GAG BINDING PROTEINS

Description

This application is a continuation of U.S. application Ser. No. 12/131,311 filed Jun. 2, 2008, which is a divisional of U.S. application Ser. No. 11/422,169 filed Jun. 5, 2006, which is a 371 of PCT/EP2004/013670 filed Dec. 2, 2004. The entire contents of the above-identified applications are hereby incorporated by reference.

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.

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 defense 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.

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.

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.

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.

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 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 [76]
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

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.

Multiple intracellular signaling pathways occur after receptor activation as a result of chemokine binding. Chemokines also interact with two types of nonsignaling 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 signaling because of high local concentration of chemokines. For maintaining a tissue bloodstream chemokine gradient, the DARC receptor functions as a sink for surplus chemokines.

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.

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).

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.

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.

Since the demonstration that heparan sulphate proteoglycans are critical for FGF signaling, 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 signaling 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.

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.

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.

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 targeted by such a strategy.

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.

Therefore, the present invention provides a method for introducing a GAG binding site into a protein characterised in that it comprises 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 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:

FIG. 1 shows a CD spectra.

FIG. 2 shows secondary structure contents of various mutants.

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

FIG. 4 shows graphics of results from fluorescence anisotropy tests of two 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.

FIG. 8 shows the results of the RANTES chemotaxis assay.

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.

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

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.

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.

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.

A protocol for introducing a GAG binding site into a protein is for example as follows:

• • Identify a region of the protein which is not essential for overall structural maintenance and which might be suitable for GAG binding
  • 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
  • Check the conformational stability of the resulting mutant protein in silico
  • Clone the wild-type protein cDNA (alternatively: purchase the cDNA)
  • Use this as template for PCR-assisted mutagenesis to introduce the above mentioned changes into the amino acid sequence
  • Subclone the mutant gene into a suitable expression system (prokaryotic or eukaryotic dependent upon biologically required post-translational modifications)
  • Expression, purification and characterisation of the mutant protein in vitro
  • Criterion for an introduced GAG binding affinity: K_d^GAG(mutant)≦10 μM.

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:

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)

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.

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 the GAG binding affinity of a respective wild-type protein.

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.

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.

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).

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 synthesis 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)).

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.

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.

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.

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.

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.

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.

According to an advantageous aspect, said GAG binding region is a C terminal α-helix. A typical 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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⁺)])+0.41 (% G+C)−(600/N), where N=chain length.

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.

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.

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.

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 suppressing 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.

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.

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.

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 (SEQ ID NOS 1070-1074 are disclosed respectively in order of appearance), 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

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″):

(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)

The reverse sequences are (5″to 3″):

(SEQ ID NO: 9)

	TTA TGA ATT CCT AGC CCT CTT
	(primer for the mutation E70R)

(SEQ ID NO: 10)

	TTA TGA ATT CTT AGC CCT CTT
	(primer for the mutation E70K)

(SEQ ID NO: 11)

	TTA TGA CTT CTC AGC CCT CTT
	(primer for the mutation N71K)

(SEQ ID NO: 12)

	TTA TGA CTT CTT AGC CCT CTT
	(primer for the mutation E70K N71K)

(SEQ ID NO: 13)

	TTA TGA CTT CCT AGC CCT CTT
	(primer for the mutation E70R N71K)

(SEQ ID NO: 14)

	TTA TGA CCT CTT AGC CCT CTT
	(primer for the mutation E70K N71R)

(SEQ ID NO: 15)

	TTA TGA CCT CCT AGC CCT CTT
	(primer for the mutation E70R N71R)

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

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 nonlinear 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.

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 Dichroism Measurements and Analysis

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.

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.

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-8wt 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-8wt 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-8wt 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.

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

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

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.

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-8wt (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-8wt, 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

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 K_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.

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 25-350. Significant improvement of ligand binding was observed, especially for heparin binding. Δ6 F17RN71R E7OK (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-8wt (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-8wt 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

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.

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-8wt, 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

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:

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 2 Mio/ml cell suspension, taking into account that only 60% of the counted cells were neutrophils.

Chemotaxis Assay:

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 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

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.

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.

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.

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.

	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

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

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 2
Chemokines and their GAG binding domains

CXC-chemokines
IL-8: 18HPK20, (R47) 60RVVEKFLKR68(residues 60-68 of SEQ ID NO: 16)
SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQR
VVEKFLKRAENS (SEQ ID NO: 16)
MGSA/GROα: 19HPK21, 45KNGR48(residues 45-48 of SEQ ID NO: 17),
60KKIIEK66(residues 60-66 of SEQ ID NO: 17)
ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVK
KIIEKMLNSDKSN (SEQ ID NO: 17)
MIP-2α/GROβ: 19HLK21,K45, 60KKIIEKMLK68(residues 60-68 of SEQ ID NO: 18)
APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKACLNPASPMVK
KIIEKMLKNGKSN (SEQ ID NO: 18)
NAP-2: 15HPK18, 42KDGR45(residues 42-45 of SEQ ID NO: 19), 57KKIVQK62(residues
57-62 of SEQ ID NO: 19)
AELRCLCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQ
KKLAGDESAD (SEQ ID NO: 19)
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)
EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLY
KKIIKKLLES (SEQ ID NO: 20)
SDF-1α: K1, 24KHLK27(residues 24-27 of SEQ ID NO: 21), 41RLK43
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQE
YLEKALN (SEQ ID NO: 21)
CC-chemokines
RANTES: (17RPLPRAH23(residues 17-23 of SEQ ID NO: 22)) 44RKNR47(residues 44-47
of SEQ ID NO: 22)
SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVEVTRKNRQVCANPEKKWVRE
YINSLEMS (SEQ ID NO: 22)
MCP-2: 18RKIPIQR24(residues 18-24 of SEQ ID NO: 23), 46KRGK49(residues 46-49
of SEQ ID NO: 23)
QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIEKTKRGKEVCADPKERWVRDSMKHLDQI
FQNLKP (SEQ ID NO: 23)
MCP-3: 22KQR24, 47KLDK50(residues 47-50 of SEQ ID NO: 24),
66KHLDKK71(residues 66-71 of SEQ ID NO: 24)
QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWV
QDFMKHLDKKTQTPKL (SEQ ID NO: 24)
MIP-1α: R17, 44KRSR47(residues 44-47 of SEQ ID NO: 25)
SLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVCADPSEEWVQK
YVSDLELSA (SEQ ID NO: 24)
MIP-1β: R18, 45KRSK48(residues 45-48 of SEQ ID NO: 26)
APMGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYDLELN
(SEQ ID NO: 26)
MPIF-1: R18, 45KKGR48(residues 45-48 of SEQ ID NO: 27)
MDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANPSDKQVQ
VCMRMLKLDTRIKTRKN (SEQ ID NO: 27)
MIP-5/HCC-2: 40KKGR43(residues 40-43 of SEQ ID NO: 28)
HFAADCCTSYISQSIPCSLMKSYFETSSECSKPGVIFLTKKGRQVCAKPSGPGVQDCMKK
LKPYSI (SEQ ID NO: 28)

	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	71	440	TKKKTRRR	447
protein	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	87	672	ERRARL	677
factor 1	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-Fli1	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	102	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-	150	139	GRKKCERY	146
receptor 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	284	121	AKRQRL	126
L47	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 membrane	294	102	DRRERE	107
protein 1	295	102	DRRERE	107
Vitamin D receptor-interacting protein	296	673	KKKKSSRL	680
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 membrane	311	100	ERKERE	105
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	321	271	HKRLRQ	276
protein	322	271	HKRLRQ	276
Kappa B and V(D)J recombination signal	323	17	PRKRLTKG	24
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	353	228	HKRMKV	233
hydrolase 12	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	404	66	ARKVKS	71
receptor	405	66	ARKVKS	71
Short transient receptor potential	406	753	FKKTRY	758
channel 7	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-	417	904	DKRKCERL	911
alpha	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	427	166	VRRHRN	171
type	428	233	WKRLKD	238
	429	166	VRRHRN	171
	430	233	WKRLKD	238
Hematopoietic PBX-interacting	431	159	LRRRRGRE	166
protein	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	487	32	GRRARP	37
factor 1	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	561	773	YRKKPHRP	780
protein	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	570	64	HRRPRG	69
protein	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
syntaxin1B2	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-	640	225	SRRSKL	230
containing 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	666	197	AKKYRD	202
protein 2	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,	710	60	GKKLKA	65
mitochondrial	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 protein 1	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	785	162	QRRRTLKN	169
protein
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	814	1333	IRKKVRRL	1340
protein	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	846	229	WKRIRF	234
TRC8	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	868	455	LRRTRI	460
synthetase	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	505	CKKKMRRK	512
4 zeta splice variant	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
immunoglobulin superfamily, member 3	893	394	SKRPKN	399
	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	906	233	ARRKRR	238
factor-1	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	8441	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 activation	960	261	RRRGKT	266
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 serine/	968	288	KKKVKN	293
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-	978	594	IRRRAVRS	601
receptor 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 (chymotryptic,	985	15	VKKVRL	20
stratum corneum)	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	375	LRKKLHKF	382
type I-beta	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 factor	1022	138	SRRRFRKI	145
	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	6	PKKPKG	11
	1067	84	GKKKKD	89
	1068	6	PKKPKG	11
	1069	84	GKKKKD	89

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