, the anisotropy signal (r) recorded at 507 nm was averaged over 60 sec.

[0086] IL-8 oligomerisation has been reported to relevantly influence the proteins GAG binding properties. Set at monomeric concentration, IL-8 bound size defined oligosaccharides 1000-fold tighter than at dimeric concentration. Therefore, the oligomerisation properties of IL-8 mutants were investigated by fluorescence anisotropy. Since the IL-8 intrinsic fluorophore (Trp57) was not sensitive enough for all of the mutants, the extrinsic fluorophore bis-ANS was used for these measurements. Again, as already noticed for the secondary structure, the mutant Δ6 E70R showed high resemblance also in the oligomerisation constant (k_oligo=350 nM) to IL-8 wt (k_oligo=379 nM). The mutant with the highest k_oligo (k_oligo=460 nM), which therefore dimerised worst, was Δ6 F17RF21R E70RN71K. Previous studies identified the β-sheets to be mainly involved in the dimerisation process, a fact, which correlates with the results for this mutants' secondary structure, which showed a very low share of β-sheet of only 11.4%. The mutant with the lowest k_oligo (k_oligo=147 nM), was found to be Δ6 F17RF21R E70K, which again showed the highest share of β-sheet structure (29.8%) of all mutants investigated. Also the impact of heparan sulphate addition was observed. As for IL-8 wt, where heparan sulphate caused a shift of the oligomerisation constant to much higher levels (k_oligo=1.075 μM), this was also found for the IL-8 mutants investigated. Δ6 F17RF21R E70K shifted from 0.147 μM to 1.162 μM, and the mutant Δ6 E70R from 0.350 μM to 1.505 μM in the presence of heparan sulphate. Some of the results obtained are demonstrated in FIGS. 3 and 4, whereby FIG. 3 shows the dependence of the fluorescence anisotropy of IL-8 mutants in PBS on the chemokine concentration and FIG. 4 shows the dependence of the fluorescence anisotropy of Δ6 F17RF21R E70K in PBS on the chemokine concentration in the presence (ten fold excess) and absence of HS ((pc=10 xy excess) protein concentration).

3.4 Isothermal Fluorescence Titration (IFT) Experiments

[0087] Dissociation constants (K_d values) are a measure for the binding affinity of a ligand to a protein and therefore concentration-dependent change in the fluorescence emission properties of the protein (fluorescence quenching) upon ligand binding was used for the determination of E_d. Since these mutants contain an intrinsic tryptophan chromophore which is located at or near the proposed GAG binding site and therefore should be sensitive to structural changes upon ligand binding, IFT experiments seemed to be suitable for this kind of investigation. Fluorescence intensity titration was performed in PBS using a protein concentration of 700 nM. The emission of the protein solution upon excitation at 282 nm was recorded over a range of 300-400 nm following the addition of an aliquot of the respective GAG ligand and an equilibration period of 60 sec.

[0088] Binding to unfractionated heparin and heparan sulphate was investigated. The mutants were set at dimeric concentration to assure sufficient sensitivity. A quenching of Trp57 fluorescence intensity upon GAG binding was registered within a range of 25-35%. Significant improvement of ligand binding was observed, especially for heparin binding. Δ6 F17RN71R E70K (K_d=14 nM) and Δ6 F17RF21R N71K (K_d=14.6 nM) showed 2600-fold better binding, and Δ6 E70K N71K (K_d=74 nM) 1760-fold better binding compared to IL-8 wt (K_d=37 μM). Good results were also obtained for heparan sulphate binding. For Δ6 F17RN71R E70K a K_d of 107 nM was found, for Δ6 F17RF21R N71K the K_d was 95 nM and the mutant Δ6 E70K N71K showed a K_d of 34 nM. As IL-8 wt binds with a K_d of 4.2 μM, the K_ds found for the mutants represent an extraordinary improvement in binding, see FIG. 5.

3.5 Unfolding Experiments

[0089] In order to obtain information about the proteins stability and whether this stability would be changed upon GAG ligand binding, unfolding experiments were undertaken. As mentioned above fluorescence techniques are very sensitive for observing quaternary structure changes and therefore are also the method of choice to investigate thermal structural changes of the protein. Measurements were undertaken as described for the IFT in which not the ligand concentration was changed but the temperature. Protein structure was observed at a concentration of 0.7 μM from temperatures of 15-85° C. in the absence and the presence of a 10 fold excess of heparan sulphate or heparin.

[0090] The emission maximum of the proteins ranged from 340 nm to 357 nm, values which are typical for a solvent exposed tryptophan residue. Beginning with the unfolding experiments at 15° C., the emission maximum of the mutants varied between 340 nm-351 nm. Compared to IL-8 wt, whose emission maximum was observed at 340 nm, this means slightly higher values. Upon an increase in temperature, the intensity of emission maximum decreased, accompanied by a shift of the maximum to either a higher or lower wavelength. The emission maximum of Δ6 E70R and Δ6 E70K N71K shifted from 352.5 nm-357 nm and 343 nm-345 nm, which is typical for a further exposure of the Trp57 residue to the solvent trough temperature increase, but interestingly the mutants Δ6 F17RN71R E70K and Δ6 F17RF21R E70R N71K showed a blue shift, ranging from 350 nm-343 nm and, less pronounced, from 350 nm-348 nm (see FIG. 6). By slowly decreasing the temperature, the process of unfolding was partially reversible regarding both the wavelength shift and changes of intensity. Addition of a 5 fold excess of heparan sulphate led to an increase of stability of the proteins, probably through complex formation. This could be observed on the one hand by a shift of the melting point to higher temperature, and on the other hand by a significantly less pronounced shift of emission maximum upon temperature increase.

Example 4

Cell-Based Assay of the Receptor-"Negative" Function of the Dominant-Negative IL-8 Mutants

[0091] In order to characterise the impaired receptor function of the IL-8 mutants with respect to neutrophil attraction, transfilter-based chemotaxis of neutrophils in response to IL-8 mutants was assayed in a microchemotaxis chamber equipped with a 5 μm PVP-free polycarbonate membrane.

Cell Preparation:

[0092] Briefly, a neutrophil fraction was prepared from freshly collected human blood. This was done by adding a 6% dextran solution to heparin-treated blood (1:2) which was then left for sedimentation for 45 min. The upper clear cell solution was collected and washed twice with HBSS w/o Ca and Mg. Cells were counted and finally diluted with HBSS at 2Mio/ml cell suspension, taking into account that only 60% of the counted cells were neutrophils.

Chemotaxis Assay:

[0093] IL-8 mutants were diluted at concentrations of 10 μg/ml, 1 μg/ml and 0.1 μg/ml and put in triplicates in the lower compartment of the chamber (26 μl per well). The freshly prepared neutrophils were seeded in the upper chamber (50 μl per well) and incubated for 30 minutes at 37° C. in a 5% CO₂ humidified incubator. After incubation, the chamber was disassembled, the upper side of the filter was washed and wiped off and cells attached to the lower side were fixed with methanol and stained with Hemacolor solutions (Merck). Cells were then counted at 400× magnifications in 4 randomly selected microscopic fields per well. Finally, the mean of three independent experiments was plotted against the chemokine concentration. In FIG. 7, the chemotaxis index for various IL-8 mutants is shown. As expected, all mutants showed significantly decreased receptor binding activity.

Example 5

Generation of Recombinant RANTES Genes, Expression, Biophysical and Activity Characterisation of the Mutants

[0094] The concept of dominant-negative "GAG-masking" chemokine mutants was also employed to RANTES, a chemokine involved in type IV hypersensitivity reactions like transplant rejection, atopic dermatitis as well as in other inflammatory disorders like arthritis, progressive glomerulonephritis and inflammatory lung disease.

[0095] The receptor binding capability was impaired by introducing into the wt protein an initiating methionine residue. Expression of the wt RANTES in E. Coli lead to the retention of this methionine residue, which renders wt RANTES to a potent inhibitor of monocyte migration, the so-called Met-RANTES. Different mutations enhancing the GAG binding affinity were introduced via PCR-based site-directed mutagenesis methods.

[0096] By these means 9 RANTES mutants have so far been cloned, expressed and purified, Met-RANTES A22K, Met-RANTES H23K, Met-RANTES T43K, Met-RANTES N46R, Met-RANTES N46K, Met-RANTES Q48K, Met-RANTES A22K/N46R, Met-RANTES V49R/E66S and Met-RANTES ¹⁵LSLA¹⁸ V49R/E66S.

[0097] Isothermal fluorescence titration experiments were carried out to measure the relative affinity constants (Kd values) of the RANTES mutants for size defined heparin. As can be seen in the table all RANTES mutant proteins showed higher affinities for this heparin, with Met-RANTES A22K, Met-RANTES H23K, Met-RANTES T43K and Met-RANTES A22K/N46R showing the most promising results.

TABLE-US-00005 Kd in nM Wt Rantes 456.2 ± 8.5 Met-Rantes V49R/E66S 345.5 ± 21.7 Rantes 15LSLA18 V49R/66S 297.3 ± 14.1 Rantes N46R 367.7 ± 11.7 Rantes N46K 257.4 ± 10.2 Rantes H23K 202.5 ± 12.8 Rantes Q48K 383.4 ± 39.6 Rantes T43K 139.2 ± 30.1 Rantes A22K 202.1 ± 9.8 Rantes A22K/N46R 164.0 ± 16.6

RANTES Chemotaxis Assay

[0098] RANTES mutant directed cell migration was investigated using the 48-well Boyden chamber system equipped with 5 μm PVP-coated polycarbonate membranes. RANTES and RANTES mutant dilutions in RPMI 1640 containing 20 mM HEPES pH 7.3 and 1 mg/ml BSA were placed in triplicates in the lower wells of the chamber. 50 μl of THP-1 cell suspensions (promonocytic cell line from the European collection of cell cultures) in the same medium at 2×10⁶ cells/ml were placed in the upper wells. After a 2 h incubation period at 37° C. in 5% CO₂ the upper surface of the filter was washed in HBSS solution. The migrated cells were fixed in methanol and stained with Hemacolor solution (Merck). Five 400× magnifications per well were counted and the mean of three independently conducted experiments was plotted against the chemokine concentration in FIG. 8. The error bars represent the standard error of the mean of the three experiments. Again, as in the case of the IL-8 mutants, all RANTES mutants showed significantly reduced receptor binding activity.

Example 6

Proteins with GAG Binding Regions

[0099] By bioinformatical and by proteomical means GAG binding proteins were characterised together with their GAG binding regions. In the following tables 2 and 3 chemokines are shown with their GAG binding regions (table 2) and examples of other proteins are given also with their GAG binding regions (table 3).

TABLE-US-00006 TABLE 2 Chemokines and their GAG binding domains CXC-chemokines IL-8: ¹⁸HPK²⁰, (R47) ⁶⁰TVVEKFLKR68 (residues 60-68 of SEQ ID NO: 16) (SEQ ID NO: 16) SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS MGSA/GROα: ¹⁹HPK²¹, ⁴⁵KNGR⁴⁸ (residues 45-48 of SEQ ID NO: 17), ⁶⁰KKIIEK⁶⁶ (residues 60-66 of SEQ ID NO: 17) (SEQ ID NO: 17) ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN MIP-2α/GROβ: ¹⁹HLK²¹, K45, ⁶⁰KKIIEKMLK⁶8 (residues 60-68 of SEQ ID NO: 18) (SEQ ID NO: 18) APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIEKMLKNGKSN NAP-2: ¹⁵HPK¹⁸, ⁴²KDGR⁴⁵ (residues 42-45 of SEQ ID NO: 19), ⁵⁷KKIVQK⁶2 (residues 57-62 of SEQ ID NO: 19) (SEQ ID NO: 19) AELRCLCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDESAD PF-4: ²⁰RPRH²³ (residues 20-23 of SEQ ID NO: 20), ⁴⁶KNGR⁴⁹ (residues 46-49 of SEQ ID NO: 20), ⁶¹KKIIKK⁶⁶ (residues 61-66 of SEQ ID NO: 20) (SEQ ID NO: 20) EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES SDF-1α: K1, ²⁴KHLK²⁷ (residues 24-27 of SEQ ID NO: 21), ⁴¹RLK⁴³ (SEQ ID NO: 21) KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN CC-chemokines RANTES: (¹⁷RPLPRAH²³ (residues 17-23 of SEQ ID NO: 22)) ⁴⁴RKNR⁴⁷ (residues 44-47 of SEQ ID NO: 22) (SEQ ID NO: 22) SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS MCP-2: ¹⁸RKIPIQR²⁴ (residues 18-24 of SEQ ID NO: 23), ⁴⁶KRGK⁴⁹ (residues 46-49 of SEQ ID NO: 23) (SEQ ID NO: 23) QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNL- KP MCP-3: ²²KQR²⁴, ⁴⁷KLDK⁵⁰ (residues 47-50 of SEQ ID NO: 24), ⁶⁶KHLDKK⁷¹ (residues 66-71 of SEQ ID NO: 24) (SEQ ID NO: 24) QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTP- KL MIP-1α: R17, ⁴⁴KRSR⁴⁷ (residues 44-47 of SEQ ID NO: 25) (SEQ ID NO: 25) SLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVCADPSEEWVQKYVSDLELSA MIP-1β: R18, ⁴⁵KRSK⁴⁸ (residues 45-48 of SEQ ID NO: 26) (SEQ ID NO: 26) APMGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYDLELN MPIF-1: R18, ⁴⁵KKGR⁴⁸ (residues 45-48 of SEQ ID NO: 27) (SEQ ID NO: 27) MDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANPSDKQVQVCMRMLKLDTRIKT- RKN MIP-5/HCC-2: ⁴⁰KKGR⁴³ (residues 40-43 of SEQ ID NO: 28) (SEQ ID NO: 28) HFAADCCTSYISQSIPCSLMKSYFETSSECSKPGVIFLTKKGRQVCAKPSGPGVQDCMKKLKPYSI

TABLE-US-00007 TABLE 3 SEQ ID NO: Peroxisome biogenesis factor 1 29 181 TRRAKE 186 30 367 QKKIRS 372 31 1263 PKRRKN 1268 32 181 TRRAKE 186 33 367 QKKIRS 372 34 1263 PKRRKN 1268 MLTK-beta 35 415 SKRRGKKV 422 36 312 ERRLKM 317 37 416 KRRGKK 421 38 312 ERRLKM 317 39 416 KRRGKK 421 BHLH factor Hes4 40 43 EKRRRARI 50 41 43 EKRRRA 48 42 43 EKRRRA 48 Protocadherin 11 43 867 MKKKKKKK 874 44 867 MKKKKK 872 45 867 MKKKKK 872 46 899 MKKKKKKK 906 47 899 MKKKKK 904 48 899 MKKKKK 904 catenin (cadherin-associated protein), 49 315 RRRLRS 320 delta 1 50 404 VRKLKG 409 51 460 LRKARD 465 52 545 RRKLRE 550 53 621 AKKGKG 626 54 787 AKKLRE 792 55 315 RRRLRS 320 56 404 VRKLKG 409 57 460 LRKARD 465 58 545 RRKLRE 550 59 621 AKKGKG 626 60 787 AKKLRE 792 Muscarinic acetylcholine receptor M5 61 221 EKRTKD 226 62 427 TKRKRV 432 63 514 WKKKKV 519 64 221 EKRTKD 226 65 427 TKRKRV 432 66 514 WKKKKV 519 Alpha-2A adrenergic receptor 67 147 PRRIKA 152 68 224 KRRTRV 229 69 147 PRRIKA 152 70 224 KRRTRV 229 IL-5 promoter REII-region-binding protein 71 440 TKKKTRRR 447 72 569 GKRRRRRG 576 73 38 ARKGKR 43 74 437 GKKTKK 442 75 444 TRRRRA 449 76 569 GKRRRR 574 77 38 ARKGKR 43 78 437 GKKTKK 442 79 444 TRRRRA 449 80 569 GKRRRR 574 Mitofusin 1 81 291 ARKQKA 296 82 395 KKKIKE 400 83 291 ARKQKA 296 84 395 KKKIKE 400 N-cym protein 85 71 VRRCKI 76 86 71 VRRCKI 76 Smad ubiquitination regulatory factor 1 87 672 ERRARL 677 88 672 ERRARL 677 CUG-BP and ETR-3 like factor 5 89 468 MKRLKV 473 90 475 LKRPKD 480 91 468 MKRLKV 473 92 475 LKRPKD 480 Ewings sarcoma EWS-Flil 93 347 QRKSKP 352 94 347 QRKSKP 352 NUF2R 95 455 LKRKMFKM 462 96 331 LKKLKT 336 97 347 VKKEKL 352 98 331 LKKLKT 336 99 347 VKKEKL 352 Kruppel-like zinc finger protein 100 22 EKRERT 27 GLIS2 101 22 EKRERT 27 FKSG32 202 15 LKRVRE 20 103 431 VRRGRI 436 104 15 LKRVRE 20 105 431 VRRGRI 436 BARH-LIKE 1 PROTEIN 106 175 LKKPRK 180 107 228 NRRTKW 233 108 175 LKKPRK 180 109 228 NRRTKW 233 Nucleolar GTP-binding protein 1 110 393 SRKKRERD 400 111 624 GKRKAGKK 631 112 48 MRKVKF 53 113 141 IKRQKQ 146 114 383 ARRKRM 388 115 393 SRKKRE 398 116 490 KKKLKI 495 117 543 ARRSRS 548 118 550 TRKRKR 555 119 586 VKKAKT 591 120 629 GKKDRR 634 121 48 MRKVKF 53 122 141 IKRQKQ 146 123 383 ARRKRM 388 124 393 SRKKRE 398 125 490 KKKLKI 495 126 543 ARRSRS 548 127 550 TRKRKR 555 128 586 VKKAKT 591 129 629 GKKDRR 634 EVG1 130 17 RRRPKT 22 131 138 ERKRKA 143 132 17 RRRPKT 22 133 138 ERKRKA 143 ASPL 134 282 PKKSKS 287 135 282 PKKSKS 287 Zinc transporter 1 136 477 EKKPRR 482 137 477 EKKPRR 482 Uveal autoantigen 138 603 EKKGRK 608 139 995 ERKFKA 1000 140 1023 VKKNKQ 1028 141 603 EKKGRK 608 142 995 ERKFKA 1000 143 1023 VKKNKQ 1028 RAB39 144 7 VRRDRV 12 145 7 VRRDRV 12 Down syndrome cell adhesion molecule 146 320 PRKVKS 325 147 387 VRKDKL 392 148 320 PRKVKS 325 149 387 VRKDKL 392 Protein-tyrosine phosphatase, non-receptor 150 139 GRKKCERY 146 type 12 151 59 VKKNRY 64 152 59 VKKNRY 64 WD-repeat protein 11 153 752 VRKIRF 757 154 752 VRKIRF 757 Gastric cancer-related protein 155 20 SRKRQTRR 27 VRG107 156 25 TRRRRN 30 157 25 TRRRRN 30 Early growth response protein 4 158 356 ARRKGRRG 363 159 452 EKKRHSKV 459 160 357 RRKGRR 362 161 357 RRKGRR 362 Vesicle transport-related protein 162 309 PKRKNKKS 316 163 226 DKKLRE 231 164 310 KRKNKK 315 165 355 VKRLKS 360 166 226 DKKLRE 231 167 310 KRKNKK 315 168 355 VKRLKS 360 UPF3X 169 140 AKKKTKKR 147 170 141 KKKTKK 146 171 217 ERRRRE 222 172 225 RKRQRE 230 173 233 RRKWKE 238 174 240 EKRKRK 245 175 296 DKREKA 301 176 373 RRRQKE 378 177 393 MKKEKD 398 178 426 VKRDRI 431 179 140 AKKKTKKRD 148 180 141 KKKTKK 146 181 217 ERRRRE 222 182 225 RKRQRE 230 183 233 RRKWKE 238 184 240 EKRKRK 245 185 296 DKREKA 301 186 373 RRRQKE 378 187 393 MKKEKD 398 188 426 VKRDRI 431 CGI-201 protein, type IV 189 49 ARRTRS 54 190 49 ARRTRS 54 RING finger protein 23 191 98 KRKIRD 103 192 98 KRKIRD 103 FKSG17 193 72 EKKARK 77 194 95 IRKSKN 100 195 72 EKKARK 77 196 95 IRKSKN 100 P83 197 681 ARKERE 686 198 681 ARKERE 686 Ovarian cancer-related protein 1 199 62 LKRDRF 67 200 62 LKRDRF 67 MHC class II transactivator CIITA 201 407 HRRPRE 412 202 741 PRKKRP 746 203 783 DRKQKV 788 204 407 HRRPRE 412 205 741 PRKKRP 746 206 783 DRKQKV 788 Platelet glycoprotein VI-2 207 275 SRRKRLRH 282 208 275 SRRKRL 280 209 275 SRRKRL 280 Ubiquitin-like 5 protein 210 11 GKKVRV 16 211 11 GKKVRV 16 Protein kinase D2 212 191 ARKRRL 196 213 191 ARKRRL 196 Homeobox protein GSH-2 214 202 GKRMRT 207 215 252 NRRVKH 257 216 202 GKRMRT 207 217 252 NRRVKH 257 ULBP3 protein 218 166 ARRMKE 171 219 201 HRKKRL 206 220 166 ARRMKE 171 221 201 HRKKRL 206 Type II iodothyronine deiodinase 222 87 SKKEKV 92 223 87 SKKEKV 92 224 299 SKRCKK 304 225 299 SKRCKK 304 Sperm antigen 226 160 LKKYKE 165 227 478 IKRLKE 483 228 160 LKKYKEKRT 168 229 160 LKKYKE 165 230 478 IKRLKE 483 UDP-GalNAc: polypeptide N- 231 4 ARKIRT 9

acetylgalactosaminyltransferase 232 44 DRRVRS 49 233 138 PRKCRQ 143 234 4 ARKIRT 9 235 44 DRRVRS 49 236 138 PRKCRQ 143 NCBE 237 62 HRRHRH 67 238 73 RKRDRE 78 239 1012 SKKKKL 1017 240 62 HRRHRH 67 241 73 RKRDRE 78 242 1012 SKKKKL 1017 WD repeat protein 243 372 LKKKEERL 379 244 384 EKKQRR 389 245 400 AKKMRP 405 246 384 EKKQRR 389 247 400 AKKMRP 405 Phosphodiesterase 11A 248 27 MRKGKQ 32 249 27 MRKGKQ 32 Probable cation-transporting ATPase 2 250 891 ERRRRPRD 898 251 306 SRKWRP 311 252 891 ERRRRP 896 253 306 SRKWRP 311 254 891 ERRRRP 896 HMG-box transcription factor TCF-3 255 420 GKKKKRKR 427 256 399 ARKERQ 404 257 420 GKKKKR 425 258 420 GKKKKRKRE 428 259 399 ARKERQ 404 260 420 GKKKKR 425 HVPS11 261 793 VRRYRE 798 262 793 VRRYRE 798 PIST 263 165 NKKEKM 170 264 165 NKKEKM 170 FYN-binding protein 265 473 KKREKE 478 266 501 KKKFKL 506 267 682 LKKLKK 687 268 696 RKKFKY 701 269 473 KKREKE 478 270 501 KKKFKL 506 271 682 LKKLKK 687 272 696 RKKFKY 701 C1orf25 273 620 GKKQKT 625 274 620 GKKQKT 625 C1orf14 275 441 LRRRKGKR 448 276 70 LRRWRR 75 277 441 LRRRKG 446 278 70 LRRWRR 75 279 441 LRRRKG 446 T-box transcription factor TBX3 280 144 DKKAKY 149 281 309 GRREKR 314 282 144 DKKAKY 149 283 309 GRREKR 314 Mitochondrial 39S ribosomal protein L47 284 121 AKRQRL 126 285 216 EKRARI 221 286 230 RKKAKI 235 287 121 AKRQRL 126 288 216 EKRARI 221 289 230 RKKAKI 235 CGI-203 290 33 VRRIRD 38 291 33 VRRIRD 38 Jagged1 292 1093 LRKRRK 1098 293 1093 LRKRRK 1098 Secretory carrier-associated 294 102 DRRERE 107 membrane protein 1 295 102 DRRERE 107 Vitamin D receptor-interacting 296 673 KKKKSSRL 680 protein complex component DRIP205 297 672 TKKKKS 677 298 954 QKRVKE 959 299 978 GKRSRT 983 300 995 PKRKKA 1000 301 1338 GKREKS 1343 302 1482 HKKHKK 1487 303 1489 KKKVKD 1494 304 672 TKKKKS 677 305 954 QKRVKE 959 306 978 GKRSRT 983 307 995 PKRKKA 1000 308 1338 GKREKS 1343 309 1482 HKKHKK 1487 310 1489 KKKVKD 1494 Secretory carrier-associated 311 100 ERKERE 105 membrane protein 2 312 100 ERKERE 105 Nogo receptor 313 420 SRKNRT 425 314 420 SRKNRT 425 FLAMINGO 1 315 169 GRRKRN 174 316 2231 ARRQRR 2236 317 169 GRRKRN 174 318 2231 ARRQRR 2236 CC-chemokine receptor 319 58 CKRLKS 63 320 58 CKRLKS 63 Prolactin regulatory element-binding protein 321 271 HKRLRQ 276 322 271 HKRLRQ 276 Kappa B and V(D)J recombination 323 17 PRKRLTKG 24 signal sequences binding protein 324 713 RKRRKEKS 720 325 903 PKKKRLRL 910 326 180 HKKERK 185 327 629 TKKTKK 634 328 712 LRKRRK 717 329 903 PKKKRL 908 330 1447 QKRVKE 1452 331 1680 SRKPRM 1685 332 180 HKKERK 185 333 629 TKKTKK 634 334 712 LRKRRK 717 335 903 PKKKRL 908 336 1447 QKRVKE 1452 337 1680 SRKPRM 1685 Breast cancer metastasis-suppressor 1 338 200 SKRKKA 205 339 229 IKKARA 234 340 200 SKRKKA 205 341 229 IKKARA 234 Forkhead box protein P3 342 414 RKKRSQRP 421 343 413 FRKKRS 418 344 413 FRKKRS 418 FAS BINDING PROTEIN 345 228 LKRKLIRL 235 346 391 RKKRRARL 398 347 358 ARRLRE 363 348 390 ERKKRR 395 349 629 CKKSRK 634 350 358 ARRLRE 363 351 390 ERKKRR 395 352 629 CKKSRK 634 Ubiquitin carboxyl-terminal hydrolase 12 353 228 HKRMKV 233 354 244 LKRFKY 249 355 228 HKRMKV 233 356 244 LKRFKY 249 KIAA0472 protein 357 110 HRKPKL 115 358 110 HRKPKL 115 PNAS-101 359 68 LKRSRP 73 360 106 PRKSRR 111 361 68 LKRSRP 73 362 106 PRKSRR 111 PNAS-26 363 118 DRRTRL 123 364 118 DRRTRL 123 Myelin transcription factor 2 365 176 GRRKSERQ 183 Sodium/potassium-transporting ATPase 366 47 SRRFRC 52 gamma chain 367 55 NKKRRQ 60 368 47 SRRFRC 52 369 55 NKKRRQ 60 Mdm4 protein 370 441 EKRPRD 446 371 464 ARRLKK 469 372 441 EKRPRD 446 373 464 ARRLKK 469 G antigen family D 2 protein 374 87 QKKIRI 92 375 87 QKKIRI 92 NipSnap2 protein 376 153 FRKARS 158 377 153 FRKARS 158 Stannin 378 73 ERKAKL 78 379 73 ERKAKL 78 Sodium bicarbonate cotransporter 380 973 EKKKKKKK 980 381 165 LRKHRH 170 382 666 LKKFKT 671 383 966 DKKKKE 971 384 973 EKKKKK 978 385 165 LRKHRH 170 386 666 LKKFKT 671 387 966 DKKKKE 971 388 973 EKKKKK 978 Myosin X 389 683 YKRYKV 688 390 828 EKKKRE 833 391 1653 LKRIRE 1658 392 1676 LKKTKC 1681 393 683 YKRYKV 688 394 828 EKKKRE 833 395 1653 LKRIRE 1658 396 1676 LKKTKC 1681 PNAS-20 397 21 RKRKSVRG 28 398 20 ERKRKS 25 399 20 ERKRKS 25 Pellino 400 36 RRKSRF 41 401 44 FKRPKA 49 402 36 RRKSRF 41 403 44 FKRPKA 49 Hyaluronan mediated motility receptor 404 66 ARKVKS 71 405 66 ARKVKS 71 Short transient receptor potential channel 7 406 753 FKKTRY 758 407 753 FKKTRY 758 Liprin-alpha2 408 825 PKKKGIKS 832 409 575 IRRPRR 580 410 748 LRKHRR 753 411 839 GKKEKA 844 412 875 DRRLKK 880 413 575 IRRPRR 580 414 748 LRKHRR 753 415 839 GKKEKA 844 416 875 DRRLKK 880 Transcription intermediary factor 1-alpha 417 904 DKRKCERL 911 418 1035 PRKKRLKS 1042 419 321 NKKGKA 326 420 1035 PRKKRL 1040 421 321 NKKGKA 326 422 1035 PRKKRL 1040 CARTILAGE INTERMEDIATE LAYER PROTEIN 423 719 QRRNKR 724 424 719 QRRNKR 724 UBX domain-containing protein 1 425 194 YRKIKL 199 426 194 YRKIKL 199 Arachidonate 12-lipoxygenase, 12R type 427 166 VRRHRN 171 428 233 WKRLKD 238 429 166 VRRHRN 171 430 233 WKRLKD 238 Hematopoietic PBX-interacting protein 431 159 LRRRRGRE 166 432 698 LKKRSGKK 705 433 159 LRRRRG 164 434 703 GKKDKH 708 435 159 LRRRRG 164 436 703 GKKDKH 708

NAG18 437 28 LKKKKK 33 438 28 LKKKKK 33 POU 5 domain protein 439 222 ARKRKR 227 440 222 ARKRKR 227 NRCAM PROTEIN 441 2 PKKKRL 7 442 887 SKRNRR 892 443 1185 IRRNKG 1190 444 1273 GKKEKE 1278 445 2 PKKKRL 7 446 887 SKRNRR 892 447 1185 IRRNKG 1190 448 1273 GKKEKE 1278 protocadherin gamma cluster 449 11 TRRSRA 16 450 11 TRRSRA 16 SKD1 protein 451 288 IRRRFEKR 295 452 251 ARRIKT 256 453 362 FKKVRG 367 454 251 ARRIKT 256 455 362 FKKVRG 367 ANTI-DEATH PROTEIN 456 58 HRKRSRRV 65 457 59 RKRSRR 64 458 59 RKRSRR 64 Centrin 3 459 14 TKRKKRRE 21 460 14 TKRKKR 19 461 14 TKRKKR 19 Ectonucleoside triphosphate 462 512 TRRKRH 517 diphosphohydrolase 3 463 512 TRRKRH 517 Homeobox protein prophet of PIT-1 464 12 PKKGRV 17 465 69 RRRHRT 74 466 119 NRRAKQ 124 467 12 PKKGRV 17 468 69 RRRHRT 74 469 119 NRRAKQ 124 PROSTAGLANDIN EP3 RECEPTOR 470 77 YRRRESKR 84 471 389 MRKRRLRE 396 472 82 SKRKKS 87 473 389 MRKRRL 394 474 82 SKRKKS 87 475 389 MRKRRL 394 Pituitary homeobox 3 476 58 LKKKQRRQ 65 477 59 KKKQRR 64 478 112 NRRAKW 117 479 118 RKRERS 123 480 59 KKKQRR 64 481 112 NRRAKW 117 482 118 RKRERS 123 HPRL-3 483 136 KRRGRI 141 484 136 KRRGRI 141 Advillin 485 812 MKKEKG 817 486 812 MKKEKG 817 Nuclear LIM interactor-interacting factor 1 487 32 GRRARP 37 488 109 LKKQRS 114 489 32 GRRARP 37 490 109 LKKQRS 114 Core histone macro-H2A.1 491 5 GKKKSTKT 12 492 114 AKKRGSKG 121 493 70 NKKGRV 75 494 132 AKKAKS 137 495 154 ARKSKK 159 496 302 DKKLKS 307 497 70 NKKGRV 75 498 132 AKKAKS 137 499 154 ARKSKK 159 500 302 DKKLKS 307 Villin-like protein 501 180 KRRRNQKL 187 502 179 EKRRRN 184 503 179 EKRRRN 184 BETA-FILAMIN 504 254 PKKARA 259 505 2002 ARRAKV 2007 506 254 PKKARA 259 507 2002 ARRAKV 2007 Tripartite motif protein TRIM31 508 290 LKKFKD 295 alpha 509 290 LKKFKD 295 Nuclear receptor co-repressor 1 510 106 SKRPRL 111 511 299 ARKQRE 304 512 330 RRKAKE 335 513 349 IRKQRE 354 514 412 QRRVKF 417 515 497 KRRGRN 502 516 580 RRKGRI 585 517 687 SRKPRE 692 518 2332 SRKSKS 2337 519 106 SKRPRL 111 520 299 ARKQRE 304 521 330 RRKAKE 335 522 349 IRKQRE 354 523 412 QRRVKF 417 524 497 KRRGRN 502 525 580 RRKGRI 585 526 687 SRKPRE 692 527 2332 SRKSKS 2337 BRAIN EXPRESSED RING FINGER PROTEIN 528 432 KRRVKS 437 529 432 KRRVKS 437 PB39 530 231 TKKIKL 236 531 231 TKKIKL 236 Sperm acrosomal protein 532 48 FRKRMEKE 55 533 24 RRKARE 29 534 135 KRKLKE 140 535 213 KKRLRQ 218 536 24 RRKARE 29 537 135 KRKLKE 140 538 213 KKRLRQ 218 VESICLE TRAFFICKING PROTEIN SEC22B 539 177 SKKYRQ 182 540 177 SKKYRQ 182 Nucleolar transcription factor 1 541 79 VRKFRT 84 542 102 GKKLKK 107 543 125 EKRAKY 130 544 147 SKKYKE 152 545 156 KKKMKY 161 546 240 KKRLKW 245 547 451 KKKAKY 456 548 523 EKKEKL 528 549 558 SKKMKF 563 550 79 VRKFRT 84 551 102 GKKLKK 107 552 125 EKRAKY 130 553 147 SKKYKE 152 554 156 KKKMKY 161 555 240 KKRLKW 245 556 451 KKKAKY 456 557 523 EKKEKL 528 558 558 SKKMKF 563 Plexin-B3 559 248 FRRRGARA 255 Junctophilin type3 560 626 QKRRYSKG 633 Plaucible mixed-lineage kinase protein 561 773 YRKKPHRP 780 562 312 ERRLKM 317 563 312 ERRLKM 317 fatty acid binding protein 4, adipocyte 564 78 DRKVKS 83 565 105 IKRKRE 110 566 78 DRKVKS 83 567 105 IKRKRE 110 exostoses (multiple) 1 568 78 SKKGRK 83 569 78 SKKGRK 83 DHHC-domain-containing cysteine-rich protein 570 64 HRRPRG 69 571 64 HRRPRG 69 Myb proto-oncogene protein 572 2 ARRPRH 7 573 292 EKRIKE 297 574 523 LKKIKQ 528 575 2 ARRPRH 7 576 292 EKRIKE 297 577 523 LKKIKQ 528 Long-chain-fatty-acid--CoA ligase 2 578 259 RRKPKP 264 579 259 RRKPKP 264 syntaxinlB2 580 260 ARRKKI 265 581 260 ARRKKI 265 Dachshund 2 582 162 ARRKRQ 167 583 516 QKRLKK 521 584 522 EKKTKR 527 585 162 ARRKRQ 167 586 516 QKRLKK 521 587 522 EKKTKR 527 DEAD/DEXH helicase DDX31 588 344 EKRKSEKA 351 589 760 TRKKRK 765 590 760 TRKKRK 765 Androgen receptor 591 628 ARKLKK 633 592 628 ARKLKK 633 Retinoic acid receptor alpha 593 364 RKRRPSRP 371 594 163 NKKKKE 168 595 363 VRKRRP 368 596 163 NKKKKE 168 597 363 VRKRRP 368 Kinesin heavy chain 598 340 WKKKYEKE 347 599 605 VKRCKQ 610 600 864 EKRLRA 869 601 605 VKRCKQ 610 602 864 EKRLRA 869 DIUBIQUITIN 603 30 VKKIKE 35 604 30 VKKIKE 35 BING1 PROTEIN 605 519 NKKFKM 524 606 564 ERRHRL 569 607 519 NKKFKM 524 608 564 ERRHRL 569 Focal adhesion kinase 1 609 664 SRRPRF 669 610 664 SRRPRF 669 EBN2 PROTEIN 611 20 TKRKKPRR 27 612 13 PKKDKL 18 613 20 TKRKKP 25 614 47 NKKNRE 52 615 64 LKKSRI 69 616 76 PKKPRE 81 617 493 SRKQRQ 498 618 566 VKRKRK 571 619 13 PKKDKL 18 620 20 TKRKKP 25 621 47 NKKNRE 52 622 64 LKKSRI 69 623 76 PKKPRE 81 624 493 SRKQRQ 498 625 566 VKRKRK 571 CO16 PROTEIN 626 33 ARRLRR 38 627 115 PRRCKW 120 628 33 ARRLRR 38 629 115 PRRCKW 120 KYNURENINE 3-MONOOXYGENASE 630 178 MKKPRF 183 631 178 MKKPRF 183 MLN 51 protein 632 4 RRRQRA 9 633 255 PRRIRK 260 634 407 ARRTRT 412 635 4 RRRQRA 9 636 255 PRRIRK 260 637 407 ARRTRT 412 MHC class II antigen 638 99 QKRGRV 104 MHC class II antigen 639 99 QKRGRV 104 Transforming acidic coiled-coil-containing 640 225 SRRSKL 230 protein 1 641 455 PKKAKS 460

642 225 SRRSKL 230 643 455 PKKAKS 460 Neuro-endocrine specific protein VGF 644 479 EKRNRK 484 645 479 EKRNRK 484 Organic cation transporter 646 230 GRRYRR 235 647 535 PRKNKE 540 648 230 GRRYRR 235 649 535 PRKNKE 540 DNA polymerase theta 650 215 KRRKHLKR 222 651 214 WKRRKH 219 652 220 LKRSRD 225 653 1340 GRKLRL 1345 654 1689 SRKRKL 1694 655 214 WKRRKH 219 656 220 LKRSRD 225 657 1340 GRKLRL 1345 658 1689 SRKRKL 1694 CDC45-related protein 659 169 MRRRQRRE 176 660 155 EKRTRL 160 661 170 RRRQRR 175 662 483 NRRCKL 488 663 155 EKRTRL 160 664 170 RRRQRR 175 665 483 NRRCKL 488 Chloride intracellular channel protein 2 666 197 AKKYRD 202 667 197 AKKYRD 202 Methyl-CpG binding protein 668 85 KRKKPSRP 92 669 83 SKKRKK 88 670 318 QKRQKC 323 671 354 YRRRKR 359 672 83 SKKRKK 88 673 318 QKRQKC 323 674 354 YRRRKR 359 Protein kinase C, eta type 675 155 RKRQRA 160 676 155 RKRQRA 160 Heterogeneous nuclear 677 71 LKKDRE 76 ribonucleoprotein H 678 169 LKKHKE 174 679 71 LKKDRE 76 680 169 LKKHKE 174 ORF2 681 11 SRRTRW 16 682 155 ERRRKF 160 683 185 LRRCRA 190 684 530 SRRSRS 535 685 537 GRRRKS 542 686 742 ERRAKQ 747 687 11 SRRTRW 16 688 155 ERRRKF 160 689 185 LRRCRA 190 690 530 SRRSRS 535 691 537 GRRRKS 542 692 742 ERRAKQ 747 F-box only protein 24 693 9 LRRRRVKR 16 694 9 LRRRRV 14 695 29 EKRGKG 34 696 9 LRRRRV 14 697 29 EKRGKG 34 Leucin rich neuronal protein 698 51 NRRLKH 56 699 51 NRRLKH 56 RER1 protein 700 181 KRRYRG 186 701 181 KRRYRG 186 Nephrocystin 702 3 ARRQRD 8 703 430 PKKPKT 435 704 557 NRRSRN 562 705 641 EKRDKE 646 706 3 ARRQRD 8 707 430 PKKPKT 435 708 557 NRRSRN 562 709 641 EKRDKE 646 Adenylate kinase isoenzyme 2, mitochondrial 710 60 GKKLKA 65 711 116 KRKEKL 121 712 60 GKKLKA 65 713 116 KRKEKL 121 Chlordecone reductase 714 245 AKKHKR 250 715 245 AKKHKR 250 Metaxin 2 716 166 KRKMKA 171 717 166 KRKMKA 171 Paired mesoderm homeobox protein1 718 89 KKKRKQRR 96 719 88 EKKKRK 93 720 94 QRRNRT 99 721 144 NRRAKF 149 722 88 EKKKRK 93 723 94 QRRNRT 99 724 144 NRRAKF 149 Ring finger protein 725 174 LKRKWIRC 181 726 8 TRKIKL 13 727 95 MRKQRE 100 728 8 TRKIKL 13 729 95 MRKQRE 100 Ataxin 7 730 55 PRRTRP 60 731 377 GRRKRF 382 732 704 GKKRKN 709 733 834 GKKRKC 839 734 55 PRRTRP 60 735 377 GRRKRF 382 736 704 GKKRKN 709 737 834 GKKRKC 839 Growth-arrest-specific protein 1 738 169 ARRRCDRD 176 SKAP55 protein 739 115 EKKSKD 120 740 115 EKKSKD 120 Serine palmitoyltransferase 1 741 232 PRKARV 237 742 232 PRKARV 237 Serine palmitoyltransferase 2 743 334 KKKYKA 339 744 450 RRRLKE 455 745 334 KKKYKA 339 746 450 RRRLKE 455 Synaptopodin 747 405 KRRQRD 410 748 405 KRRQRD 410 Alpha-tectorin 749 1446 TRRCRC 1451 750 2080 IRRKRL 2085 751 1446 TRRCRC 1451 752 2080 IRRKRL 2085 LONG FORM TRANSCRIPTION FACTOR C-MAF 753 291 QKRRTLKN 298 Usher syndrome type IIa protein 754 1285 MRRLRS 1290 755 1285 MRRLRS 1290 MSin3A associated polypeptide p30 756 95 QKKVKI 100 757 124 NRRKRK 129 758 158 LRRYKR 163 759 95 QKKVKI 100 760 124 NRRKRK 129 761 158 LRRYKR 163 Ig delta chain C region 762 142 KKKEKE 147 763 142 KKKEKE 147 THYROID HORMONE RECEPTOR-ASSOCIATED 764 383 AKRKADRE 390 PROTEIN COMPLEX COMPONENT TRAP100 765 833 KKRHRE 838 766 833 KKRHRE 838 P60 katanin 767 369 LRRRLEKR 376 768 326 SRRVKA 331 769 326 SRRVKA 331 Transcription factor jun-D 770 286 RKRKLERI 293 771 273 RKRLRN 278 772 285 CRKRKL 290 773 273 RKRLRN 278 774 285 CRKRKL 290 Sterol/retinol dehydrogenase 775 152 VRKARG 157 776 152 VRKARG 157 Glycogen [starch] synthase, liver 777 554 DRRFRS 559 778 578 SRRQRI 583 779 554 DRRFRS 559 780 578 SRRQRI 583 Estrogen-related receptor gamma 781 173 TKRRRK 178 782 353 VKKYKS 358 783 173 TKRRRK 178 784 353 VKKYKS 358 Neural retina-specific leucine zipper protein 785 162 QRRRTLKN 169 Cytosolic phospholipase A2-gamma 786 514 NKKKILRE 521 787 31 LKKLRI 36 788 218 FKKGRL 223 789 428 CRRHKI 433 790 31 LKKLRI 36 Cytosolic phospholipase A2-gamma 791 218 FKKGRL 223 792 428 CRRHKI 433 GLE1 793 415 AKKIKM 420 794 415 AKKIKM 420 Multiple exostoses type II protein 795 296 VRKRCHKH 303 EXT2.I 796 659 RKKFKC 664 797 659 RKKFKC 664 Cyclic-AMP-dependent transcription 798 86 EKKARS 91 factor ATF-7 799 332 GRRRRT 337 800 344 ERRQRF 349 801 86 EKKARS 91 802 332 GRRRRT 337 803 344 ERRQRF 349 Protein kinase/endoribonulcease 804 886 LRKFRT 891 805 886 LRKFRT 891 Transcription factor E2F6 806 23 RRRCRD 28 807 59 VKRPRF 64 808 98 VRKRRV 103 809 117 EKKSKN 122 810 23 RRRCRD 28 811 59 VKRPRF 64 812 98 VRKRRV 103 813 117 EKKSKN 122 MAP kinase-activating death domain protein 814 1333 IRKKVRRL 1340 815 160 KRRAKA 165 816 943 MKKVRR 948 817 1034 DKRKRS 1039 818 1334 RKKVRR 1339 819 1453 TKKCRE 1458 820 160 KRRAKA 165 821 943 MKKVRR 948 822 1034 DKRKRS 1039 823 1334 RKKVRR 1339 824 1453 TKKCRE 1458 Orphan nuclear receptor PXR 825 126 KRKKSERT 133 826 87 TRKTRR 92 827 125 IKRKKS 130 828 87 TRKTRR 92 829 125 IKRKKS 130 LENS EPITHELIUM-DERIVED GROWTH FACTOR 830 149 RKRKAEKQ 156 831 286 KKRKGGRN 293 832 145 ARRGRK 150 833 178 PKRGRP 183 834 285 EKKRKG 290 835 313 DRKRKQ 318 836 400 LKKIRR 405 837 337 VKKVEKKRE 345 838 145 ARRGRK 150 839 178 PKRGRP 183 840 285 EKKRKG 290 841 313 DRKRKQ 318 842 400 LKKIRR 405 LIM homeobox protein cofactor 843 255 TKRRKRKN 262 844 255 TKRRKR 260 845 255 TKRRKR 260

MULTIPLE MEMBRANE SPANNING RECEPTOR TRC8 846 229 WKRIRF 234 847 229 WKRIRF 234 Transcription factor SUPT3H 848 172 DKKKLRRL 179 849 169 MRKDKK 174 850 213 NKRQKI 218 851 169 MRKDKK 174 852 213 NKRQKI 218 GEMININ 853 50 KRKHRN 55 854 104 EKRRKA 109 855 50 KRKHRN 55 856 104 EKRRKA 109 Cell cycle-regulated factor p78 857 165 EKKKVSKA 172 858 124 IKRKKF 129 859 188 TKRVKK 193 860 381 DRRQKR 386 861 124 IKRKKF 129 862 188 TKRVKK 193 863 381 DRRQKR 386 lymphocyte antigen 6 complex, locus D 864 61 QRKGRK 66 865 85 ARRLRA 90 866 61 QRKGRK 66 867 85 ARRLRA 90 Delta 1-pyrroline-5-carboxylate synthetase 868 455 LRRTRI 460 869 455 LRRTRI 460 B CELL LINKER PROTEIN BLNK 870 36 IKKLKV 41 871 36 IKKLKV 41 B CELL LINKER PROTEIN BLNK-S 872 36 IKKLKV 41 873 36 IKKLKV 41 fetal Alzheimer antigen 874 5 ARRRRKRR 12 875 16 PRRRRRRT 23 876 93 WKKKTSRP 100 877 5 ARRRRK 10 878 16 PRRRRR 21 879 26 PRRPRI 31 880 35 TRRMRW 40 881 5 ARRRRK 10 882 16 PRRRRR 21 883 26 PRRPRI 31 884 35 TRRMRW 40 Transient receptor potential channel 885 4 zeta splice variant 505 CKKKMRRK 512 886 506 KKKMRR 511 887 676 HRRSKQ 681 888 506 KKKMRR 511 889 676 HRRSKQ 681 Myofibrillogenesis regulator MR-2 890 65 RKRGKN 70 891 65 RKRGKN 70 SH2 domain-containing phosphatase 892 269 IKKRSLRS 276 anchor protein 2c 893 394 SKRPKN 399 immunoglobulin superfamily, member 3 894 394 SKRPKN 399 Meis (mouse) homolog 3 895 112 PRRSRR 117 896 120 WRRTRG 125 897 112 PRRSRR 117 898 120 WRRTRG 125 Deleted in azoospermia 2 899 105 GKKLKL 110 900 114 IRKQKL 119 901 105 GKKLKL 110 902 114 IRKQKL 119 Centaurin gamma3 903 543 NRKKHRRK 550 904 544 RKKHRR 549 905 544 RKKHRR 549 Pre-B-cell leukemia transcription factor-1 906 233 ARRKRR 238 907 286 NKRIRY 291 908 233 ARRKRR 238 909 286 NKRIRY 291 60S ribosomal protein L13a 910 112 DKKKRM 117 911 158 KRKEKA 163 912 167 YRKKKQ 172 913 112 DKKKRM 117 914 158 KRKEKA 163 915 167 YRKKKQ 172 WD40-and FYVE-domain containing protein 3 916 388 IKRLKI 393 917 388 IKRLKI 393 LENG1 protein 918 34 RKRRGLRS 41 919 84 SRKKTRRM 91 920 1 MRRSRA 6 921 33 ERKRRG 38 922 85 RKKTRR 90 923 1 MRRSRA 6 924 33 ERKRRG 38 925 85 RKKTRR 90 MRIP2 926 375 NKKKHLKK 382 G protein-coupled receptor 927 430 EKKKLKRH 437 928 290 WKKKRA 295 929 395 RKKAKF 400 930 431 KKKLKR 436 931 290 WKKKRA 295 932 395 RKKAKF 400 933 431 KKKLKR 436 934 143 LKKFRQ 148 935 228 LRKIRT 233 936 143 LKKFRQ 148 937 228 LRKIRT 233 938 232 QKRRRHRA 239 939 232 QKRRRH 237 940 232 QKRRRH 237 Sperm ion channel 941 402 QKRKTGRL 409 A-kinase anchoring protein 942 2232 KRKKLVRD 2239 943 2601 EKRRRERE 2608 944 2788 EKKKKNKT 2795 945 370 RKKNKG 375 946 1763 SKKSKE 1768 947 2200 EKKVRL 2205 948 2231 LKRKKL 2236 949 2601 EKRRRE 2606 950 2785 EKKEKK 2790 951 1992 QKKDVVKRQ 2000 952 370 RKKNKG 375 953 1763 SKKSKE 1768 954 2200 EKKVRL 2205 955 2231 LKRKKL 2236 956 2601 EKRRRE 2606 957 2785 EKKEKK 2790 Lymphocyte-specific protein LSP1 958 315 GKRYKF 320 959 315 GKRYKF 320 similar to signaling lymphocytic 960 261 RRRGKT 266 activation molecule (H. sapiens) 961 261 RRRGKT 266 Dermatan-4-sulfotransferase-1 962 242 VRRYRA 247 963 242 VRRYRA 247 Moesin 964 291 MRRRKP 296 965 325 EKKKRE 330 966 291 MRRRKP 296 967 325 EKKKRE 330 A-Raf proto-oncogene 968 288 KKKVKN 293 serine/threonine-protein kinase 969 358 LRKTRH 363 970 288 KKKVKN 293 971 358 LRKTRH 363 Cytochrome P450 2C18 972 117 GKRWKE 122 973 117 GKRWKE 122 974 117 GKRWKE 122 975 156 LRKTKA 161 976 117 GKRWKE 122 977 156 LRKTKA 161 Protein tyrosine phosphatase, non-receptor 978 594 IRRRAVRS 601 type 3 979 263 FKRKKF 268 980 388 IRKPRH 393 981 874 VRKMRD 879 982 263 FKRKKF 268 983 388 IRKPRH 393 984 874 VRKMRD 879 similar to kallikrein 7 985 (chymotryptic, stratum corneum) 15 VKKVRL 20 986 15 VKKVRL 20 Hormone sensitive lipase 987 703 ARRLRN 708 988 703 ARRLRN 708 40S ribosomal protein S30 989 25 KKKKTGRA 32 990 23 EKKKKK 28 991 23 EKKKKK 28 Zinc finger protein 91 992 617 LRRHKR 622 993 617 LRRHKR 622 NNP-1 protein 994 320 NRKRLYKV 327 995 387 ERKRSRRR 394 996 432 QRRRTPRP 439 997 454 EKKKKRRE 461 998 29 VRKLRK 34 999 355 GRRQKK 360 1000 361 TKKQKR 366 1001 388 RKRSRR 393 1002 454 EKKKKR 459 1003 29 VRKLRK 34 1004 355 GRRQKK 360 1005 361 TKKQKR 366 1006 388 RKRSRR 393 1007 454 EKKKKR 459 Methionyl-tRNA synthetase 1008 725 WKRIKG 730 1009 725 WKRIKG 730 ELMO2 1010 560 NRRRQERF 567 Meningioma-expressed antigen 6/11 1011 432 RKRAKD 437 1012 432 RKRAKD 437 Inositol polyphosphate 4-phosphatase 1013 type I-beta 375 LRKKLHKF 382 1014 829 ARKNKN 834 1015 829 ARKNKN 834 1016 815 SKKRKN 820 1017 815 SKKRKN 820 C7ORF12 1018 40 SRRYRG 45 1019 338 HRKNKP 343 1020 40 SRRYRG 45 1021 338 HRKNKP 343 Rap guanine nucleotide exchange 1022 138 SRRRFRKI 145 factor 1023 1071 QRKKRWRS 1078 1024 1099 HKKRARRS 1106 1025 139 RRRFRK 144 1026 661 SKKVKA 666 1027 930 LKRMKI 935 1028 1071 QRKKRW 1076 1029 1100 KKRARR 1105 1030 1121 ARKVKQ 1126 1031 139 RRRFRK 144 1032 661 SKKVKA 666 1033 930 LKRMKI 935 1034 1071 QRKKRW 1076 1035 1100 KKRARR 1105 1036 1121 ARKVKQ 1126 Sigma 1C adaptin 1037 27 ERKKITRE 34 Alsin 1038 883 GRKRKE 888 1039 883 GRKRKE 888 NOPAR2 1040 14 LKRPRL 19 1041 720 VKREKP 725 1042 14 LKRPRL 19 1043 720 VKREKP 725 AT-binding transcription factor 1 1044 294 SKRPKT 299 1045 961 EKKNKL 966 1046 1231 NKRPRT 1236 1047 1727 DKRLRT 1732 1048 2032 QKRFRT 2037 1049 2087 EKKSKL 2092 1050 2317 QRKDKD 2322 1051 2343 PKKEKG 2348

1052 294 SKRPKT 299 1053 961 EKKNKL 966 1054 1231 NKRPRT 1236 1055 1727 DKRLRT 1732 1056 2032 QKRFRT 2037 1057 2087 EKKSKL 2092 1058 2317 QRKDKD 2322 1059 2343 PKKEKG 2348 Suppressin 1060 232 YKRRKK 237 1061 232 YKRRKK 237 Midline 1 protein 1062 100 TRRERA 105 1063 494 HRKLKV 499 1064 100 TRRERA 105 1065 494 HRKLKV 499 High mobility group protein 2a 1066 6PKKPKG11 1067 84GKKKKD89 1068 6PKKPKG11 1069 84GKKKKD89

[0100] This application claims priority to A 1952/2003 filed on Dec. 4, 2003, the entirety of which is hereby incorporated by reference.

Sequence CWU 1

107416PRTHomo sapiens 1Lys Ser Leu Ser Leu Ser1 527PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 2Ser Lys Ser Lys Lys Leu Ser1 536PRTHomo sapiens 3Trp Ile Ala Ser His Thr1 546PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 4Trp Lys Ala Lys His Lys1 558PRTHomo sapiens 5Met Leu Asp Ala Glu Arg Leu Lys1 568PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 6Met Lys Lys Ala Lys Arg Leu Lys1 5731DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 7caccatgtgt cagtgtataa agacatactc c 31855DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 8caccatgtgt cagtgtataa agacatactc caaacctagg caccccaaaa ggata 55921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 9ttatgaattc ctagccctct t 211021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 10ttatgaattc ttagccctct t 211121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 11ttatgacttc tcagccctct t 211221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 12ttatgacttc ttagccctct t 211321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 13ttatgacttc ctagccctct t 211421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 14ttatgacctc ttagccctct t 211521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 15ttatgacctc ctagccctct t 211672PRTHomo sapiens 16Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro1 5 10 15Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro 20 25 30His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu 35 40 45Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys 50 55 60Phe Leu Lys Arg Ala Glu Asn Ser65 701773PRTHomo sapiens 17Ala Ser Val Ala Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu Gln1 5 10 15Gly Ile His Pro Lys Asn Ile Gln Ser Val Asn Val Lys Ser Pro Gly 20 25 30Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Arg 35 40 45Lys Ala Cys Leu Asn Pro Ala Ser Pro Ile Val Lys Lys Ile Ile Glu 50 55 60Lys Met Leu Asn Ser Asp Lys Ser Asn65 701873PRTHomo sapiens 18Ala Pro Leu Ala Thr Glu Leu Arg Cys Gln Cys Leu Gln Thr Leu Gln1 5 10 15Gly Ile His Leu Lys Asn Ile Gln Ser Val Lys Val Lys Ser Pro Gly 20 25 30Pro His Cys Ala Gln Thr Glu Val Ile Ala Thr Leu Lys Asn Gly Gln 35 40 45Lys Ala Cys Leu Asn Pro Ala Ser Pro Met Val Lys Lys Ile Ile Glu 50 55 60Lys Met Leu Lys Asn Gly Lys Ser Asn65 701970PRTHomo sapiens 19Ala Glu Leu Arg Cys Leu Cys Ile Lys Thr Thr Ser Gly Ile His Pro1 5 10 15Lys Asn Ile Gln Ser Leu Glu Val Ile Gly Lys Gly Thr His Cys Asn 20 25 30Gln Val Glu Val Ile Ala Thr Leu Lys Asp Gly Arg Lys Ile Cys Leu 35 40 45Asp Pro Asp Ala Pro Arg Ile Lys Lys Ile Val Gln Lys Lys Leu Ala 50 55 60Gly Asp Glu Ser Ala Asp65 702070PRTHomo sapiens 20Glu Ala Glu Glu Asp Gly Asp Leu Gln Cys Leu Cys Val Lys Thr Thr1 5 10 15Ser Gln Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile Lys Ala 20 25 30Gly Pro His Cys Pro Thr Ala Gln Leu Ile Ala Thr Leu Lys Asn Gly 35 40 45Arg Lys Ile Cys Leu Asp Leu Gln Ala Pro Leu Tyr Lys Lys Ile Ile 50 55 60Lys Lys Leu Leu Glu Ser65 702167PRTHomo sapiens 21Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser1 5 10 15His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 30Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60Ala Leu Asn652268PRTHomo sapiens 22Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala1 5 10 15Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly 20 25 30Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln 35 40 45Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser 50 55 60Leu Glu Met Ser652376PRTHomo sapiens 23Gln Pro Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile1 5 10 15Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr 20 25 30Asn Ile Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly 35 40 45Lys Glu Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met 50 55 60Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro65 70 752476PRTHomo sapiens 24Gln Pro Val Gly Ile Asn Thr Ser Thr Thr Cys Cys Tyr Arg Phe Ile1 5 10 15Asn Lys Lys Ile Pro Lys Gln Arg Leu Glu Ser Tyr Arg Arg Thr Thr 20 25 30Ser Ser His Cys Pro Arg Glu Ala Val Ile Phe Lys Thr Lys Leu Asp 35 40 45Lys Glu Ile Cys Ala Asp Pro Thr Gln Lys Trp Val Gln Asp Phe Met 50 55 60Lys His Leu Asp Lys Lys Thr Gln Thr Pro Lys Leu65 70 752569PRTHomo sapiens 25Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser Tyr Thr Ser1 5 10 15Arg Gln Ile Pro Gln Asn Phe Ile Ala Asp Tyr Phe Glu Thr Ser Ser 20 25 30Gln Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Arg Ser Arg Gln 35 40 45Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gln Lys Tyr Val Ser Asp 50 55 60Leu Glu Leu Ser Ala652669PRTHomo sapiens 26Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser Tyr Thr1 5 10 15Ala Arg Lys Leu Pro Arg Asn Phe Val Val Asp Tyr Tyr Glu Thr Ser 20 25 30Ser Leu Cys Ser Gln Pro Ala Val Val Phe Gln Thr Lys Arg Ser Lys 35 40 45Gln Val Cys Ala Asp Pro Ser Glu Ser Trp Val Gln Glu Tyr Val Tyr 50 55 60Asp Leu Glu Leu Asn652777PRTHomo sapiens 27Met Asp Arg Phe His Ala Thr Ser Ala Asp Cys Cys Ile Ser Tyr Thr1 5 10 15Pro Arg Ser Ile Pro Cys Ser Leu Leu Glu Ser Tyr Phe Glu Thr Asn 20 25 30Ser Glu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg 35 40 45Arg Phe Cys Ala Asn Pro Ser Asp Lys Gln Val Gln Val Cys Met Arg 50 55 60Met Leu Lys Leu Asp Thr Arg Ile Lys Thr Arg Lys Asn65 70 752866PRTHomo sapiens 28His Phe Ala Ala Asp Cys Cys Thr Ser Tyr Ile Ser Gln Ser Ile Pro1 5 10 15Cys Ser Leu Met Lys Ser Tyr Phe Glu Thr Ser Ser Glu Cys Ser Lys 20 25 30Pro Gly Val Ile Phe Leu Thr Lys Lys Gly Arg Gln Val Cys Ala Lys 35 40 45Pro Ser Gly Pro Gly Val Gln Asp Cys Met Lys Lys Leu Lys Pro Tyr 50 55 60Ser Ile65296PRTHomo sapiens 29Thr Arg Arg Ala Lys Glu1 5306PRTHomo sapiens 30Gln Lys Lys Ile Arg Ser1 5316PRTHomo sapiens 31Pro Lys Arg Arg Lys Asn1 5326PRTHomo sapiens 32Thr Arg Arg Ala Lys Glu1 5336PRTHomo sapiens 33Gln Lys Lys Ile Arg Ser1 5346PRTHomo sapiens 34Pro Lys Arg Arg Lys Asn1 5358PRTHomo sapiens 35Ser Lys Arg Arg Gly Lys Lys Val1 5366PRTHomo sapiens 36Glu Arg Arg Leu Lys Met1 5376PRTHomo sapiens 37Lys Arg Arg Gly Lys Lys1 5386PRTHomo sapiens 38Glu Arg Arg Leu Lys Met1 5396PRTHomo sapiens 39Lys Arg Arg Gly Lys Lys1 5408PRTHomo sapiens 40Glu Lys Arg Arg Arg Ala Arg Ile1 5416PRTHomo sapiens 41Glu Lys Arg Arg Arg Ala1 5426PRTHomo sapiens 42Glu Lys Arg Arg Arg Ala1 5438PRTHomo sapiens 43Met Lys Lys Lys Lys Lys Lys Lys1 5446PRTHomo sapiens 44Met Lys Lys Lys Lys Lys1 5456PRTHomo sapiens 45Met Lys Lys Lys Lys Lys1 5468PRTHomo sapiens 46Met Lys Lys Lys Lys Lys Lys Lys1 5476PRTHomo sapiens 47Met Lys Lys Lys Lys Lys1 5486PRTHomo sapiens 48Met Lys Lys Lys Lys Lys1 5496PRTHomo sapiens 49Arg Arg Arg Leu Arg Ser1 5506PRTHomo sapiens 50Val Arg Lys Leu Lys Gly1 5516PRTHomo sapiens 51Leu Arg Lys Ala Arg Asp1 5526PRTHomo sapiens 52Arg Arg Lys Leu Arg Glu1 5536PRTHomo sapiens 53Ala Lys Lys Gly Lys Gly1 5546PRTHomo sapiens 54Ala Lys Lys Leu Arg Glu1 5556PRTHomo sapiens 55Arg Arg Arg Leu Arg Ser1 5566PRTHomo sapiens 56Val Arg Lys Leu Lys Gly1 5576PRTHomo sapiens 57Leu Arg Lys Ala Arg Asp1 5586PRTHomo sapiens 58Arg Arg Lys Leu Arg Glu1 5596PRTHomo sapiens 59Ala Lys Lys Gly Lys Gly1 5606PRTHomo sapiens 60Ala Lys Lys Leu Arg Glu1 5616PRTHomo sapiens 61Glu Lys Arg Thr Lys Asp1 5626PRTHomo sapiens 62Thr Lys Arg Lys Arg Val1 5636PRTHomo sapiens 63Trp Lys Lys Lys Lys Val1 5646PRTHomo sapiens 64Glu Lys Arg Thr Lys Asp1 5656PRTHomo sapiens 65Thr Lys Arg Lys Arg Val1 5666PRTHomo sapiens 66Trp Lys Lys Lys Lys Val1 5676PRTHomo sapiens 67Pro Arg Arg Ile Lys Ala1 5686PRTHomo sapiens 68Lys Arg Arg Thr Arg Val1 5696PRTHomo sapiens 69Pro Arg Arg Ile Lys Ala1 5706PRTHomo sapiens 70Lys Arg Arg Thr Arg Val1 5718PRTHomo sapiens 71Thr Lys Lys Lys Thr Arg Arg Arg1 5728PRTHomo sapiens 72Gly Lys Arg Arg Arg Arg Arg Gly1 5736PRTHomo sapiens 73Ala Arg Lys Gly Lys Arg1 5746PRTHomo sapiens 74Gly Lys Lys Thr Lys Lys1 5756PRTHomo sapiens 75Thr Arg Arg Arg Arg Ala1 5766PRTHomo sapiens 76Gly Lys Arg Arg Arg Arg1 5776PRTHomo sapiens 77Ala Arg Lys Gly Lys Arg1 5786PRTHomo sapiens 78Gly Lys Lys Thr Lys Lys1 5796PRTHomo sapiens 79Thr Arg Arg Arg Arg Ala1 5806PRTHomo sapiens 80Gly Lys Arg Arg Arg Arg1 5816PRTHomo sapiens 81Ala Arg Lys Gln Lys Ala1 5826PRTHomo sapiens 82Lys Lys Lys Ile Lys Glu1 5836PRTHomo sapiens 83Ala Arg Lys Gln Lys Ala1 5846PRTHomo sapiens 84Lys Lys Lys Ile Lys Glu1 5856PRTHomo sapiens 85Val Arg Arg Cys Lys Ile1 5866PRTHomo sapiens 86Val Arg Arg Cys Lys Ile1 5876PRTHomo sapiens 87Glu Arg Arg Ala Arg Leu1 5886PRTHomo sapiens 88Glu Arg Arg Ala Arg Leu1 5896PRTHomo sapiens 89Met Lys Arg Leu Lys Val1 5906PRTHomo sapiens 90Leu Lys Arg Pro Lys Asp1 5916PRTHomo sapiens 91Met Lys Arg Leu Lys Val1 5926PRTHomo sapiens 92Leu Lys Arg Pro Lys Asp1 5936PRTHomo sapiens 93Gln Arg Lys Ser Lys Pro1 5946PRTHomo sapiens 94Gln Arg Lys Ser Lys Pro1 5958PRTHomo sapiens 95Leu Lys Arg Lys Met Phe Lys Met1 5966PRTHomo sapiens 96Leu Lys Lys Leu Lys Thr1 5976PRTHomo sapiens 97Val Lys Lys Glu Lys Leu1 5986PRTHomo sapiens 98Leu Lys Lys Leu Lys Thr1 5996PRTHomo sapiens 99Val Lys Lys Glu Lys Leu1 51006PRTHomo sapiens 100Glu Lys Arg Glu Arg Thr1 51016PRTHomo sapiens 101Glu Lys Arg Glu Arg Thr1 51026PRTHomo sapiens 102Leu Lys Arg Val Arg Glu1 51036PRTHomo sapiens 103Val Arg Arg Gly Arg Ile1 51046PRTHomo sapiens 104Leu Lys Arg Val Arg Glu1 51056PRTHomo sapiens 105Val Arg Arg Gly Arg Ile1 51066PRTHomo sapiens 106Leu Lys Lys Pro Arg Lys1 51076PRTHomo sapiens 107Asn Arg Arg Thr Lys Trp1 51086PRTHomo sapiens 108Leu Lys Lys Pro Arg Lys1 51096PRTHomo sapiens 109Asn Arg Arg Thr Lys Trp1 51108PRTHomo sapiens 110Ser Arg Lys Lys Arg Glu Arg Asp1 51118PRTHomo sapiens 111Gly Lys Arg Lys Ala Gly Lys Lys1 51126PRTHomo sapiens 112Met Arg Lys Val Lys Phe1 51136PRTHomo sapiens 113Ile Lys Arg Gln Lys Gln1 51146PRTHomo sapiens 114Ala Arg Arg Lys Arg Met1 51156PRTHomo sapiens 115Ser Arg Lys Lys Arg Glu1 51166PRTHomo sapiens 116Lys Lys Lys Leu Lys Ile1 51176PRTHomo sapiens 117Ala Arg Arg Ser Arg Ser1 51186PRTHomo sapiens 118Thr Arg Lys Arg Lys Arg1 51196PRTHomo sapiens 119Val Lys Lys Ala Lys Thr1 51206PRTHomo sapiens 120Gly Lys Lys Asp Arg Arg1 51216PRTHomo sapiens 121Met Arg Lys Val Lys Phe1 51226PRTHomo sapiens 122Ile Lys Arg Gln Lys Gln1 51236PRTHomo sapiens 123Ala Arg Arg Lys Arg Met1 51246PRTHomo sapiens 124Ser Arg Lys Lys Arg Glu1 51256PRTHomo sapiens 125Lys Lys Lys Leu Lys Ile1 51266PRTHomo sapiens 126Ala Arg Arg Ser Arg Ser1 51276PRTHomo sapiens 127Thr Arg Lys Arg Lys Arg1 51286PRTHomo sapiens 128Val Lys Lys Ala Lys Thr1 51296PRTHomo sapiens 129Gly Lys Lys Asp Arg Arg1 51306PRTHomo sapiens 130Arg Arg Arg Pro Lys Thr1 51316PRTHomo sapiens 131Glu Arg Lys Arg Lys Ala1 51326PRTHomo sapiens 132Arg Arg Arg Pro Lys Thr1 51336PRTHomo sapiens 133Glu Arg Lys Arg Lys Ala1 51346PRTHomo sapiens 134Pro Lys Lys Ser Lys Ser1 51356PRTHomo sapiens 135Pro Lys Lys Ser Lys Ser1 51366PRTHomo sapiens 136Glu Lys Lys Pro Arg Arg1 51376PRTHomo sapiens 137Glu Lys Lys Pro Arg Arg1 51386PRTHomo sapiens 138Glu Lys Lys Gly Arg Lys1 51396PRTHomo sapiens 139Glu Arg Lys Phe Lys Ala1 51406PRTHomo sapiens 140Val Lys Lys Asn Lys Gln1 51416PRTHomo sapiens 141Glu Lys Lys Gly Arg Lys1 51426PRTHomo sapiens 142Glu Arg Lys Phe Lys Ala1 51436PRTHomo sapiens 143Val Lys Lys Asn Lys Gln1 51446PRTHomo sapiens 144Val Arg Arg Asp Arg Val1 51456PRTHomo sapiens 145Val Arg Arg Asp Arg Val1 51466PRTHomo sapiens 146Pro Arg Lys Val Lys Ser1 51476PRTHomo sapiens 147Val Arg Lys Asp Lys Leu1 51486PRTHomo sapiens 148Pro Arg Lys Val Lys Ser1 51496PRTHomo sapiens 149Val Arg Lys Asp Lys Leu1 51508PRTHomo sapiens 150Gly Arg Lys Lys Cys Glu Arg Tyr1 51516PRTHomo sapiens 151Val Lys Lys Asn Arg Tyr1 51526PRTHomo sapiens 152Val Lys Lys Asn Arg Tyr1 51536PRTHomo sapiens 153Val Arg Lys Ile Arg Phe1 51546PRTHomo sapiens 154Val Arg Lys Ile Arg Phe1 51558PRTHomo sapiens 155Ser Arg Lys Arg Gln Thr Arg Arg1 51566PRTHomo sapiens 156Thr Arg Arg Arg Arg Asn1 51576PRTHomo sapiens 157Thr Arg Arg Arg Arg Asn1 51588PRTHomo sapiens 158Ala Arg Arg Lys Gly Arg Arg Gly1 51598PRTHomo sapiens 159Glu Lys Lys Arg His Ser Lys Val1 51606PRTHomo sapiens 160Arg Arg Lys Gly Arg Arg1 51616PRTHomo sapiens 161Arg Arg Lys Gly Arg Arg1 51628PRTHomo sapiens 162Pro Lys Arg Lys Asn Lys Lys Ser1 51636PRTHomo sapiens 163Asp Lys Lys Leu Arg Glu1 51646PRTHomo sapiens 164Lys Arg Lys Asn Lys Lys1 51656PRTHomo sapiens 165Val Lys Arg Leu Lys Ser1 51666PRTHomo sapiens 166Asp Lys Lys Leu Arg Glu1

51676PRTHomo sapiens 167Lys Arg Lys Asn Lys Lys1 51686PRTHomo sapiens 168Val Lys Arg Leu Lys Ser1 51698PRTHomo sapiens 169Ala Lys Lys Lys Thr Lys Lys Arg1 51706PRTHomo sapiens 170Lys Lys Lys Thr Lys Lys1 51716PRTHomo sapiens 171Glu Arg Arg Arg Arg Glu1 51726PRTHomo sapiens 172Arg Lys Arg Gln Arg Glu1 51736PRTHomo sapiens 173Arg Arg Lys Trp Lys Glu1 51746PRTHomo sapiens 174Glu Lys Arg Lys Arg Lys1 51756PRTHomo sapiens 175Asp Lys Arg Glu Lys Ala1 51766PRTHomo sapiens 176Arg Arg Arg Gln Lys Glu1 51776PRTHomo sapiens 177Met Lys Lys Glu Lys Asp1 51786PRTHomo sapiens 178Val Lys Arg Asp Arg Ile1 51799PRTHomo sapiens 179Ala Lys Lys Lys Thr Lys Lys Arg Asp1 51806PRTHomo sapiens 180Lys Lys Lys Thr Lys Lys1 51816PRTHomo sapiens 181Glu Arg Arg Arg Arg Glu1 51826PRTHomo sapiens 182Arg Lys Arg Gln Arg Glu1 51836PRTHomo sapiens 183Arg Arg Lys Trp Lys Glu1 51846PRTHomo sapiens 184Glu Lys Arg Lys Arg Lys1 51856PRTHomo sapiens 185Asp Lys Arg Glu Lys Ala1 51866PRTHomo sapiens 186Arg Arg Arg Gln Lys Glu1 51876PRTHomo sapiens 187Met Lys Lys Glu Lys Asp1 51886PRTHomo sapiens 188Val Lys Arg Asp Arg Ile1 51896PRTHomo sapiens 189Ala Arg Arg Thr Arg Ser1 51906PRTHomo sapiens 190Ala Arg Arg Thr Arg Ser1 51916PRTHomo sapiens 191Lys Arg Lys Ile Arg Asp1 51926PRTHomo sapiens 192Lys Arg Lys Ile Arg Asp1 51936PRTHomo sapiens 193Glu Lys Lys Ala Arg Lys1 51946PRTHomo sapiens 194Ile Arg Lys Ser Lys Asn1 51956PRTHomo sapiens 195Glu Lys Lys Ala Arg Lys1 51966PRTHomo sapiens 196Ile Arg Lys Ser Lys Asn1 51976PRTHomo sapiens 197Ala Arg Lys Glu Arg Glu1 51986PRTHomo sapiens 198Ala Arg Lys Glu Arg Glu1 51996PRTHomo sapiens 199Leu Lys Arg Asp Arg Phe1 52006PRTHomo sapiens 200Leu Lys Arg Asp Arg Phe1 52016PRTHomo sapiens 201His Arg Arg Pro Arg Glu1 52026PRTHomo sapiens 202Pro Arg Lys Lys Arg Pro1 52036PRTHomo sapiens 203Asp Arg Lys Gln Lys Val1 52046PRTHomo sapiens 204His Arg Arg Pro Arg Glu1 52056PRTHomo sapiens 205Pro Arg Lys Lys Arg Pro1 52066PRTHomo sapiens 206Asp Arg Lys Gln Lys Val1 52078PRTHomo sapiens 207Ser Arg Arg Lys Arg Leu Arg His1 52086PRTHomo sapiens 208Ser Arg Arg Lys Arg Leu1 52096PRTHomo sapiens 209Ser Arg Arg Lys Arg Leu1 52106PRTHomo sapiens 210Gly Lys Lys Val Arg Val1 52116PRTHomo sapiens 211Gly Lys Lys Val Arg Val1 52126PRTHomo sapiens 212Ala Arg Lys Arg Arg Leu1 52136PRTHomo sapiens 213Ala Arg Lys Arg Arg Leu1 52146PRTHomo sapiens 214Gly Lys Arg Met Arg Thr1 52156PRTHomo sapiens 215Asn Arg Arg Val Lys His1 52166PRTHomo sapiens 216Gly Lys Arg Met Arg Thr1 52176PRTHomo sapiens 217Asn Arg Arg Val Lys His1 52186PRTHomo sapiens 218Ala Arg Arg Met Lys Glu1 52196PRTHomo sapiens 219His Arg Lys Lys Arg Leu1 52206PRTHomo sapiens 220Ala Arg Arg Met Lys Glu1 52216PRTHomo sapiens 221His Arg Lys Lys Arg Leu1 52226PRTHomo sapiens 222Ser Lys Lys Glu Lys Val1 52236PRTHomo sapiens 223Ser Lys Lys Glu Lys Val1 52246PRTHomo sapiens 224Ser Lys Arg Cys Lys Lys1 52256PRTHomo sapiens 225Ser Lys Arg Cys Lys Lys1 52266PRTHomo sapiens 226Leu Lys Lys Tyr Lys Glu1 52276PRTHomo sapiens 227Ile Lys Arg Leu Lys Glu1 52289PRTHomo sapiens 228Leu Lys Lys Tyr Lys Glu Lys Arg Thr1 52296PRTHomo sapiens 229Leu Lys Lys Tyr Lys Glu1 52306PRTHomo sapiens 230Ile Lys Arg Leu Lys Glu1 52316PRTHomo sapiens 231Ala Arg Lys Ile Arg Thr1 52326PRTHomo sapiens 232Asp Arg Arg Val Arg Ser1 52336PRTHomo sapiens 233Pro Arg Lys Cys Arg Gln1 52346PRTHomo sapiens 234Ala Arg Lys Ile Arg Thr1 52356PRTHomo sapiens 235Asp Arg Arg Val Arg Ser1 52366PRTHomo sapiens 236Pro Arg Lys Cys Arg Gln1 52376PRTHomo sapiens 237His Arg Arg His Arg His1 52386PRTHomo sapiens 238Arg Lys Arg Asp Arg Glu1 52396PRTHomo sapiens 239Ser Lys Lys Lys Lys Leu1 52406PRTHomo sapiens 240His Arg Arg His Arg His1 52416PRTHomo sapiens 241Arg Lys Arg Asp Arg Glu1 52426PRTHomo sapiens 242Ser Lys Lys Lys Lys Leu1 52438PRTHomo sapiens 243Leu Lys Lys Lys Glu Glu Arg Leu1 52446PRTHomo sapiens 244Glu Lys Lys Gln Arg Arg1 52456PRTHomo sapiens 245Ala Lys Lys Met Arg Pro1 52466PRTHomo sapiens 246Glu Lys Lys Gln Arg Arg1 52476PRTHomo sapiens 247Ala Lys Lys Met Arg Pro1 52486PRTHomo sapiens 248Met Arg Lys Gly Lys Gln1 52496PRTHomo sapiens 249Met Arg Lys Gly Lys Gln1 52508PRTHomo sapiens 250Glu Arg Arg Arg Arg Pro Arg Asp1 52516PRTHomo sapiens 251Ser Arg Lys Trp Arg Pro1 52526PRTHomo sapiens 252Glu Arg Arg Arg Arg Pro1 52536PRTHomo sapiens 253Ser Arg Lys Trp Arg Pro1 52546PRTHomo sapiens 254Glu Arg Arg Arg Arg Pro1 52558PRTHomo sapiens 255Gly Lys Lys Lys Lys Arg Lys Arg1 52566PRTHomo sapiens 256Ala Arg Lys Glu Arg Gln1 52576PRTHomo sapiens 257Gly Lys Lys Lys Lys Arg1 52589PRTHomo sapiens 258Gly Lys Lys Lys Lys Arg Lys Arg Glu1 52596PRTHomo sapiens 259Ala Arg Lys Glu Arg Gln1 52606PRTHomo sapiens 260Gly Lys Lys Lys Lys Arg1 52616PRTHomo sapiens 261Val Arg Arg Tyr Arg Glu1 52626PRTHomo sapiens 262Val Arg Arg Tyr Arg Glu1 52636PRTHomo sapiens 263Asn Lys Lys Glu Lys Met1 52646PRTHomo sapiens 264Asn Lys Lys Glu Lys Met1 52656PRTHomo sapiens 265Lys Lys Arg Glu Lys Glu1 52666PRTHomo sapiens 266Lys Lys Lys Phe Lys Leu1 52676PRTHomo sapiens 267Leu Lys Lys Leu Lys Lys1 52686PRTHomo sapiens 268Arg Lys Lys Phe Lys Tyr1 52696PRTHomo sapiens 269Lys Lys Arg Glu Lys Glu1 52706PRTHomo sapiens 270Lys Lys Lys Phe Lys Leu1 52716PRTHomo sapiens 271Leu Lys Lys Leu Lys Lys1 52726PRTHomo sapiens 272Arg Lys Lys Phe Lys Tyr1 52736PRTHomo sapiens 273Gly Lys Lys Gln Lys Thr1 52746PRTHomo sapiens 274Gly Lys Lys Gln Lys Thr1 52758PRTHomo sapiens 275Leu Arg Arg Arg Lys Gly Lys Arg1 52766PRTHomo sapiens 276Leu Arg Arg Trp Arg Arg1 52776PRTHomo sapiens 277Leu Arg Arg Arg Lys Gly1 52786PRTHomo sapiens 278Leu Arg Arg Trp Arg Arg1 52796PRTHomo sapiens 279Leu Arg Arg Arg Lys Gly1 52806PRTHomo sapiens 280Asp Lys Lys Ala Lys Tyr1 52816PRTHomo sapiens 281Gly Arg Arg Glu Lys Arg1 52826PRTHomo sapiens 282Asp Lys Lys Ala Lys Tyr1 52836PRTHomo sapiens 283Gly Arg Arg Glu Lys Arg1 52846PRTHomo sapiens 284Ala Lys Arg Gln Arg Leu1 52856PRTHomo sapiens 285Glu Lys Arg Ala Arg Ile1 52866PRTHomo sapiens 286Arg Lys Lys Ala Lys Ile1 52876PRTHomo sapiens 287Ala Lys Arg Gln Arg Leu1 52886PRTHomo sapiens 288Glu Lys Arg Ala Arg Ile1 52896PRTHomo sapiens 289Arg Lys Lys Ala Lys Ile1 52906PRTHomo sapiens 290Val Arg Arg Ile Arg Asp1 52916PRTHomo sapiens 291Val Arg Arg Ile Arg Asp1 52926PRTHomo sapiens 292Leu Arg Lys Arg Arg Lys1 52936PRTHomo sapiens 293Leu Arg Lys Arg Arg Lys1 52946PRTHomo sapiens 294Asp Arg Arg Glu Arg Glu1 52956PRTHomo sapiens 295Asp Arg Arg Glu Arg Glu1 52968PRTHomo sapiens 296Lys Lys Lys Lys Ser Ser Arg Leu1 52976PRTHomo sapiens 297Thr Lys Lys Lys Lys Ser1 52986PRTHomo sapiens 298Gln Lys Arg Val Lys Glu1 52996PRTHomo sapiens 299Gly Lys Arg Ser Arg Thr1 53006PRTHomo sapiens 300Pro Lys Arg Lys Lys Ala1 53016PRTHomo sapiens 301Gly Lys Arg Glu Lys Ser1 53026PRTHomo sapiens 302His Lys Lys His Lys Lys1 53036PRTHomo sapiens 303Lys Lys Lys Val Lys Asp1 53046PRTHomo sapiens 304Thr Lys Lys Lys Lys Ser1 53056PRTHomo sapiens 305Gln Lys Arg Val Lys Glu1 53066PRTHomo sapiens 306Gly Lys Arg Ser Arg Thr1 53076PRTHomo sapiens 307Pro Lys Arg Lys Lys Ala1 53086PRTHomo sapiens 308Gly Lys Arg Glu Lys Ser1 53096PRTHomo sapiens 309His Lys Lys His Lys Lys1 53106PRTHomo sapiens 310Lys Lys Lys Val Lys Asp1 53116PRTHomo sapiens 311Glu Arg Lys Glu Arg Glu1 53126PRTHomo sapiens 312Glu Arg Lys Glu Arg Glu1 53136PRTHomo sapiens 313Ser Arg Lys Asn Arg Thr1 53146PRTHomo sapiens 314Ser Arg Lys Asn Arg Thr1 53156PRTHomo sapiens 315Gly Arg Arg Lys Arg Asn1 53166PRTHomo sapiens 316Ala Arg Arg Gln Arg Arg1 53176PRTHomo sapiens 317Gly Arg Arg Lys Arg Asn1 53186PRTHomo sapiens 318Ala Arg Arg Gln Arg Arg1 53196PRTHomo sapiens 319Cys Lys Arg Leu Lys Ser1 53206PRTHomo sapiens 320Cys Lys Arg Leu Lys Ser1 53216PRTHomo sapiens 321His Lys Arg Leu Arg Gln1 53226PRTHomo sapiens 322His Lys Arg Leu Arg Gln1 53238PRTHomo sapiens 323Pro Arg Lys Arg Leu Thr Lys Gly1 53248PRTHomo sapiens 324Arg Lys Arg Arg Lys Glu Lys Ser1 53258PRTHomo sapiens 325Pro Lys Lys Lys Arg Leu Arg Leu1 53266PRTHomo sapiens 326His Lys Lys Glu Arg Lys1 53276PRTHomo sapiens 327Thr Lys Lys Thr Lys Lys1 53286PRTHomo sapiens 328Leu Arg Lys Arg Arg Lys1 53296PRTHomo sapiens 329Pro Lys Lys Lys Arg Leu1 53306PRTHomo sapiens 330Gln Lys Arg Val Lys Glu1 53316PRTHomo sapiens 331Ser Arg Lys Pro Arg Met1 53326PRTHomo sapiens 332His Lys Lys Glu Arg Lys1 53336PRTHomo sapiens 333Thr Lys Lys Thr Lys Lys1 53346PRTHomo sapiens 334Leu Arg Lys Arg Arg Lys1 53356PRTHomo sapiens 335Pro Lys Lys Lys Arg Leu1 53366PRTHomo sapiens 336Gln Lys Arg Val Lys Glu1 53376PRTHomo sapiens 337Ser Arg Lys Pro Arg Met1 53386PRTHomo sapiens 338Ser Lys Arg Lys Lys Ala1 53396PRTHomo sapiens 339Ile Lys Lys Ala Arg Ala1 53406PRTHomo sapiens 340Ser Lys Arg Lys Lys Ala1 53416PRTHomo sapiens 341Ile Lys Lys Ala Arg Ala1 53428PRTHomo sapiens 342Arg Lys Lys Arg Ser Gln Arg Pro1 53436PRTHomo sapiens 343Phe Arg Lys Lys Arg Ser1 53446PRTHomo sapiens 344Phe Arg Lys Lys Arg Ser1 53458PRTHomo sapiens 345Leu Lys Arg Lys Leu Ile Arg Leu1 53468PRTHomo sapiens 346Arg Lys Lys Arg Arg Ala Arg Leu1 53476PRTHomo sapiens 347Ala Arg Arg Leu Arg Glu1 53486PRTHomo sapiens 348Glu Arg Lys Lys Arg Arg1 53496PRTHomo sapiens 349Cys Lys Lys Ser Arg Lys1 53506PRTHomo sapiens 350Ala Arg Arg Leu Arg Glu1 53516PRTHomo sapiens 351Glu Arg Lys Lys Arg Arg1 53526PRTHomo sapiens 352Cys Lys Lys Ser Arg Lys1 53536PRTHomo sapiens 353His Lys Arg Met Lys Val1 53546PRTHomo sapiens 354Leu Lys Arg Phe Lys Tyr1 53556PRTHomo sapiens 355His Lys Arg Met Lys Val1 53566PRTHomo sapiens 356Leu Lys Arg Phe Lys Tyr1 53576PRTHomo sapiens 357His Arg Lys Pro Lys Leu1 53586PRTHomo sapiens 358His Arg Lys Pro Lys Leu1 53596PRTHomo sapiens 359Leu Lys Arg Ser Arg Pro1 53606PRTHomo sapiens 360Pro Arg Lys Ser Arg Arg1 53616PRTHomo sapiens 361Leu Lys Arg Ser Arg Pro1 53626PRTHomo sapiens 362Pro Arg Lys Ser Arg Arg1 53636PRTHomo sapiens 363Asp Arg Arg Thr Arg Leu1 53646PRTHomo sapiens 364Asp Arg Arg Thr Arg Leu1 53658PRTHomo sapiens 365Gly Arg Arg Lys Ser Glu Arg Gln1 53666PRTHomo sapiens 366Ser Arg Arg Phe Arg Cys1 53676PRTHomo sapiens 367Asn Lys Lys Arg Arg Gln1 53686PRTHomo sapiens 368Ser Arg Arg Phe Arg Cys1 53696PRTHomo sapiens 369Asn Lys Lys Arg Arg Gln1 53706PRTHomo sapiens 370Glu Lys Arg Pro Arg Asp1 53716PRTHomo sapiens 371Ala Arg Arg Leu Lys Lys1 53726PRTHomo sapiens 372Glu Lys Arg Pro Arg Asp1 53736PRTHomo sapiens 373Ala Arg Arg Leu Lys Lys1 53746PRTHomo sapiens 374Gln Lys Lys Ile Arg Ile1 53756PRTHomo sapiens 375Gln Lys Lys Ile Arg Ile1 53766PRTHomo sapiens 376Phe Arg Lys Ala Arg Ser1 53776PRTHomo sapiens 377Phe Arg Lys Ala Arg Ser1 53786PRTHomo sapiens 378Glu Arg Lys Ala Lys Leu1 53796PRTHomo sapiens 379Glu Arg Lys Ala Lys Leu1 53808PRTHomo sapiens 380Glu Lys Lys Lys Lys Lys Lys Lys1 53816PRTHomo sapiens 381Leu Arg Lys His Arg His1 53826PRTHomo sapiens 382Leu Lys Lys Phe Lys Thr1 53836PRTHomo sapiens 383Asp Lys Lys Lys Lys Glu1 53846PRTHomo sapiens 384Glu Lys Lys Lys Lys Lys1 53856PRTHomo sapiens 385Leu Arg Lys His Arg His1 53866PRTHomo sapiens 386Leu Lys Lys Phe Lys Thr1 53876PRTHomo sapiens 387Asp Lys Lys Lys Lys Glu1 53886PRTHomo sapiens 388Glu Lys Lys Lys Lys Lys1 53896PRTHomo sapiens 389Tyr Lys Arg Tyr Lys Val1 53906PRTHomo sapiens 390Glu Lys Lys Lys Arg Glu1 53916PRTHomo sapiens 391Leu Lys Arg Ile Arg Glu1 53926PRTHomo sapiens 392Leu Lys Lys Thr Lys Cys1 53936PRTHomo sapiens 393Tyr Lys Arg Tyr Lys Val1 53946PRTHomo sapiens 394Glu Lys Lys Lys Arg Glu1 53956PRTHomo sapiens 395Leu Lys Arg Ile Arg Glu1 53966PRTHomo sapiens 396Leu Lys Lys Thr Lys Cys1 53978PRTHomo sapiens 397Arg Lys Arg Lys Ser Val Arg Gly1 53986PRTHomo sapiens 398Glu Arg Lys Arg Lys Ser1 53996PRTHomo sapiens 399Glu Arg Lys Arg Lys Ser1 54006PRTHomo sapiens 400Arg Arg Lys Ser Arg Phe1 54016PRTHomo sapiens 401Phe Lys Arg Pro Lys Ala1 54026PRTHomo sapiens 402Arg Arg Lys Ser Arg Phe1 54036PRTHomo sapiens 403Phe Lys Arg Pro Lys Ala1 54046PRTHomo sapiens 404Ala Arg Lys Val Lys Ser1 54056PRTHomo sapiens 405Ala Arg Lys Val Lys Ser1 54066PRTHomo sapiens 406Phe Lys Lys Thr Arg Tyr1 54076PRTHomo sapiens 407Phe Lys Lys Thr Arg Tyr1 54088PRTHomo sapiens 408Pro Lys Lys Lys Gly Ile Lys Ser1 54096PRTHomo sapiens 409Ile Arg Arg Pro Arg Arg1 54106PRTHomo sapiens 410Leu Arg Lys His Arg Arg1 54116PRTHomo sapiens 411Gly Lys Lys Glu Lys Ala1 54126PRTHomo sapiens 412Asp Arg Arg Leu Lys Lys1 54136PRTHomo sapiens 413Ile Arg Arg Pro Arg Arg1 54146PRTHomo sapiens 414Leu Arg Lys His Arg Arg1 54156PRTHomo sapiens 415Gly Lys Lys Glu Lys Ala1 54166PRTHomo sapiens 416Asp Arg Arg Leu Lys Lys1 54178PRTHomo sapiens 417Asp Lys Arg Lys Cys Glu Arg Leu1

54188PRTHomo sapiens 418Pro Arg Lys Lys Arg Leu Lys Ser1 54196PRTHomo sapiens 419Asn Lys Lys Gly Lys Ala1 54206PRTHomo sapiens 420Pro Arg Lys Lys Arg Leu1 54216PRTHomo sapiens 421Asn Lys Lys Gly Lys Ala1 54226PRTHomo sapiens 422Pro Arg Lys Lys Arg Leu1 54236PRTHomo sapiens 423Gln Arg Arg Asn Lys Arg1 54246PRTHomo sapiens 424Gln Arg Arg Asn Lys Arg1 54256PRTHomo sapiens 425Tyr Arg Lys Ile Lys Leu1 54266PRTHomo sapiens 426Tyr Arg Lys Ile Lys Leu1 54276PRTHomo sapiens 427Val Arg Arg His Arg Asn1 54286PRTHomo sapiens 428Trp Lys Arg Leu Lys Asp1 54296PRTHomo sapiens 429Val Arg Arg His Arg Asn1 54306PRTHomo sapiens 430Trp Lys Arg Leu Lys Asp1 54318PRTHomo sapiens 431Leu Arg Arg Arg Arg Gly Arg Glu1 54328PRTHomo sapiens 432Leu Lys Lys Arg Ser Gly Lys Lys1 54336PRTHomo sapiens 433Leu Arg Arg Arg Arg Gly1 54346PRTHomo sapiens 434Gly Lys Lys Asp Lys His1 54356PRTHomo sapiens 435Leu Arg Arg Arg Arg Gly1 54366PRTHomo sapiens 436Gly Lys Lys Asp Lys His1 54376PRTHomo sapiens 437Leu Lys Lys Lys Lys Lys1 54386PRTHomo sapiens 438Leu Lys Lys Lys Lys Lys1 54396PRTHomo sapiens 439Ala Arg Lys Arg Lys Arg1 54406PRTHomo sapiens 440Ala Arg Lys Arg Lys Arg1 54416PRTHomo sapiens 441Pro Lys Lys Lys Arg Leu1 54426PRTHomo sapiens 442Ser Lys Arg Asn Arg Arg1 54436PRTHomo sapiens 443Ile Arg Arg Asn Lys Gly1 54446PRTHomo sapiens 444Gly Lys Lys Glu Lys Glu1 54456PRTHomo sapiens 445Pro Lys Lys Lys Arg Leu1 54466PRTHomo sapiens 446Ser Lys Arg Asn Arg Arg1 54476PRTHomo sapiens 447Ile Arg Arg Asn Lys Gly1 54486PRTHomo sapiens 448Gly Lys Lys Glu Lys Glu1 54496PRTHomo sapiens 449Thr Arg Arg Ser Arg Ala1 54506PRTHomo sapiens 450Thr Arg Arg Ser Arg Ala1 54518PRTHomo sapiens 451Ile Arg Arg Arg Phe Glu Lys Arg1 54526PRTHomo sapiens 452Ala Arg Arg Ile Lys Thr1 54536PRTHomo sapiens 453Phe Lys Lys Val Arg Gly1 54546PRTHomo sapiens 454Ala Arg Arg Ile Lys Thr1 54556PRTHomo sapiens 455Phe Lys Lys Val Arg Gly1 54568PRTHomo sapiens 456His Arg Lys Arg Ser Arg Arg Val1 54576PRTHomo sapiens 457Arg Lys Arg Ser Arg Arg1 54586PRTHomo sapiens 458Arg Lys Arg Ser Arg Arg1 54598PRTHomo sapiens 459Thr Lys Arg Lys Lys Arg Arg Glu1 54606PRTHomo sapiens 460Thr Lys Arg Lys Lys Arg1 54616PRTHomo sapiens 461Thr Lys Arg Lys Lys Arg1 54626PRTHomo sapiens 462Thr Arg Arg Lys Arg His1 54636PRTHomo sapiens 463Thr Arg Arg Lys Arg His1 54646PRTHomo sapiens 464Pro Lys Lys Gly Arg Val1 54656PRTHomo sapiens 465Arg Arg Arg His Arg Thr1 54666PRTHomo sapiens 466Asn Arg Arg Ala Lys Gln1 54676PRTHomo sapiens 467Pro Lys Lys Gly Arg Val1 54686PRTHomo sapiens 468Arg Arg Arg His Arg Thr1 54696PRTHomo sapiens 469Asn Arg Arg Ala Lys Gln1 54708PRTHomo sapiens 470Tyr Arg Arg Arg Glu Ser Lys Arg1 54718PRTHomo sapiens 471Met Arg Lys Arg Arg Leu Arg Glu1 54726PRTHomo sapiens 472Ser Lys Arg Lys Lys Ser1 54736PRTHomo sapiens 473Met Arg Lys Arg Arg Leu1 54746PRTHomo sapiens 474Ser Lys Arg Lys Lys Ser1 54756PRTHomo sapiens 475Met Arg Lys Arg Arg Leu1 54768PRTHomo sapiens 476Leu Lys Lys Lys Gln Arg Arg Gln1 54776PRTHomo sapiens 477Lys Lys Lys Gln Arg Arg1 54786PRTHomo sapiens 478Asn Arg Arg Ala Lys Trp1 54796PRTHomo sapiens 479Arg Lys Arg Glu Arg Ser1 54806PRTHomo sapiens 480Lys Lys Lys Gln Arg Arg1 54816PRTHomo sapiens 481Asn Arg Arg Ala Lys Trp1 54826PRTHomo sapiens 482Arg Lys Arg Glu Arg Ser1 54836PRTHomo sapiens 483Lys Arg Arg Gly Arg Ile1 54846PRTHomo sapiens 484Lys Arg Arg Gly Arg Ile1 54856PRTHomo sapiens 485Met Lys Lys Glu Lys Gly1 54866PRTHomo sapiens 486Met Lys Lys Glu Lys Gly1 54876PRTHomo sapiens 487Gly Arg Arg Ala Arg Pro1 54886PRTHomo sapiens 488Leu Lys Lys Gln Arg Ser1 54896PRTHomo sapiens 489Gly Arg Arg Ala Arg Pro1 54906PRTHomo sapiens 490Leu Lys Lys Gln Arg Ser1 54918PRTHomo sapiens 491Gly Lys Lys Lys Ser Thr Lys Thr1 54928PRTHomo sapiens 492Ala Lys Lys Arg Gly Ser Lys Gly1 54936PRTHomo sapiens 493Asn Lys Lys Gly Arg Val1 54946PRTHomo sapiens 494Ala Lys Lys Ala Lys Ser1 54956PRTHomo sapiens 495Ala Arg Lys Ser Lys Lys1 54966PRTHomo sapiens 496Asp Lys Lys Leu Lys Ser1 54976PRTHomo sapiens 497Asn Lys Lys Gly Arg Val1 54986PRTHomo sapiens 498Ala Lys Lys Ala Lys Ser1 54996PRTHomo sapiens 499Ala Arg Lys Ser Lys Lys1 55006PRTHomo sapiens 500Asp Lys Lys Leu Lys Ser1 55018PRTHomo sapiens 501Lys Arg Arg Arg Asn Gln Lys Leu1 55026PRTHomo sapiens 502Glu Lys Arg Arg Arg Asn1 55036PRTHomo sapiens 503Glu Lys Arg Arg Arg Asn1 55046PRTHomo sapiens 504Pro Lys Lys Ala Arg Ala1 55056PRTHomo sapiens 505Ala Arg Arg Ala Lys Val1 55066PRTHomo sapiens 506Pro Lys Lys Ala Arg Ala1 55076PRTHomo sapiens 507Ala Arg Arg Ala Lys Val1 55086PRTHomo sapiens 508Leu Lys Lys Phe Lys Asp1 55096PRTHomo sapiens 509Leu Lys Lys Phe Lys Asp1 55106PRTHomo sapiens 510Ser Lys Arg Pro Arg Leu1 55116PRTHomo sapiens 511Ala Arg Lys Gln Arg Glu1 55126PRTHomo sapiens 512Arg Arg Lys Ala Lys Glu1 55136PRTHomo sapiens 513Ile Arg Lys Gln Arg Glu1 55146PRTHomo sapiens 514Gln Arg Arg Val Lys Phe1 55156PRTHomo sapiens 515Lys Arg Arg Gly Arg Asn1 55166PRTHomo sapiens 516Arg Arg Lys Gly Arg Ile1 55176PRTHomo sapiens 517Ser Arg Lys Pro Arg Glu1 55186PRTHomo sapiens 518Ser Arg Lys Ser Lys Ser1 55196PRTHomo sapiens 519Ser Lys Arg Pro Arg Leu1 55206PRTHomo sapiens 520Ala Arg Lys Gln Arg Glu1 55216PRTHomo sapiens 521Arg Arg Lys Ala Lys Glu1 55226PRTHomo sapiens 522Ile Arg Lys Gln Arg Glu1 55236PRTHomo sapiens 523Gln Arg Arg Val Lys Phe1 55246PRTHomo sapiens 524Lys Arg Arg Gly Arg Asn1 55256PRTHomo sapiens 525Arg Arg Lys Gly Arg Ile1 55266PRTHomo sapiens 526Ser Arg Lys Pro Arg Glu1 55276PRTHomo sapiens 527Ser Arg Lys Ser Lys Ser1 55286PRTHomo sapiens 528Lys Arg Arg Val Lys Ser1 55296PRTHomo sapiens 529Lys Arg Arg Val Lys Ser1 55306PRTHomo sapiens 530Thr Lys Lys Ile Lys Leu1 55316PRTHomo sapiens 531Thr Lys Lys Ile Lys Leu1 55328PRTHomo sapiens 532Phe Arg Lys Arg Met Glu Lys Glu1 55336PRTHomo sapiens 533Arg Arg Lys Ala Arg Glu1 55346PRTHomo sapiens 534Lys Arg Lys Leu Lys Glu1 55356PRTHomo sapiens 535Lys Lys Arg Leu Arg Gln1 55366PRTHomo sapiens 536Arg Arg Lys Ala Arg Glu1 55376PRTHomo sapiens 537Lys Arg Lys Leu Lys Glu1 55386PRTHomo sapiens 538Lys Lys Arg Leu Arg Gln1 55396PRTHomo sapiens 539Ser Lys Lys Tyr Arg Gln1 55406PRTHomo sapiens 540Ser Lys Lys Tyr Arg Gln1 55416PRTHomo sapiens 541Val Arg Lys Phe Arg Thr1 55426PRTHomo sapiens 542Gly Lys Lys Leu Lys Lys1 55436PRTHomo sapiens 543Glu Lys Arg Ala Lys Tyr1 55446PRTHomo sapiens 544Ser Lys Lys Tyr Lys Glu1 55456PRTHomo sapiens 545Lys Lys Lys Met Lys Tyr1 55466PRTHomo sapiens 546Lys Lys Arg Leu Lys Trp1 55476PRTHomo sapiens 547Lys Lys Lys Ala Lys Tyr1 55486PRTHomo sapiens 548Glu Lys Lys Glu Lys Leu1 55496PRTHomo sapiens 549Ser Lys Lys Met Lys Phe1 55506PRTHomo sapiens 550Val Arg Lys Phe Arg Thr1 55516PRTHomo sapiens 551Gly Lys Lys Leu Lys Lys1 55526PRTHomo sapiens 552Glu Lys Arg Ala Lys Tyr1 55536PRTHomo sapiens 553Ser Lys Lys Tyr Lys Glu1 55546PRTHomo sapiens 554Lys Lys Lys Met Lys Tyr1 55556PRTHomo sapiens 555Lys Lys Arg Leu Lys Trp1 55566PRTHomo sapiens 556Lys Lys Lys Ala Lys Tyr1 55576PRTHomo sapiens 557Glu Lys Lys Glu Lys Leu1 55586PRTHomo sapiens 558Ser Lys Lys Met Lys Phe1 55598PRTHomo sapiens 559Phe Arg Arg Arg Gly Ala Arg Ala1 55608PRTHomo sapiens 560Gln Lys Arg Arg Tyr Ser Lys Gly1 55618PRTHomo sapiens 561Tyr Arg Lys Lys Pro His Arg Pro1 55626PRTHomo sapiens 562Glu Arg Arg Leu Lys Met1 55636PRTHomo sapiens 563Glu Arg Arg Leu Lys Met1 55646PRTHomo sapiens 564Asp Arg Lys Val Lys Ser1 55656PRTHomo sapiens 565Ile Lys Arg Lys Arg Glu1 55666PRTHomo sapiens 566Asp Arg Lys Val Lys Ser1 55676PRTHomo sapiens 567Ile Lys Arg Lys Arg Glu1 55686PRTHomo sapiens 568Ser Lys Lys Gly Arg Lys1 55696PRTHomo sapiens 569Ser Lys Lys Gly Arg Lys1 55706PRTHomo sapiens 570His Arg Arg Pro Arg Gly1 55716PRTHomo sapiens 571His Arg Arg Pro Arg Gly1 55726PRTHomo sapiens 572Ala Arg Arg Pro Arg His1 55736PRTHomo sapiens 573Glu Lys Arg Ile Lys Glu1 55746PRTHomo sapiens 574Leu Lys Lys Ile Lys Gln1 55756PRTHomo sapiens 575Ala Arg Arg Pro Arg His1 55766PRTHomo sapiens 576Glu Lys Arg Ile Lys Glu1 55776PRTHomo sapiens 577Leu Lys Lys Ile Lys Gln1 55786PRTHomo sapiens 578Arg Arg Lys Pro Lys Pro1 55796PRTHomo sapiens 579Arg Arg Lys Pro Lys Pro1 55806PRTHomo sapiens 580Ala Arg Arg Lys Lys Ile1 55816PRTHomo sapiens 581Ala Arg Arg Lys Lys Ile1 55826PRTHomo sapiens 582Ala Arg Arg Lys Arg Gln1 55836PRTHomo sapiens 583Gln Lys Arg Leu Lys Lys1 55846PRTHomo sapiens 584Glu Lys Lys Thr Lys Arg1 55856PRTHomo sapiens 585Ala Arg Arg Lys Arg Gln1 55866PRTHomo sapiens 586Gln Lys Arg Leu Lys Lys1 55876PRTHomo sapiens 587Glu Lys Lys Thr Lys Arg1 55888PRTHomo sapiens 588Glu Lys Arg Lys Ser Glu Lys Ala1 55896PRTHomo sapiens 589Thr Arg Lys Lys Arg Lys1 55906PRTHomo sapiens 590Thr Arg Lys Lys Arg Lys1 55916PRTHomo sapiens 591Ala Arg Lys Leu Lys Lys1 55926PRTHomo sapiens 592Ala Arg Lys Leu Lys Lys1 55938PRTHomo sapiens 593Arg Lys Arg Arg Pro Ser Arg Pro1 55946PRTHomo sapiens 594Asn Lys Lys Lys Lys Glu1 55956PRTHomo sapiens 595Val Arg Lys Arg Arg Pro1 55966PRTHomo sapiens 596Asn Lys Lys Lys Lys Glu1 55976PRTHomo sapiens 597Val Arg Lys Arg Arg Pro1 55988PRTHomo sapiens 598Trp Lys Lys Lys Tyr Glu Lys Glu1 55996PRTHomo sapiens 599Val Lys Arg Cys Lys Gln1 56006PRTHomo sapiens 600Glu Lys Arg Leu Arg Ala1 56016PRTHomo sapiens 601Val Lys Arg Cys Lys Gln1 56026PRTHomo sapiens 602Glu Lys Arg Leu Arg Ala1 56036PRTHomo sapiens 603Val Lys Lys Ile Lys Glu1 56046PRTHomo sapiens 604Val Lys Lys Ile Lys Glu1 56056PRTHomo sapiens 605Asn Lys Lys Phe Lys Met1 56066PRTHomo sapiens 606Glu Arg Arg His Arg Leu1 56076PRTHomo sapiens 607Asn Lys Lys Phe Lys Met1 56086PRTHomo sapiens 608Glu Arg Arg His Arg Leu1 56096PRTHomo sapiens 609Ser Arg Arg Pro Arg Phe1 56106PRTHomo sapiens 610Ser Arg Arg Pro Arg Phe1 56118PRTHomo sapiens 611Thr Lys Arg Lys Lys Pro Arg Arg1 56126PRTHomo sapiens 612Pro Lys Lys Asp Lys Leu1 56136PRTHomo sapiens 613Thr Lys Arg Lys Lys Pro1 56146PRTHomo sapiens 614Asn Lys Lys Asn Arg Glu1 56156PRTHomo sapiens 615Leu Lys Lys Ser Arg Ile1 56166PRTHomo sapiens 616Pro Lys Lys Pro Arg Glu1 56176PRTHomo sapiens 617Ser Arg Lys Gln Arg Gln1 56186PRTHomo sapiens 618Val Lys Arg Lys Arg Lys1 56196PRTHomo sapiens 619Pro Lys Lys Asp Lys Leu1 56206PRTHomo sapiens 620Thr Lys Arg Lys Lys Pro1 56216PRTHomo sapiens 621Asn Lys Lys Asn Arg Glu1 56226PRTHomo sapiens 622Leu Lys Lys Ser Arg Ile1 56236PRTHomo sapiens 623Pro Lys Lys Pro Arg Glu1 56246PRTHomo sapiens 624Ser Arg Lys Gln Arg Gln1 56256PRTHomo sapiens 625Val Lys Arg Lys Arg Lys1 56266PRTHomo sapiens 626Ala Arg Arg Leu Arg Arg1 56276PRTHomo sapiens 627Pro Arg Arg Cys Lys Trp1 56286PRTHomo sapiens 628Ala Arg Arg Leu Arg Arg1 56296PRTHomo sapiens 629Pro Arg Arg Cys Lys Trp1 56306PRTHomo sapiens 630Met Lys Lys Pro Arg Phe1 56316PRTHomo sapiens 631Met Lys Lys Pro Arg Phe1 56326PRTHomo sapiens 632Arg Arg Arg Gln Arg Ala1 56336PRTHomo sapiens 633Pro Arg Arg Ile Arg Lys1 56346PRTHomo sapiens 634Ala Arg Arg Thr Arg Thr1 56356PRTHomo sapiens 635Arg Arg Arg Gln Arg Ala1 56366PRTHomo sapiens 636Pro Arg Arg Ile Arg Lys1 56376PRTHomo sapiens 637Ala Arg Arg Thr Arg Thr1 56386PRTHomo sapiens 638Gln Lys Arg Gly Arg Val1 56396PRTHomo sapiens 639Gln Lys Arg Gly Arg Val1 56406PRTHomo sapiens 640Ser Arg Arg Ser Lys Leu1 56416PRTHomo sapiens 641Pro Lys Lys Ala Lys Ser1 56426PRTHomo sapiens 642Ser Arg Arg Ser Lys Leu1 56436PRTHomo sapiens 643Pro Lys Lys Ala Lys Ser1 56446PRTHomo sapiens 644Glu Lys Arg Asn Arg Lys1 56456PRTHomo sapiens 645Glu Lys Arg Asn Arg Lys1 56466PRTHomo sapiens 646Gly Arg Arg Tyr Arg Arg1 56476PRTHomo sapiens 647Pro Arg Lys Asn Lys Glu1 56486PRTHomo sapiens 648Gly Arg Arg Tyr Arg Arg1 56496PRTHomo sapiens 649Pro Arg Lys Asn Lys Glu1 56508PRTHomo sapiens 650Lys Arg Arg Lys His Leu Lys Arg1 56516PRTHomo sapiens 651Trp Lys Arg Arg Lys His1 56526PRTHomo sapiens 652Leu Lys Arg Ser Arg Asp1 56536PRTHomo sapiens 653Gly Arg Lys Leu Arg Leu1 56546PRTHomo sapiens 654Ser Arg Lys Arg Lys Leu1 56556PRTHomo sapiens 655Trp Lys Arg Arg Lys His1 56566PRTHomo sapiens 656Leu Lys Arg Ser Arg Asp1 56576PRTHomo sapiens 657Gly Arg Lys Leu Arg Leu1 56586PRTHomo sapiens 658Ser Arg Lys Arg Lys Leu1 56598PRTHomo sapiens 659Met Arg Arg Arg Gln Arg Arg Glu1 56606PRTHomo sapiens 660Glu Lys Arg Thr Arg Leu1 56616PRTHomo sapiens 661Arg Arg Arg Gln Arg Arg1 56626PRTHomo sapiens 662Asn Arg Arg Cys Lys Leu1 56636PRTHomo sapiens 663Glu Lys Arg Thr Arg Leu1 56646PRTHomo sapiens 664Arg Arg Arg Gln Arg Arg1 56656PRTHomo sapiens 665Asn Arg Arg Cys Lys Leu1 56666PRTHomo sapiens 666Ala Lys Lys Tyr Arg Asp1 56676PRTHomo sapiens 667Ala Lys Lys Tyr Arg Asp1 56688PRTHomo sapiens 668Lys Arg Lys Lys Pro Ser Arg Pro1

56696PRTHomo sapiens 669Ser Lys Lys Arg Lys Lys1 56706PRTHomo sapiens 670Gln Lys Arg Gln Lys Cys1 56716PRTHomo sapiens 671Tyr Arg Arg Arg Lys Arg1 56726PRTHomo sapiens 672Ser Lys Lys Arg Lys Lys1 56736PRTHomo sapiens 673Gln Lys Arg Gln Lys Cys1 56746PRTHomo sapiens 674Tyr Arg Arg Arg Lys Arg1 56756PRTHomo sapiens 675Arg Lys Arg Gln Arg Ala1 56766PRTHomo sapiens 676Arg Lys Arg Gln Arg Ala1 56776PRTHomo sapiens 677Leu Lys Lys Asp Arg Glu1 56786PRTHomo sapiens 678Leu Lys Lys His Lys Glu1 56796PRTHomo sapiens 679Leu Lys Lys Asp Arg Glu1 56806PRTHomo sapiens 680Leu Lys Lys His Lys Glu1 56816PRTHomo sapiens 681Ser Arg Arg Thr Arg Trp1 56826PRTHomo sapiens 682Glu Arg Arg Arg Lys Phe1 56836PRTHomo sapiens 683Leu Arg Arg Cys Arg Ala1 56846PRTHomo sapiens 684Ser Arg Arg Ser Arg Ser1 56856PRTHomo sapiens 685Gly Arg Arg Arg Lys Ser1 56866PRTHomo sapiens 686Glu Arg Arg Ala Lys Gln1 56876PRTHomo sapiens 687Ser Arg Arg Thr Arg Trp1 56886PRTHomo sapiens 688Glu Arg Arg Arg Lys Phe1 56896PRTHomo sapiens 689Leu Arg Arg Cys Arg Ala1 56906PRTHomo sapiens 690Ser Arg Arg Ser Arg Ser1 56916PRTHomo sapiens 691Gly Arg Arg Arg Lys Ser1 56926PRTHomo sapiens 692Glu Arg Arg Ala Lys Gln1 56938PRTHomo sapiens 693Leu Arg Arg Arg Arg Val Lys Arg1 56946PRTHomo sapiens 694Leu Arg Arg Arg Arg Val1 56956PRTHomo sapiens 695Glu Lys Arg Gly Lys Gly1 56966PRTHomo sapiens 696Leu Arg Arg Arg Arg Val1 56976PRTHomo sapiens 697Glu Lys Arg Gly Lys Gly1 56986PRTHomo sapiens 698Asn Arg Arg Leu Lys His1 56996PRTHomo sapiens 699Asn Arg Arg Leu Lys His1 57006PRTHomo sapiens 700Lys Arg Arg Tyr Arg Gly1 57016PRTHomo sapiens 701Lys Arg Arg Tyr Arg Gly1 57026PRTHomo sapiens 702Ala Arg Arg Gln Arg Asp1 57036PRTHomo sapiens 703Pro Lys Lys Pro Lys Thr1 57046PRTHomo sapiens 704Asn Arg Arg Ser Arg Asn1 57056PRTHomo sapiens 705Glu Lys Arg Asp Lys Glu1 57066PRTHomo sapiens 706Ala Arg Arg Gln Arg Asp1 57076PRTHomo sapiens 707Pro Lys Lys Pro Lys Thr1 57086PRTHomo sapiens 708Asn Arg Arg Ser Arg Asn1 57096PRTHomo sapiens 709Glu Lys Arg Asp Lys Glu1 57106PRTHomo sapiens 710Gly Lys Lys Leu Lys Ala1 57116PRTHomo sapiens 711Lys Arg Lys Glu Lys Leu1 57126PRTHomo sapiens 712Gly Lys Lys Leu Lys Ala1 57136PRTHomo sapiens 713Lys Arg Lys Glu Lys Leu1 57146PRTHomo sapiens 714Ala Lys Lys His Lys Arg1 57156PRTHomo sapiens 715Ala Lys Lys His Lys Arg1 57166PRTHomo sapiens 716Lys Arg Lys Met Lys Ala1 57176PRTHomo sapiens 717Lys Arg Lys Met Lys Ala1 57188PRTHomo sapiens 718Lys Lys Lys Arg Lys Gln Arg Arg1 57196PRTHomo sapiens 719Glu Lys Lys Lys Arg Lys1 57206PRTHomo sapiens 720Gln Arg Arg Asn Arg Thr1 57216PRTHomo sapiens 721Asn Arg Arg Ala Lys Phe1 57226PRTHomo sapiens 722Glu Lys Lys Lys Arg Lys1 57236PRTHomo sapiens 723Gln Arg Arg Asn Arg Thr1 57246PRTHomo sapiens 724Asn Arg Arg Ala Lys Phe1 57258PRTHomo sapiens 725Leu Lys Arg Lys Trp Ile Arg Cys1 57266PRTHomo sapiens 726Thr Arg Lys Ile Lys Leu1 57276PRTHomo sapiens 727Met Arg Lys Gln Arg Glu1 57286PRTHomo sapiens 728Thr Arg Lys Ile Lys Leu1 57296PRTHomo sapiens 729Met Arg Lys Gln Arg Glu1 57306PRTHomo sapiens 730Pro Arg Arg Thr Arg Pro1 57316PRTHomo sapiens 731Gly Arg Arg Lys Arg Phe1 57326PRTHomo sapiens 732Gly Lys Lys Arg Lys Asn1 57336PRTHomo sapiens 733Gly Lys Lys Arg Lys Cys1 57346PRTHomo sapiens 734Pro Arg Arg Thr Arg Pro1 57356PRTHomo sapiens 735Gly Arg Arg Lys Arg Phe1 57366PRTHomo sapiens 736Gly Lys Lys Arg Lys Asn1 57376PRTHomo sapiens 737Gly Lys Lys Arg Lys Cys1 57388PRTHomo sapiens 738Ala Arg Arg Arg Cys Asp Arg Asp1 57396PRTHomo sapiens 739Glu Lys Lys Ser Lys Asp1 57406PRTHomo sapiens 740Glu Lys Lys Ser Lys Asp1 57416PRTHomo sapiens 741Pro Arg Lys Ala Arg Val1 57426PRTHomo sapiens 742Pro Arg Lys Ala Arg Val1 57436PRTHomo sapiens 743Lys Lys Lys Tyr Lys Ala1 57446PRTHomo sapiens 744Arg Arg Arg Leu Lys Glu1 57456PRTHomo sapiens 745Lys Lys Lys Tyr Lys Ala1 57466PRTHomo sapiens 746Arg Arg Arg Leu Lys Glu1 57476PRTHomo sapiens 747Lys Arg Arg Gln Arg Asp1 57486PRTHomo sapiens 748Lys Arg Arg Gln Arg Asp1 57496PRTHomo sapiens 749Thr Arg Arg Cys Arg Cys1 57506PRTHomo sapiens 750Ile Arg Arg Lys Arg Leu1 57516PRTHomo sapiens 751Thr Arg Arg Cys Arg Cys1 57526PRTHomo sapiens 752Ile Arg Arg Lys Arg Leu1 57538PRTHomo sapiens 753Gln Lys Arg Arg Thr Leu Lys Asn1 57546PRTHomo sapiens 754Met Arg Arg Leu Arg Ser1 57556PRTHomo sapiens 755Met Arg Arg Leu Arg Ser1 57566PRTHomo sapiens 756Gln Lys Lys Val Lys Ile1 57576PRTHomo sapiens 757Asn Arg Arg Lys Arg Lys1 57586PRTHomo sapiens 758Leu Arg Arg Tyr Lys Arg1 57596PRTHomo sapiens 759Gln Lys Lys Val Lys Ile1 57606PRTHomo sapiens 760Asn Arg Arg Lys Arg Lys1 57616PRTHomo sapiens 761Leu Arg Arg Tyr Lys Arg1 57626PRTHomo sapiens 762Lys Lys Lys Glu Lys Glu1 57636PRTHomo sapiens 763Lys Lys Lys Glu Lys Glu1 57648PRTHomo sapiens 764Ala Lys Arg Lys Ala Asp Arg Glu1 57656PRTHomo sapiens 765Lys Lys Arg His Arg Glu1 57666PRTHomo sapiens 766Lys Lys Arg His Arg Glu1 57678PRTHomo sapiens 767Leu Arg Arg Arg Leu Glu Lys Arg1 57686PRTHomo sapiens 768Ser Arg Arg Val Lys Ala1 57696PRTHomo sapiens 769Ser Arg Arg Val Lys Ala1 57708PRTHomo sapiens 770Arg Lys Arg Lys Leu Glu Arg Ile1 57716PRTHomo sapiens 771Arg Lys Arg Leu Arg Asn1 57726PRTHomo sapiens 772Cys Arg Lys Arg Lys Leu1 57736PRTHomo sapiens 773Arg Lys Arg Leu Arg Asn1 57746PRTHomo sapiens 774Cys Arg Lys Arg Lys Leu1 57756PRTHomo sapiens 775Val Arg Lys Ala Arg Gly1 57766PRTHomo sapiens 776Val Arg Lys Ala Arg Gly1 57776PRTHomo sapiens 777Asp Arg Arg Phe Arg Ser1 57786PRTHomo sapiens 778Ser Arg Arg Gln Arg Ile1 57796PRTHomo sapiens 779Asp Arg Arg Phe Arg Ser1 57806PRTHomo sapiens 780Ser Arg Arg Gln Arg Ile1 57816PRTHomo sapiens 781Thr Lys Arg Arg Arg Lys1 57826PRTHomo sapiens 782Val Lys Lys Tyr Lys Ser1 57836PRTHomo sapiens 783Thr Lys Arg Arg Arg Lys1 57846PRTHomo sapiens 784Val Lys Lys Tyr Lys Ser1 57858PRTHomo sapiens 785Gln Arg Arg Arg Thr Leu Lys Asn1 57868PRTHomo sapiens 786Asn Lys Lys Lys Ile Leu Arg Glu1 57876PRTHomo sapiens 787Leu Lys Lys Leu Arg Ile1 57886PRTHomo sapiens 788Phe Lys Lys Gly Arg Leu1 57896PRTHomo sapiens 789Cys Arg Arg His Lys Ile1 57906PRTHomo sapiens 790Leu Lys Lys Leu Arg Ile1 57916PRTHomo sapiens 791Phe Lys Lys Gly Arg Leu1 57926PRTHomo sapiens 792Cys Arg Arg His Lys Ile1 57936PRTHomo sapiens 793Ala Lys Lys Ile Lys Met1 57946PRTHomo sapiens 794Ala Lys Lys Ile Lys Met1 57958PRTHomo sapiens 795Val Arg Lys Arg Cys His Lys His1 57966PRTHomo sapiens 796Arg Lys Lys Phe Lys Cys1 57976PRTHomo sapiens 797Arg Lys Lys Phe Lys Cys1 57986PRTHomo sapiens 798Glu Lys Lys Ala Arg Ser1 57996PRTHomo sapiens 799Gly Arg Arg Arg Arg Thr1 58006PRTHomo sapiens 800Glu Arg Arg Gln Arg Phe1 58016PRTHomo sapiens 801Glu Lys Lys Ala Arg Ser1 58026PRTHomo sapiens 802Gly Arg Arg Arg Arg Thr1 58036PRTHomo sapiens 803Glu Arg Arg Gln Arg Phe1 58046PRTHomo sapiens 804Leu Arg Lys Phe Arg Thr1 58056PRTHomo sapiens 805Leu Arg Lys Phe Arg Thr1 58066PRTHomo sapiens 806Arg Arg Arg Cys Arg Asp1 58076PRTHomo sapiens 807Val Lys Arg Pro Arg Phe1 58086PRTHomo sapiens 808Val Arg Lys Arg Arg Val1 58096PRTHomo sapiens 809Glu Lys Lys Ser Lys Asn1 58106PRTHomo sapiens 810Arg Arg Arg Cys Arg Asp1 58116PRTHomo sapiens 811Val Lys Arg Pro Arg Phe1 58126PRTHomo sapiens 812Val Arg Lys Arg Arg Val1 58136PRTHomo sapiens 813Glu Lys Lys Ser Lys Asn1 58148PRTHomo sapiens 814Ile Arg Lys Lys Val Arg Arg Leu1 58156PRTHomo sapiens 815Lys Arg Arg Ala Lys Ala1 58166PRTHomo sapiens 816Met Lys Lys Val Arg Arg1 58176PRTHomo sapiens 817Asp Lys Arg Lys Arg Ser1 58186PRTHomo sapiens 818Arg Lys Lys Val Arg Arg1 58196PRTHomo sapiens 819Thr Lys Lys Cys Arg Glu1 58206PRTHomo sapiens 820Lys Arg Arg Ala Lys Ala1 58216PRTHomo sapiens 821Met Lys Lys Val Arg Arg1 58226PRTHomo sapiens 822Asp Lys Arg Lys Arg Ser1 58236PRTHomo sapiens 823Arg Lys Lys Val Arg Arg1 58246PRTHomo sapiens 824Thr Lys Lys Cys Arg Glu1 58258PRTHomo sapiens 825Lys Arg Lys Lys Ser Glu Arg Thr1 58266PRTHomo sapiens 826Thr Arg Lys Thr Arg Arg1 58276PRTHomo sapiens 827Ile Lys Arg Lys Lys Ser1 58286PRTHomo sapiens 828Thr Arg Lys Thr Arg Arg1 58296PRTHomo sapiens 829Ile Lys Arg Lys Lys Ser1 58308PRTHomo sapiens 830Arg Lys Arg Lys Ala Glu Lys Gln1 58318PRTHomo sapiens 831Lys Lys Arg Lys Gly Gly Arg Asn1 58326PRTHomo sapiens 832Ala Arg Arg Gly Arg Lys1 58336PRTHomo sapiens 833Pro Lys Arg Gly Arg Pro1 58346PRTHomo sapiens 834Glu Lys Lys Arg Lys Gly1 58356PRTHomo sapiens 835Asp Arg Lys Arg Lys Gln1 58366PRTHomo sapiens 836Leu Lys Lys Ile Arg Arg1 58379PRTHomo sapiens 837Val Lys Lys Val Glu Lys Lys Arg Glu1 58386PRTHomo sapiens 838Ala Arg Arg Gly Arg Lys1 58396PRTHomo sapiens 839Pro Lys Arg Gly Arg Pro1 58406PRTHomo sapiens 840Glu Lys Lys Arg Lys Gly1 58416PRTHomo sapiens 841Asp Arg Lys Arg Lys Gln1 58426PRTHomo sapiens 842Leu Lys Lys Ile Arg Arg1 58438PRTHomo sapiens 843Thr Lys Arg Arg Lys Arg Lys Asn1 58446PRTHomo sapiens 844Thr Lys Arg Arg Lys Arg1 58456PRTHomo sapiens 845Thr Lys Arg Arg Lys Arg1 58466PRTHomo sapiens 846Trp Lys Arg Ile Arg Phe1 58476PRTHomo sapiens 847Trp Lys Arg Ile Arg Phe1 58488PRTHomo sapiens 848Asp Lys Lys Lys Leu Arg Arg Leu1 58496PRTHomo sapiens 849Met Arg Lys Asp Lys Lys1 58506PRTHomo sapiens 850Asn Lys Arg Gln Lys Ile1 58516PRTHomo sapiens 851Met Arg Lys Asp Lys Lys1 58526PRTHomo sapiens 852Asn Lys Arg Gln Lys Ile1 58536PRTHomo sapiens 853Lys Arg Lys His Arg Asn1 58546PRTHomo sapiens 854Glu Lys Arg Arg Lys Ala1 58556PRTHomo sapiens 855Lys Arg Lys His Arg Asn1 58566PRTHomo sapiens 856Glu Lys Arg Arg Lys Ala1 58578PRTHomo sapiens 857Glu Lys Lys Lys Val Ser Lys Ala1 58586PRTHomo sapiens 858Ile Lys Arg Lys Lys Phe1 58596PRTHomo sapiens 859Thr Lys Arg Val Lys Lys1 58606PRTHomo sapiens 860Asp Arg Arg Gln Lys Arg1 58616PRTHomo sapiens 861Ile Lys Arg Lys Lys Phe1 58626PRTHomo sapiens 862Thr Lys Arg Val Lys Lys1 58636PRTHomo sapiens 863Asp Arg Arg Gln Lys Arg1 58646PRTHomo sapiens 864Gln Arg Lys Gly Arg Lys1 58656PRTHomo sapiens 865Ala Arg Arg Leu Arg Ala1 58666PRTHomo sapiens 866Gln Arg Lys Gly Arg Lys1 58676PRTHomo sapiens 867Ala Arg Arg Leu Arg Ala1 58686PRTHomo sapiens 868Leu Arg Arg Thr Arg Ile1 58696PRTHomo sapiens 869Leu Arg Arg Thr Arg Ile1 58706PRTHomo sapiens 870Ile Lys Lys Leu Lys Val1 58716PRTHomo sapiens 871Ile Lys Lys Leu Lys Val1 58726PRTHomo sapiens 872Ile Lys Lys Leu Lys Val1 58736PRTHomo sapiens 873Ile Lys Lys Leu Lys Val1 58748PRTHomo sapiens 874Ala Arg Arg Arg Arg Lys Arg Arg1 58758PRTHomo sapiens 875Pro Arg Arg Arg Arg Arg Arg Thr1 58768PRTHomo sapiens 876Trp Lys Lys Lys Thr Ser Arg Pro1 58776PRTHomo sapiens 877Ala Arg Arg Arg Arg Lys1 58786PRTHomo sapiens 878Pro Arg Arg Arg Arg Arg1 58796PRTHomo sapiens 879Pro Arg Arg Pro Arg Ile1 58806PRTHomo sapiens 880Thr Arg Arg Met Arg Trp1 58816PRTHomo sapiens 881Ala Arg Arg Arg Arg Lys1 58826PRTHomo sapiens 882Pro Arg Arg Arg Arg Arg1 58836PRTHomo sapiens 883Pro Arg Arg Pro Arg Ile1 58846PRTHomo sapiens 884Thr Arg Arg Met Arg Trp1 58858PRTHomo sapiens 885Cys Lys Lys Lys Met Arg Arg Lys1 58866PRTHomo sapiens 886Lys Lys Lys Met Arg Arg1 58876PRTHomo sapiens 887His Arg Arg Ser Lys Gln1 58886PRTHomo sapiens 888Lys Lys Lys Met Arg Arg1 58896PRTHomo sapiens 889His Arg Arg Ser Lys Gln1 58906PRTHomo sapiens 890Arg Lys Arg Gly Lys Asn1 58916PRTHomo sapiens 891Arg Lys Arg Gly Lys Asn1 58928PRTHomo sapiens 892Ile Lys Lys Arg Ser Leu Arg Ser1 58936PRTHomo sapiens 893Ser Lys Arg Pro Lys Asn1 58946PRTHomo sapiens 894Ser Lys Arg Pro Lys Asn1 58956PRTHomo sapiens 895Pro Arg Arg Ser Arg Arg1 58966PRTHomo sapiens 896Trp Arg Arg Thr Arg Gly1 58976PRTHomo sapiens 897Pro Arg Arg Ser Arg Arg1 58986PRTHomo sapiens 898Trp Arg Arg Thr Arg Gly1 58996PRTHomo sapiens 899Gly Lys Lys Leu Lys Leu1 59006PRTHomo sapiens 900Ile Arg Lys Gln Lys Leu1 59016PRTHomo sapiens 901Gly Lys Lys Leu Lys Leu1 59026PRTHomo sapiens 902Ile Arg Lys Gln Lys Leu1 59038PRTHomo sapiens 903Asn Arg Lys Lys His Arg Arg Lys1 59046PRTHomo sapiens 904Arg Lys Lys His Arg Arg1 59056PRTHomo sapiens 905Arg Lys Lys His Arg Arg1 59066PRTHomo sapiens 906Ala Arg Arg Lys Arg Arg1 59076PRTHomo sapiens 907Asn Lys Arg Ile Arg Tyr1 59086PRTHomo sapiens 908Ala Arg Arg Lys Arg Arg1 59096PRTHomo sapiens 909Asn Lys Arg Ile Arg Tyr1 59106PRTHomo sapiens 910Asp Lys Lys Lys Arg Met1 59116PRTHomo sapiens 911Lys Arg Lys Glu Lys Ala1 59126PRTHomo sapiens 912Tyr Arg Lys Lys Lys Gln1 59136PRTHomo sapiens 913Asp Lys Lys Lys Arg Met1 59146PRTHomo sapiens 914Lys Arg Lys Glu Lys Ala1 59156PRTHomo sapiens 915Tyr Arg Lys Lys Lys Gln1 59166PRTHomo sapiens 916Ile Lys Arg Leu Lys Ile1 59176PRTHomo sapiens 917Ile Lys Arg Leu Lys Ile1 59188PRTHomo sapiens 918Arg Lys Arg Arg Gly Leu Arg Ser1 59198PRTHomo sapiens 919Ser Arg Lys Lys Thr Arg Arg Met1

59206PRTHomo sapiens 920Met Arg Arg Ser Arg Ala1 59216PRTHomo sapiens 921Glu Arg Lys Arg Arg Gly1 59226PRTHomo sapiens 922Arg Lys Lys Thr Arg Arg1 59236PRTHomo sapiens 923Met Arg Arg Ser Arg Ala1 59246PRTHomo sapiens 924Glu Arg Lys Arg Arg Gly1 59256PRTHomo sapiens 925Arg Lys Lys Thr Arg Arg1 59268PRTHomo sapiens 926Asn Lys Lys Lys His Leu Lys Lys1 59278PRTHomo sapiens 927Glu Lys Lys Lys Leu Lys Arg His1 59286PRTHomo sapiens 928Trp Lys Lys Lys Arg Ala1 59296PRTHomo sapiens 929Arg Lys Lys Ala Lys Phe1 59306PRTHomo sapiens 930Lys Lys Lys Leu Lys Arg1 59316PRTHomo sapiens 931Trp Lys Lys Lys Arg Ala1 59326PRTHomo sapiens 932Arg Lys Lys Ala Lys Phe1 59336PRTHomo sapiens 933Lys Lys Lys Leu Lys Arg1 59346PRTHomo sapiens 934Leu Lys Lys Phe Arg Gln1 59356PRTHomo sapiens 935Leu Arg Lys Ile Arg Thr1 59366PRTHomo sapiens 936Leu Lys Lys Phe Arg Gln1 59376PRTHomo sapiens 937Leu Arg Lys Ile Arg Thr1 59388PRTHomo sapiens 938Gln Lys Arg Arg Arg His Arg Ala1 59396PRTHomo sapiens 939Gln Lys Arg Arg Arg His1 59406PRTHomo sapiens 940Gln Lys Arg Arg Arg His1 59418PRTHomo sapiens 941Gln Lys Arg Lys Thr Gly Arg Leu1 59428PRTHomo sapiens 942Lys Arg Lys Lys Leu Val Arg Asp1 59438PRTHomo sapiens 943Glu Lys Arg Arg Arg Glu Arg Glu1 59448PRTHomo sapiens 944Glu Lys Lys Lys Lys Asn Lys Thr1 59456PRTHomo sapiens 945Arg Lys Lys Asn Lys Gly1 59466PRTHomo sapiens 946Ser Lys Lys Ser Lys Glu1 59476PRTHomo sapiens 947Glu Lys Lys Val Arg Leu1 59486PRTHomo sapiens 948Leu Lys Arg Lys Lys Leu1 59496PRTHomo sapiens 949Glu Lys Arg Arg Arg Glu1 59506PRTHomo sapiens 950Glu Lys Lys Glu Lys Lys1 59519PRTHomo sapiens 951Gln Lys Lys Asp Val Val Lys Arg Gln1 59526PRTHomo sapiens 952Arg Lys Lys Asn Lys Gly1 59536PRTHomo sapiens 953Ser Lys Lys Ser Lys Glu1 59546PRTHomo sapiens 954Glu Lys Lys Val Arg Leu1 59556PRTHomo sapiens 955Leu Lys Arg Lys Lys Leu1 59566PRTHomo sapiens 956Glu Lys Arg Arg Arg Glu1 59576PRTHomo sapiens 957Glu Lys Lys Glu Lys Lys1 59586PRTHomo sapiens 958Gly Lys Arg Tyr Lys Phe1 59596PRTHomo sapiens 959Gly Lys Arg Tyr Lys Phe1 59606PRTHomo sapiens 960Arg Arg Arg Gly Lys Thr1 59616PRTHomo sapiens 961Arg Arg Arg Gly Lys Thr1 59626PRTHomo sapiens 962Val Arg Arg Tyr Arg Ala1 59636PRTHomo sapiens 963Val Arg Arg Tyr Arg Ala1 59646PRTHomo sapiens 964Met Arg Arg Arg Lys Pro1 59656PRTHomo sapiens 965Glu Lys Lys Lys Arg Glu1 59666PRTHomo sapiens 966Met Arg Arg Arg Lys Pro1 59676PRTHomo sapiens 967Glu Lys Lys Lys Arg Glu1 59686PRTHomo sapiens 968Lys Lys Lys Val Lys Asn1 59696PRTHomo sapiens 969Leu Arg Lys Thr Arg His1 59706PRTHomo sapiens 970Lys Lys Lys Val Lys Asn1 59716PRTHomo sapiens 971Leu Arg Lys Thr Arg His1 59726PRTHomo sapiens 972Gly Lys Arg Trp Lys Glu1 59736PRTHomo sapiens 973Gly Lys Arg Trp Lys Glu1 59746PRTHomo sapiens 974Gly Lys Arg Trp Lys Glu1 59756PRTHomo sapiens 975Leu Arg Lys Thr Lys Ala1 59766PRTHomo sapiens 976Gly Lys Arg Trp Lys Glu1 59776PRTHomo sapiens 977Leu Arg Lys Thr Lys Ala1 59788PRTHomo sapiens 978Ile Arg Arg Arg Ala Val Arg Ser1 59796PRTHomo sapiens 979Phe Lys Arg Lys Lys Phe1 59806PRTHomo sapiens 980Ile Arg Lys Pro Arg His1 59816PRTHomo sapiens 981Val Arg Lys Met Arg Asp1 59826PRTHomo sapiens 982Phe Lys Arg Lys Lys Phe1 59836PRTHomo sapiens 983Ile Arg Lys Pro Arg His1 59846PRTHomo sapiens 984Val Arg Lys Met Arg Asp1 59856PRTHomo sapiens 985Val Lys Lys Val Arg Leu1 59866PRTHomo sapiens 986Val Lys Lys Val Arg Leu1 59876PRTHomo sapiens 987Ala Arg Arg Leu Arg Asn1 59886PRTHomo sapiens 988Ala Arg Arg Leu Arg Asn1 59898PRTHomo sapiens 989Lys Lys Lys Lys Thr Gly Arg Ala1 59906PRTHomo sapiens 990Glu Lys Lys Lys Lys Lys1 59916PRTHomo sapiens 991Glu Lys Lys Lys Lys Lys1 59926PRTHomo sapiens 992Leu Arg Arg His Lys Arg1 59936PRTHomo sapiens 993Leu Arg Arg His Lys Arg1 59948PRTHomo sapiens 994Asn Arg Lys Arg Leu Tyr Lys Val1 59958PRTHomo sapiens 995Glu Arg Lys Arg Ser Arg Arg Arg1 59968PRTHomo sapiens 996Gln Arg Arg Arg Thr Pro Arg Pro1 59978PRTHomo sapiens 997Glu Lys Lys Lys Lys Arg Arg Glu1 59986PRTHomo sapiens 998Val Arg Lys Leu Arg Lys1 59996PRTHomo sapiens 999Gly Arg Arg Gln Lys Lys1 510006PRTHomo sapiens 1000Thr Lys Lys Gln Lys Arg1 510016PRTHomo sapiens 1001Arg Lys Arg Ser Arg Arg1 510026PRTHomo sapiens 1002Glu Lys Lys Lys Lys Arg1 510036PRTHomo sapiens 1003Val Arg Lys Leu Arg Lys1 510046PRTHomo sapiens 1004Gly Arg Arg Gln Lys Lys1 510056PRTHomo sapiens 1005Thr Lys Lys Gln Lys Arg1 510066PRTHomo sapiens 1006Arg Lys Arg Ser Arg Arg1 510076PRTHomo sapiens 1007Glu Lys Lys Lys Lys Arg1 510086PRTHomo sapiens 1008Trp Lys Arg Ile Lys Gly1 510096PRTHomo sapiens 1009Trp Lys Arg Ile Lys Gly1 510108PRTHomo sapiens 1010Asn Arg Arg Arg Gln Glu Arg Phe1 510116PRTHomo sapiens 1011Arg Lys Arg Ala Lys Asp1 510126PRTHomo sapiens 1012Arg Lys Arg Ala Lys Asp1 510138PRTHomo sapiens 1013Leu Arg Lys Lys Leu His Lys Phe1 510146PRTHomo sapiens 1014Ala Arg Lys Asn Lys Asn1 510156PRTHomo sapiens 1015Ala Arg Lys Asn Lys Asn1 510166PRTHomo sapiens 1016Ser Lys Lys Arg Lys Asn1 510176PRTHomo sapiens 1017Ser Lys Lys Arg Lys Asn1 510186PRTHomo sapiens 1018Ser Arg Arg Tyr Arg Gly1 510196PRTHomo sapiens 1019His Arg Lys Asn Lys Pro1 510206PRTHomo sapiens 1020Ser Arg Arg Tyr Arg Gly1 510216PRTHomo sapiens 1021His Arg Lys Asn Lys Pro1 510228PRTHomo sapiens 1022Ser Arg Arg Arg Phe Arg Lys Ile1 510238PRTHomo sapiens 1023Gln Arg Lys Lys Arg Trp Arg Ser1 510248PRTHomo sapiens 1024His Lys Lys Arg Ala Arg Arg Ser1 510256PRTHomo sapiens 1025Arg Arg Arg Phe Arg Lys1 510266PRTHomo sapiens 1026Ser Lys Lys Val Lys Ala1 510276PRTHomo sapiens 1027Leu Lys Arg Met Lys Ile1 510286PRTHomo sapiens 1028Gln Arg Lys Lys Arg Trp1 510296PRTHomo sapiens 1029Lys Lys Arg Ala Arg Arg1 510306PRTHomo sapiens 1030Ala Arg Lys Val Lys Gln1 510316PRTHomo sapiens 1031Arg Arg Arg Phe Arg Lys1 510326PRTHomo sapiens 1032Ser Lys Lys Val Lys Ala1 510336PRTHomo sapiens 1033Leu Lys Arg Met Lys Ile1 510346PRTHomo sapiens 1034Gln Arg Lys Lys Arg Trp1 510356PRTHomo sapiens 1035Lys Lys Arg Ala Arg Arg1 510366PRTHomo sapiens 1036Ala Arg Lys Val Lys Gln1 510378PRTHomo sapiens 1037Glu Arg Lys Lys Ile Thr Arg Glu1 510386PRTHomo sapiens 1038Gly Arg Lys Arg Lys Glu1 510396PRTHomo sapiens 1039Gly Arg Lys Arg Lys Glu1 510406PRTHomo sapiens 1040Leu Lys Arg Pro Arg Leu1 510416PRTHomo sapiens 1041Val Lys Arg Glu Lys Pro1 510426PRTHomo sapiens 1042Leu Lys Arg Pro Arg Leu1 510436PRTHomo sapiens 1043Val Lys Arg Glu Lys Pro1 510446PRTHomo sapiens 1044Ser Lys Arg Pro Lys Thr1 510456PRTHomo sapiens 1045Glu Lys Lys Asn Lys Leu1 510466PRTHomo sapiens 1046Asn Lys Arg Pro Arg Thr1 510476PRTHomo sapiens 1047Asp Lys Arg Leu Arg Thr1 510486PRTHomo sapiens 1048Gln Lys Arg Phe Arg Thr1 510496PRTHomo sapiens 1049Glu Lys Lys Ser Lys Leu1 510506PRTHomo sapiens 1050Gln Arg Lys Asp Lys Asp1 510516PRTHomo sapiens 1051Pro Lys Lys Glu Lys Gly1 510526PRTHomo sapiens 1052Ser Lys Arg Pro Lys Thr1 510536PRTHomo sapiens 1053Glu Lys Lys Asn Lys Leu1 510546PRTHomo sapiens 1054Asn Lys Arg Pro Arg Thr1 510556PRTHomo sapiens 1055Asp Lys Arg Leu Arg Thr1 510566PRTHomo sapiens 1056Gln Lys Arg Phe Arg Thr1 510576PRTHomo sapiens 1057Glu Lys Lys Ser Lys Leu1 510586PRTHomo sapiens 1058Gln Arg Lys Asp Lys Asp1 510596PRTHomo sapiens 1059Pro Lys Lys Glu Lys Gly1 510606PRTHomo sapiens 1060Tyr Lys Arg Arg Lys Lys1 510616PRTHomo sapiens 1061Tyr Lys Arg Arg Lys Lys1 510626PRTHomo sapiens 1062Thr Arg Arg Glu Arg Ala1 510636PRTHomo sapiens 1063His Arg Lys Leu Lys Val1 510646PRTHomo sapiens 1064Thr Arg Arg Glu Arg Ala1 510656PRTHomo sapiens 1065His Arg Lys Leu Lys Val1 510666PRTHomo sapiens 1066Pro Lys Lys Pro Lys Gly1 510676PRTHomo sapiens 1067Gly Lys Lys Lys Lys Asp1 510686PRTHomo sapiens 1068Pro Lys Lys Pro Lys Gly1 510696PRTHomo sapiens 1069Gly Lys Lys Lys Lys Asp1 510704PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1070Arg Arg Lys Arg110714PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1071Phe Phe Lys Lys110724PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1072Lys Lys Lys Lys110734PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1073Arg Arg Arg Lys110744PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1074Arg Arg Lys Asn1

Claims

1. A method of making a modified GAG binding protein by modifying a GAG binding site of the GAG binding protein, wherein the GAG binding site is modified by a method comprising the steps of: a) identifying a region in the protein which is not essential for structure maintenance; and b) introducing at least one basic amino acid into the site and/or deleting at least one bulky and/or acidic amino acid in the site; whereby the GAG binding site has a GAG binding affinity of Kd≦10 μM.

2. A method according to claim 1, wherein the GAG binding site has a GAG binding affinity of ≦1 μM.

3. A method according to claim 1, wherein the GAG binding site has a GAG binding affinity of ≦0.1 μM.

4. A method according to claim 1, wherein the GAG binding affinity is higher by a factor of minimum 5 compared with wild-type GAG binding protein.

5. A method according to claim 1, wherein at least one basic amino acid selected from the group consisting of Arg, Lys, and His is inserted into the GAG binding region.

6. A method according to claim 1, wherein the protein is a chemokine, preferably IL-8, RANTES or MCP-1.

7. A method according to claim 6, wherein the GAG binding region is a C terminal a-helix.

8. A method according to claim 7, wherein positions 17, 21, 70, and/or 71 are substituted by Arg, Lys, His, Asn, and/or Gln.

9. A method according to claim 1, wherein the increased GAG binding affinity is an increased binding affinity to heparan sulfate and/or heparin.

10. A method according to claim 1, wherein a further biologically active region is modified thereby inhibiting or down-regulating a further biological activity of the protein.

11. A method according to claim 10, wherein the further biologically active region is modified by deletion, insertion, and/or substitution, preferably with alanine, a sterically and/or electrostatically similar residue.

12. A method according to claim 10, wherein the protein is a chemokine and the further biological activity is leukocyte activation.

13. A method according to claim 12, wherein the protein is IL-8 and the further biologically active region is located within the first 10 N-terminal amino acids.

14. A method according to claim 13, wherein the protein is a mutant with the first 6 N-terminal amino acids deleted.

15. A method according to claim 14, wherein the protein is a mutant, and wherein the protein is modified such that Arg is at position 17, Lys is at position 70, and Arg is at position 71; Arg is at position 17, Arg is at position 70, and Lys is at position 71; or Lys is at positions 70 and 71.

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