# Patent

## Title
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.

## Background
This application is a divisional of U.S. application Ser. No. 11/422,169 filed on Jun. 5, 2006, now U.S. Pat. No. 7,585,937 which is a continuation of PCT/EP2004/013670 filed on 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 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.
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 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.
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 (6-0 and 3-0 sulphation of the glucosamine and 2-0 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 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.
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 Kd≦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 Kd. One possibility is to determine the dissociation constant (Kd) 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: KdGAG (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. Kd≦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 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 (Kd-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 Kd (mutant)<Kd (wild-type), preferably by at least 100%. Specifically improved modified proteins have—compared with wild-type Kd—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 Kd 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 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.
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, 2nd 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 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.
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′):









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)






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 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.
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
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-8 wt only one mutant (Δ6 E70R) showed nearly similar structure whereas the others differed mainly in their α-helix which ranged from 17.20 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.
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 (koligo=350 nM) to IL-8 wt (koligo=379 nM). The mutant with the highest koligo (koligo=460 μM), 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 koligo (koligo=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 (koligo=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 (Kd 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 Kd. 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 25-35%. Significant improvement of ligand binding was observed, especially for heparin binding. Δ6 F17RN71R E70K (Kd=14 nM) and Δ6 F17RF21R N71K (Kd=14.6 nM) showed 2600-fold better binding, and Δ6 E70K N71K (Kd=74 nM) 1760-fold better binding compared to IL-8 wt (Kd=37 μM). Good results were also obtained for heparan sulphate binding. For A6 F17RN71R E70K a Kd of 107 nM was found, for Δ6 F17RF21R N71K the Kd was 95 nM and the mutant AG E70K N71K showed a Kd of 34 nM. As IL-8 wt binds with a Kd of 4.2 μM, the Kds 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-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
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 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
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)


SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELC


LDPKENWVQRVVEKFLKRAENS


(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)


ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKA


CLNPASPIVKKIIEKMLNSDKSN


(SEQ ID NO: 17)


 


MIP-2α/GROβ:


19HLK21, K45, 60KKIIEKMLK68


(residues 60-68 of SEQ ID NO: 18)


APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKA


CLNPASPMVKKIIEKMLKNGKSN


(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)


AELRCLCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDP


DAPRIKKIVQKKLAGDESAD


(SEQ ID NO: 19)


 


PF-4:


20RPRH23


(residues 20-23 of SEQ ID NO: 20),


46KNGR49


(residues 46-49 of SEQ ID NO: 20),


61 KKIIKK66


(residues 61-66 of SEQ ID NO: 20)


EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRK


ICLDLQAPLYKKIIKKLLES


(SEQ ID NO: 20)


 


SDF-1α:


K1, 24KHLK27


(residues 24-27 of SEQ ID NO: 21),


41RLK43


KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVC


IDPKLKWIQEYLEKALN


(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)


SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC


ANPEKKWVREYINSLEMS


(SEQ ID NO: 22)


 


MCP-2:


18RKIPIQR24


(residues 18-24 of SEQ ID NO: 23),


46KRGK49


(residues 46-49 of SEQ ID NO: 23)


QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKE


VCADPKERWVRDSMKHLDQIFQNLKP


(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)


QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKE


ICADPTQKWVQDFMKHLDKKTQTPKL


(SEQ ID NO: 24)


 


MIP-1α:


R17, 44KRSR47


(residues 44-47 of SEQ ID NO: 25)


SLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVC


ADPSEEWVQKYVSDLELSA


(SEQ ID NO: 25)


 


MIP-1β:


R18, 45KRSK48


(residues 45-48 of SEQ ID NO: 26)


APMGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQV


CADPSESWVQEYVYDLELN


(SEQ ID NO: 26)


 


MPIF-1:


R18, 45KKGR48


(residues 45-48 of SEQ ID NO: 27)


MDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRF


CANPSDKQVQVCMRMLKLDTRIKTRKN


(SEQ ID NO: 27)


 


MIP-5/HCC-2:


40KKGR43


(residues 40-43 of SEQ ID NO: 28)


HFAADCCTSYISQSIPCSLMKSYFETSSECSKPGVIFLTKKGRQVCAKPS


GPGVQDCMKKLKPYSI


(SEQ ID NO: 28)



















TABLE 3






SEQ ID





NO:


















Peroxisome biogenesis
29
 181 TRRAKE 186



factor 1
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-
49
 315 RRRLRS 320


associated protein),
50
 404 VRKLKG 409


delta 1
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
61
 221 EKRTKD 226


receptor MS
62
 427 TKRKRV 432



63
 514 WKKKKV 519



64
 221 EKRTKD 226



65
 427 TKRKRV 432



66
 514 WKKKKV 519


 


Alpha-2A adrenergi
67
 147 PRRIKA 152


receptor
68
 224 KRRTRV 229



69
 147 FRRIKA 152



70
 224 KRRTRV 229


 


IL-S promoter
71
 440 TKKKTRRR 447


REII-region-binding
72
 569 GKRRRRRG 576


protein
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
87
 672 ERRARL 677


regulatory factor 1
88
 672 ERRARL 677


 


CUG-BP and ETR-3 like
89
 468 MKRLKV 473


factor 5
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
100
  22 EKRERT 27


protein GLIS2
101
  22 EKEERT 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
110
 393 SRKKRERD 400


protein
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
146
320 PRKVKS 325


adhesion molecule
147
 387 VRKDKL 392



148
 320 PRKVKS 325



149
 387 VRKDKL 392


 


Proteintyrosine
150
 139 GRKKCERY 146


phosphatase, non-
151
  59 VKKNRY 64


receptor type 12
152
  59 VKKNRY 64


 


WD-repeat protein 11
153
 752 VRKIRF 757



154
 752 VRKIRF 757


 


Gastric cancer-related
155
  20 SRKRQTRR 27


protein VRG107
156
  25 TRRRRN 30



157
  25 TRRRRN 30


 


Early growth response
158
 356 ARRKGRRG 363


protein 4
159
 452 EKKRHSKV 459



160
 357 RRKGRR 362



161
 357 RRKGRR 362


 


Vesicle transport-
162
 309 PKRKNKKS 316


related protein
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
199
  62 LKRDRF 67


protein 1
200
  62 LKRDRF 67


 


MHC class II
201
 407 HRRPRE 412


transactivator CIITA
202
 741 PRKKRP 746



203
 783 DRKQKV 788



204
 407 HRRPRE 412



205
 741 PRKKRP 746



206
 783 DRKQKV 788


 


Platelet glycoprotein
207
 275 SRRKRLRN 282


VI-2
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
222
  87 SKKEKV 92


deiodinase
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
231
   4 ARKIRT 9


N-acetylgalactosaminyl-
232
  44 DRRVRS 49


transferase
233
 138 PRKCRQ 143



234
   4 ARKIRT 9



235
  44 DRRVRS 49



236
 138 PRKCRQ 143


 


NCBE
237
  62 HRRHRN 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-
250
891 ERRRRPRD 898


transporting ATPase 2
251
 306 SRKWRP 311



252
 891 ERRRRP 896



253
 306 SRKWRP 311



254
 891 ERRRRP 896


 


HMG-box transcription
255
 420 GKKKKRKR 427


factor TCF-3
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


 


Clorf25
273
 620 GKKQKT 625



274
 620 GKKQKT 625


 


Clorf14
275
 441 LRRRKGKR 448



276
  70 LRRWRR 75



277
 441 LRRRKG 446



278
  70 LRRWRR 75



279
 441 LRRRKG 446


 


T-box transcription
280
 144 DKKAKY 149


factor TBX3
281
 309 GRREKR 314



282
 144 DKKAKY 149



283
 309 GRREKR 314


 


Mitochondrial 39S
284
 121 AKRQRL 126


ribosomal protein 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


 


Jaggedl
292
1093 LRKRRK 1098



293
1093 LRKRRK 1098


Secretory carrier-
294
 102 DRRERE 107


associated membrane
295
 102 DRRERE 107


protein 1


 


Vitamin D receptor-
296
 673 KKKKSSRL 680


interacting protein
297
 672 TKKKKS 677


complex component
298
 954 QKRVKE 959


DRIP205
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
 999 PKRKKA 1000



308
1338 GKREKS 1343



309
1482 HKKHKK 1487



310
1489 KKKVKD 1494


 


Secretory carrier-
311
 100 ERKERE 105


associated membrane
312
 100 ERKERE 105


protein 2


 


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
321
 271 HKRLRQ 276


element-binding protien
322
 271 HKRLRQ 276


 


Kappa B and V(D)J
323
  17 PRKRLTKG 24


recombination signal
324
 713 RKRRKEKS 720


sequences binding
325
 903 PKKKRLRL 910


protien 
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
338
 200 SKRKKA 205


metastasis-suppressor 1
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-
353
  28 HKRMKV 233


terminal hydrolase 12
354
 244 LKRFKY 249



355
 228 HKRMKV 233



356
 244 LKRFKY 249


 


K1AA0472 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
365
 176 GRRKSERQ 183


factor 2


 


Sodium/potassium-
366
  47 SRRFRC 52


transporting ATPase
367
  55 NKKRRQ 60


gamma chain
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
374
  87 QKKIRI 92


protein
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
380
 973 EKKKKKKK 980


cotransporter
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
404
  66 ARKVKS 71


motility receptor
405
  66 ARKVKS 71


 


Short transient receptor
406
 753 FKKTRY 758


potential 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
417
 904 DKRKCERL 911


intermediary factor 1-
418
1035 PRKKRLKS 1042


alpha
419
 321 NKKGKA 326



420
1035 PRKKRL 1040



421
 321 NKKGKA 326



422
1035 PRKKRL 1040


 


CARTILAGE INTERMEDIATE
423
 719 QRRNKR 724


LAYER PROTEIN
424
 719 QRRNKR 724


 


UBX domain-containing
425
 194 YRKIKL 199


protien 1
426
 194 YRKIKL 199


 


Arachidonate 12-
427
 166 VRRHRN 171


lipoxygenase, 12R type 
428
 233 WKRLKD 238



429
 166 VRRHRN 171



430
 233 WKRLKD 238


 


Hematopoietic PBX-
431
 159 LRRRRGRE 166


interacting 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
449
  11 TRRSRA 16


cluster
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
462
 512 TRRKRH 517


triphosphate
463
 512 TRRKRH 517


diphosphohydrolase 3


 


Homeobox protein prophet
464
  12 PKKGRV 17


of PIT-1
465
  69 RRRHRT 74



466
 119 NRRAKQ 124



467
  12 PKKGRV 17



468
  69 RRRHRT 74



469
 119 NRRAKQ 124


 


PROSTAGLANDIN EP3
470
  77 YRRRESKR 84


RCEPTOR
471
 389 MRKRRLRE 396



472
  82 SKRKKS 87



473
 389 MRKRRL394



474
  82 SKRKKS 87



475
 389 MRKRRL394


 


Pituitary homeobox 3
476
  58 LKKKQRRQ 65



477
  59 KKKQRR 64



478
 112 NRRAXW 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-
487
  32 GRRARP 37


interacting 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 ANKAKS 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 FKKARA 259



507
2002 ARRAKV 2007


 


Tripartite motif protein
508
 290 LKKFKD 295


TRIM31 alpha
509
 290 LKKFKD 295


 


Nuclear receptor co-
510
 106 SKRPRL 111


repressor 1
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
528
 432 KRRVKS 437


FINGER PROTEIN
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
539
 177 SKKYRQ 182


PROTEIN SEC22B
540
 177 SKKYRQ 182


 


Nucleolar transcription
541
  79 VRKFRT 84


factor 1
542
 102 GKKLKK 107



543
 125 EKRAKY 130



544
 147 SKKYKE 152



545
 156 KKKNKY 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 type 3
560
 626 QKRRYSKG 633


 


Plaucible mixed-lineage
561
 773 YRKKPHRP 780


kinase protein
562
 312 ERRLKM 317



563
 312 ERRLKM 317


 


fatty acid binding
564
  78 DRKVKS 83


protein 4, adipocyte
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
570
  64 HRRPRG 69


cysteine-rich protein
571
  64 HRRPRG 69


 


Myb proto-oncogene
572
   2 ARRPRH 7


protein
573
 292 EKRIKE 297



574
 523 LKKIKQ 528



575
   2 ARRPRH 7



576
 292 EKRIKE 297



577
 523 LKKIKQ 528


 


Longchain-fatty-acid--
578
 259 RRKPKP 264


COA ligase 2
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 APRKRQ 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
593
 364 RKRRPSRP 371


alpha
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-
630
 178 MKKPRF 183


MONOOXYGENASE
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
640
 225 SRRSKL 230


colied-coli-containing
641
 455 PKKAKS 460


protein 1
642
 225 SRRSKL 230



643
 455 PKKAKS 460


 


Neuro-endocrine specific
644
 479 EKRNRK 484


protein VGF
645
 479 EKRNRK 484


 


Organic cation
646
 230 GRRYRR 235


transporter
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 GRKLRIA 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
666
 197 AKKYRD 202


channel protein 2
667
 197 AKKYRD 202


 


Methyl-CpG binding
668
  85 KRKKPSRP 92


protein
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
675
 155 RKRQRA 160


type
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
698
  51 NRRLKH 56


protein
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
710
  60 GKKLKA 65


isoenzyme 2,
711
 116 KRKEKL 121


mitochondrial
712
  60 GKKLKA 65



713
 116 KRKEKL 121


 


Chiordecone reductase
714
 245 AKKHKR 250



715
 245 AKKHKR 250


 


Metaxin 2
716
 166 KRKNKA 171



717
 166 KRKMKA 171


 


Paired mesoderm
718
  89 KKKRKQRR 96


homebox protein 1
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 LKRKWZLRC 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
738
 169 ARRRCDRD 176


protein 1


 


SKAP55 protein
739
 115 EKKSKD 120



740
 115 EKKSKD 120


 


Serine palmitoyl-
741
 232 PRKARV 237


transferase 1
742
 232 PRKARV 237


 


Serine palmitoyl-
743
 334 KKKYKA 339


transferase 2
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
753
 291 QKRRTLKN 298


FACTOR C-MAF


 


Usher syndrome type
754
1285 MRRLRS 1290


IIa protein
755
1285 MRRLRS 1290


 


MSin3A associated
756
  95 QKKVKI 100


polypeptied p30
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
764
 383 AKRKADRE 390


RECEPTOR-ASSOCIATED
765
 833 KKRHRE 838


PROTEIN COMPLEX
766
 833 KKRHRE 838


COMPONENT TRAP100


 


P60 katanin
767
 369 LRRRLEKR 376



768
 326 SRRVKA 331



769
 326 SRRVKA 331


Transcription factor
770
 286 RKRKLERI 293


jun-D
771
 273 RKRLRN 278



772
 285 CRKRKL 290



773
 273 RKRLRN 278



774
 285 CRKRKL 290


 


Sterol/retinol
775
 152 VRKARG 157


dehydrogenase
776
 152 VRKARG 157


 


Glycogen [starch]
777
 554 DRRFRS 559


synthase liver
778
 578 SRRQRI 583



779
 554 DRRFRS 559



780
 578 SRRQRI 583


 


Estrogen-related
781
 173 TKRRRK 178


receptor gamma
782
 353 VKKYKS 358



783
 173 TKRRRK 178



784
 353 VKKYKS 358


 


Neural retina-specific
785
 162 QRRRTLKN 169


leucine zipper protein


 


Cytosolic phospholipase
786
 514 NKKKILRE 521


A2-gamma
787
  31 LKKLRI 36



788
 218 FKKGRL 223



789
 428 CRRHKI 433



790
  31 LKKLRI 36


 


Cytosolic phospholipase
791
 218 FKKGRL 223


A2-gamma
792
 428 CRRHKI 433


 


GLE1
793
 415 AKKIKM 420



794
 415 AKKIKM 420


 


Multiple exostoses type
795
 296 VRKRCHKH 303


II protein EAXT.2
796
 659 RKKFKC 664



797
 659 RKKFKC 664


 


Cyclic-AMP-dependent
798
  86 EKKARS 91


transcription factor
799
 332 GRRRRT 337


ATF-7
800
 344 ERRQRF 349



801
  86 EKKARS 91



802
 332 GRRRRT 337



803
 344 ERRQRF 349


 


Protein kinase/
804
 886 LRKFRT 891


endoribonulcease
805
 886 LRKFRT 891


 


Transcription factor
806
  23 RRRCRD 28


E2F6
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
814
1333 IRKKVRRL 1340


death domain 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
825
 126 KRKKSERT 133


PXR
826
  87 TRKTRR 92



827
 125 IKRKKS 130



828
  87 TRKTRR 92



829
 125 IKRKKS 130


 


LENS EPITHELIUM-DERIVED
830
 149 RKRKAEKQ 156


GROWTH FACTOR
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
843
 255 TKRRKRKN 262


cofactor
844
 255 TKRRKR 260



845
 255 TKRRKR 260


MULTIPLE MEMBRANE
846
 229 WKRIRF 234


SPANNING RECEPTOR TRC8
847
 229 WKRIRF 234


 


Transcription factor
848
 172 DKKKLRRL 179


SUPT3H
849
 169 MRKDKK 174



850
 213 NKRQKI 218



851
 169 MRKDKK 174



852
 213 NKRQKI 218


 


GEMININ
853
  50 KRKHFN 55



854
 104 EKRRKA 109



855
  50 KRKHRN 55



856
 104 EKRRKA 109


 


Cell cycle-regulated
857
 165 EKKKVSKA 172


factor p78
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
864
  61 QRKGRK 66


complex, locus D
865
  85 ARRLRA 90



866
  61 QRKGRK 66



867
  85 ARRLRA 90


 


Delta 1-pyrroline-5-
868
 455 LRRTRI 460


carboxylate synthetase
869
 455 LRRTRI 460


 


B CELL LINKER PROTEIN
870
  36 IKKLKV 41


BLINK
871
  36 IKKLKV 41


 


B CELL LINKER PROTEIN
872
  36 IKKLKV 41


BLINK-S
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
885
 505 CKKKMRRK 512


potential channel zeta
886
 506 KKKMRR 511


splice variant
887
 676 HRRSKQ 681



888
 506 KKKNRR 511



889
 676 HRRSKQ 681


 


Myofibrillogenesis
890
  65 RKRGKN 70


regulator MR-2
891
  65 RKRGKN 70


 


SH2 domain-containing
892
 269 IKKRSLRS 276


phosphatase anchor


protein 2c


 


immunoglobulin super-
893
 394 SKRPKN 399


family, 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
906
 233 ARRKRR 238


transcription factor-1
907
 286 NKRIRY 291



908
 233 ARRKRR 238



909
 286 NKRIRY 291


 


60S ribosomal protein
910
 112 DKKKRM 117


L13a
911
 158 KRKEKA 163



912
 167 YRKKKQ 172



913
 112 DKKKRM 117



914
 158 KRKEKA 163



915
 167 YRKKKQ 172


 


WD40-and FYVE-domain
916
 388 IKRLKI 393


containing protein 3
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
927
 430 EKKKLKRH 437


receptor
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
942
2232 KRKKLVRD 2239


protein
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
958
 315 GKRYKF 320


protein LSP1
959
 315 GKRYKF 320


 


similar to signaling
960
 261 RRRGKT 266


lymphocytic activation
961
 261 RRRGKT 266


molecule (H. sapiens)


 


Dermatan-4-
962
 242 VRRYRA 247


sulfotransferase-1
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
969
 358 LRKTRH 363


kinase
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
978
 594 IRRRAVRS 601


phosphatase non-receptor
979
 263 FKRKKF 268


type 3
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
  15 VKKVRL 20


(chymotryptic, stratum
986
  15 VKKVRL 20


corneum)


 


Hormone sensitive lipase
987
  703 ARRLRN 708



988
  703 ARRLRN 708


 


40S ribosomal protein
989
  25 KKKKTGRA 32


S30
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
1008
 725 WKRIKG 730


synthetase
1009
 725 WKRIKG 730


 


ELMO2
1010
 560 NRRRQERF 567


 


Meningioma-expressed
1011
 432 RKEAKD 437


antigen 6/11
1012
 432 RKRAKD 437


 


Inositol polyphosphate
1013
 375 LRKKLHKF 382


4-phosphatase type
1014
 829 ARKNKN 834


I-beta
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
1022
 138 SRRRFRKI 145


exchange 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
1044
 294 SKRPKT 299


factor 1
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
1066
   6 PKKPKG 11


protein 2a
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.

## Claims

1. A method of making modified human Interleukin-8 (IL-8) protein, wherein the method comprises:
substituting one or more amino acids in SEQ ID NO. 16 of human IL-8, wherein the amino acid substitutions are selected from the group consisting of:
(A) Arg, Lys or His substitution at position 70, and
(B) Arg, Lys or His substitution at position 71.

2. The method of making modified human IL-8 according to claim 1, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 70, and
(B) Arg, Lys or His substitution at position 71.

3. The method of making modified human IL-8 according to claim 1, wherein the amino acid substitutions are selected from the group consisting of:
(C) Arg, Lys or His substitution at position 17, and
(D) Arg, Lys or His substitution at position 21.

4. The method of making modified human IL-8 according to claim 2, wherein the amino acid substitutions are selected from the group consisting of:
(C) Arg, Lys or His substitution at position 17, and
(D) Arg, Lys or His substitution at position 21.

5. The method of making modified human IL-8 according to claim 1, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 17, and
(B) Arg, Lys or His substitution at position 21.

6. The method of making modified human IL-8 according to claim 2, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 17, and
(B) Arg, Lys or His substitution at position 21.

7. The method of making modified human IL-8 according to claim 1, wherein the modified human IL-8 is further modified by deleting amino acids at positions 1 through 6 of SEQ ID NO: 16.

8. The method of making modified human IL-8 according to claim 2, wherein the modified human IL-8 is further modified by deleting amino acids at positions 1 through 6 of SEQ ID NO: 16.

9. A method of making modified human Interleukin-8 (IL-8) protein, wherein the method comprises:
(I) substituting one or more amino acids in SEQ ID NO. 16 of human IL-8, wherein the amino acid substitutions are selected from the group consisting of:
(A) Arg, Lys or His substitution at position 70,
(B) Arg, Lys or His substitution at position 71,
(C) Arg, Lys or His substitution at position 17, and
(D) Arg, Lys or His substitution at position 21;

and
(II) deleting amino acids at positions 1 through 6 of SEQ ID NO. 16.

10. The method of making modified human IL-8 according to claim 9, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 70, and
(B) Arg, Lys or His substitution at position 71.

11. The method of making modified human IL-8 according to claim 9, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 70,
(B) Arg, Lys or His substitution at position 71, and
(C) Arg, Lys or His substitution at position 17.

12. The method of making modified human IL-8 according to claim 9, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 70,
(B) Arg, Lys or His substitution at position 71, and
(C) Arg, Lys or His substitution at position 21.

13. A method of making modified human Interleukin-8 (IL-8) protein, wherein the method comprises:
substituting one or more amino acids in SEQ ID NO. 16 of human IL-8, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 70,
(B) Arg, Lys or His substitution at position 71,
(C) Arg, Lys or His substitution at position 17, and
(D) Arg, Lys or His substitution at position 21.

14. The method of making modified human IL-8 according to claim 13, wherein the modified human Interleukin-8 is further modified by deleting amino acids at positions 1 through 6 of SEQ ID NO: 16.

15. A method of making modified human Interleukin-8 (IL-8) protein, wherein the method comprises:
substituting one or more amino acids in SEQ ID NO. 16 of human IL-8, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 70, and
at least one selected from the group consisting of:
(B) Arg, Lys or His substitution at position 17, and
(C) Arg, Lys or His substitution at position 21.

16. A method of making modified human Interleukin-8 (IL-8) protein, wherein the method comprises:
substituting one or more amino acids in SEQ ID NO. 16 of human IL-8, wherein the amino acid substitutions are:
(A) Arg, Lys or His substitution at position 71,
and at least one selected from the group consisting of:
(B) Arg, Lys or His substitution at position 17, and
(C) Arg, Lys or His substitution at position 21.