US20110144305A1 - Gag binding protein - Google Patents

Gag binding protein Download PDF

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US20110144305A1
US20110144305A1 US12/437,121 US43712109A US2011144305A1 US 20110144305 A1 US20110144305 A1 US 20110144305A1 US 43712109 A US43712109 A US 43712109A US 2011144305 A1 US2011144305 A1 US 2011144305A1
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gag binding
gag
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Andreas J. Kungl
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Protaffin Biotechnologie AG
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    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
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Definitions

  • 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 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.
  • 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 ⁇ -strands and connecting loops found between the second and the fourth cysteine residue, as well as a carboxy-terminal ⁇ -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.
  • the first two of the four cysteines are separated by a single amino acid (CXC)
  • CXC single amino acid
  • the ⁇ -chemokines the corresponding cysteine residues are adjacent to each other (CC).
  • the ⁇ -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).
  • the ⁇ -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 ⁇ -chemokines.
  • MCP monocyte chemoattractant proteins
  • eotaxin the five monocyte chemoattractant proteins
  • the remaining ⁇ -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 ⁇ -chemokines in general, do not act on neutrophils but attract monocytes, eosinophils, basophils and lymphocytes with variable selectivity.
  • 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 ⁇ - or C-chemokine.
  • fractalkine has to be mentioned as the only representative of the ⁇ - 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).
  • 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.
  • 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.
  • GAGs glycosaminoglycans
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 is species-specific and is, moreover, dependent upon the nature of the HSPG-expressing tissue.
  • Heparan sulphate is the most abundant member of the glycosaminoglycan (GAG) family of linear polysaccharides which also includes heparin, chondroitin sulphate, dermatan sulphate and keratan sulphate.
  • GAG glycosaminoglycan
  • Naturally occurring HS is characterised by a linear chain of 20-100 disaccharide units composed of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA) which can be modified to include N- and O-sulphation (6-O and 3-O sulphation of the glucosamine and 2-O sulphation of the uronic acid) as well as epimerisation of ⁇ -D-gluronic acid to ⁇ -L-iduronic acid (IdoA).
  • GlcNAc N-acetyl-D-glucosamine
  • GlcA D-glucuronic acid
  • IdoA epimerisation of ⁇ -D-gluronic acid to ⁇ -L-iduronic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 remains controversial although all chemokines have a clear structural basis for multimerisation. Dimerisation through association of the ⁇ -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.
  • CXC-family e.g. IL-8
  • CC-chemokine family e.g. RANTES
  • Interleukin-8 is a key molecule involved in neutrophil attraction during chronic and acute inflammation.
  • IL-8 Interleukin-8
  • 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 ⁇ , 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 targetted 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).
  • 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.
  • the present invention provides a method for introducing a GAG binding site into a protein characterised in that it comprises the steps:
  • 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.
  • Figure shows graphics of results from fluorescence anisotropy tests of two mutants.
  • Figure shows the graphic of results from isothermal fluorescence titrations.
  • Figure shows the graphic of results from unfolding experiments of various mutants.
  • Figure shows chemotaxis index of IL-8 mutants.
  • Figure shows the results of the RANTES chemotaxis assay.
  • 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.
  • pool 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.
  • the resulting GAG binding site is free of bulky and acidic amino acids, meaning that all bulky and acidic amino acids are removed.
  • the GAG binding affinity is determined—for the scope of protection of the present application—over the dissociation constant K d .
  • One possibility is to determine the dissociation constant (K d ) values of any given protein by the structural change in ligand binding.
  • K d dissociation constant
  • 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:
  • 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:
  • a further aspect of the present invention is a protein obtainable by the inventive method as described above.
  • the inventive protein therefore comprises a—compared to the wild-type protein—new GAG binding site as defined above and will therefore act as competitor with natural GAG binding proteins, in particular since the GAG binding affinity of the inventive protein is very high, e.g. K d ⁇ 10 ⁇ M.
  • 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.
  • 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.
  • at least 3, still preferred 4, most preferred 5, basic amino acids are present in the GAG binding region.
  • 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.
  • 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.
  • 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.
  • GAG binding region is defined as a region which binds to GAG with a dissociation constant (K d -value) of under 100 ⁇ M, preferably under 50 ⁇ M, still preferred under 20 ⁇ M, as determined by isothermal fluorescence titration (see examples below).
  • the GAG binding region can be modified by substitution, insertion and/or deletion.
  • 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.
  • 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.
  • 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.
  • the GAG binding affinity of said modified protein is increased compared to the GAG binding affinity of the respective wild-type protein, can be determined as mentioned above with the help of, for example, fluorescence titration experiments which determine the dissociation constants.
  • the criterion for improved GAG binding affinity will be K d (mutant) ⁇ K d (wild-type), preferably by at least 100%.
  • Specifically improved modified proteins have—compared with wild-type K d —a GAG binding affinity which is higher by a factor of minimum 5, preferably of minimum 10, still preferred of minimum 100.
  • the increased GAG binding affinity will therefore preferably show a K d of under 10 ⁇ M, preferred under 1 ⁇ M, still preferred under 0.1 ⁇ M.
  • the modified protein 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.
  • 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.
  • inserted relates to an insertion as such as well as substituting any non-basic amino acid with arginine, lysine or histidine.
  • 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.
  • 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).
  • said GAG binding region is a C terminal ⁇ -helix.
  • a typicial chemical monomer is organised around a triple stranded anti-parallel ⁇ -sheet overlaid by a C-terminal ⁇ -helix. It has been shown that this C-terminal ⁇ -helix in chemokines is to a major part involved in the GAG binding, so that modification in this C-terminal ⁇ -helix in order to increase the amount of basic amino acids results in a modified chemokine with an increased GAG binding affinity.
  • positions 17, 21, 70, and/or 71 in IL-8 are substituted by Arg, Lys, His, Asn and/or Gln.
  • Arg, Lys, His, Asn and/or Gln positions 17, 21, 70, and/or 71 in IL-8 are substituted by Arg, Lys, His, Asn and/or Gln.
  • Arg or Lys or His or Asn or Gln positions 17, 21, 70, and/or 71 in IL-8 are substituted by Arg, Lys, His, Asn and/or Gln.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • receptor competition assays using fluorescently labelled wt chemokines, calcium influx, and cell migration (performed on native leukocytes or on 7TM stably-transfected cell lines).
  • further biologically active regions are, in addition to further receptor binding sites (as in the growth factor family), enzymatic sites (as in hydrolase
  • 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.
  • 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.
  • said protein is a chemokine and said further biological activity is leukocyte activation.
  • 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.
  • 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.
  • said protein is an IL-8 mutant selected from the group consisting of del6F17RE70KN71R, del6F17RE70RN71K and del6E70KN71K.
  • IL-8 mutants 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.
  • 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 ⁇ -helix as can be seen on a three dimensional model of a protein.
  • 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.
  • the isolated polynucleic acid molecule hybridises to the above defined inventive polynucleic acid molecule under stringent conditions.
  • complementary duplexes form between the two DNA or RNA molecules, either by perfectly being matched or also comprising mismatched bases (see Sambrook et al., Molecular Cloning: A laboratory manual, 2 nd ed., Cold Spring Harbor, N.Y. 1989).
  • Probes greater in length than about 50 nucleotides may accommodate up to 25 to 30% mismatched bases. Smaller probes will accommodate fewer mismatches.
  • the tendency of a target and probe to form duplexes containing mismatched base pairs is controlled by the stringency of the hybridisation conditions which itself is a function of factors, such as the concentration of salt or formamide in the hybridisation buffer, the temperature of the hybridisation and the post-hybridisation wash conditions.
  • 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.
  • stringent hybridisation usually is achieved by hybridising at 5 to 10° C. below the Tm.
  • 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.
  • a recombinant cell as well as any therefrom descendant cell comprises the vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • FIG. 8 shows the results of the RANTES chemotaxis assay.
  • PCR Polymerase chain reaction
  • PCR products were purified, cloned into the pCR®T7/NT-TOPO®TA (Invitrogen) vector and transformed into TOP10F competent E. coli (Invitrogen).
  • pCR®T7/NT-TOPO®TA Invitrogen
  • TOP10F competent E. coli Invitrogen
  • a confirmation of the sequence was carried out by double-stranded DNA sequencing using a ABI PRISM CE1 Sequencer.
  • 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 ⁇ g/ml Ampicillin at 37° C. until an OD 600 of about 0.8 was reached.
  • Induction of protein expression was accomplished by addition of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • 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).
  • 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.
  • 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.
  • CD Circular Dichroism
  • 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.
  • IL-8 oligomerisation has been reported to relevantly influence the proteins GAG binding properties.
  • k oligo 460 nM
  • Dissociation constants are a measure for the binding affinity of a ligand to a protein and therefore concentration-dependent change in the fluorescence emission properties of the protein (fluorescence quenching) upon ligand binding was used for the determination of E d . Since these mutants contain an intrinsic tryptophan chromophore which is located at or near the proposed GAG binding site and therefore should be sensitive to structural changes upon ligand binding, IFT experiments seemed to be suitable for this kind of investigation. Fluorescence intensity titration was performed in PBS using a protein concentration of 700 nM. The emission of the protein solution upon excitation at 282 nm was recorded over a range of 300-400 nm following the addition of an aliquot of the respective GAG ligand and an equilibration period of 60 sec.
  • 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.
  • transfilter-based chemotaxis of neutrophils in response to IL-8 mutants was assayed in a microchemotaxis chamber equipped with a 5 ⁇ m PVP-free polycarbonate membrane.
  • a neutrophil fraction was prepared from freshly collected human blood. This was done by adding a 6% dextran solution to heparin-treated blood (1:2) which was then left for sedimentation for 45 min. The upper clear cell solution was collected and washed twice with HBSS w/o Ca and Mg. Cells were counted and finally diluted with HBSS at 2Mio/ml cell suspension, taking into account that only 60% of the counted cells were neutrophils.
  • IL-8 mutants were diluted at concentrations of 10 ⁇ g/ml, 1 ⁇ g/ml and 0.1 ⁇ g/ml and put in triplicates in the lower compartment of the chamber (26 ⁇ l per well).
  • the freshly prepared neutrophils were seeded in the upper chamber (50 ⁇ l per well) and incubated for 30 minutes at 37° C. in a 5% CO 2 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.
  • GAG-masking chemokine mutants were 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.
  • RANTES mutant directed cell migration was investigated using the 48-well Boyden chamber system equipped with 5 ⁇ m 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 ⁇ l of THP-1 cell suspensions (promonocytic cell line from the European collection of cell cultures) in the same medium at 2 ⁇ 10 6 cells/ml were placed in the upper wells. After a 2 h incubation period at 37° C. in 5% CO 2 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.
  • GAG binding proteins were characterised together with their GAG binding regions.
  • 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).
  • CXC-chemokines IL-8 18 HPK 20 , (R47) 60 TVVEKFLKR68 (residues 60-68 of SEQ ID NO: 16) (SEQ ID NO: 16) SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS MGSA/GRO ⁇ : 19 HPK 21 , 45 KNGR 48 (residues 45-48 of SEQ ID NO: 17), 60 KKIIEK 66 (residues 60-66 of SEQ ID NO: 17) (SEQ ID NO: 17) ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN MIP-2 ⁇ /GRO ⁇ : 19 HLK 21 , K45, 60 KKIIEKMLK 68 (residue

Abstract

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

Description

  • This application is a divisional of U.S. application Ser. No. 11/422,169 filed on Jun. 5, 2006, which is a 371 of PCT/EP2004/013670 filed on Dec. 2, 2004. The entire contents of the above-identified applications are hereby incorporated by reference.
  • 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 [761
    CC- MCP-1 CCR2 Basophils; Monocytes; Asthma [77];
    Chemokine Activated T cells; Glomerulonephritis [78];
    Dentritic cells; Natural Atheroscleosis [79];
    killer cells Inflammatory bowel
    disease [80];
    Psoriasis [81];
    Bacterial and viral
    meningitis [82, 83]
    RANTES CCR1 Eosinophils; Monocytes; Asthma [84];
    Activated T cells; Glomerulonephritis [85]
    Dentritic cells
    CCR3 Eosinophils; Basophils;
    Dentritic cells
    CCR5 Monocytes; Activated T
    cells; Dentritic cells;
    Natural killer cells
  • 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-O and 3-O sulphation of the glucosamine and 2-O sulphation of the uronic acid) as well as epimerisation of β-D-gluronic acid to α-L-iduronic acid (IdoA).
  • Clusters of N- and O-sulphated sugar residues, separated by regions of low sulphation, are assumed to be mainly responsible for the numerous protein binding and regulatory properties of HS. In addition to the electrostatic interactions of the HS sulphate groups with basic amino acids, van der Waals and hydrophobic interactions are also thought to be involved in protein binding. Furthermore, the presence of the conformationally flexible iduronate residues seems to favour GAG binding to proteins. Other factors such as the spacing between the protein binding sites play also a critical role in protein-GAG binding interactions: For example γ-interferon dimerisation induced by HS requires GAG chains with two protein binding sequences separated by a 7 kDa region with low sulphation. Additional sequences are sometimes required for full biological activity of some ligands: in order to support FGF-2 signal transduction, HS must have both the minimum binding sequence as well as additional residues that are supposed to interact with the FGF receptor.
  • Heparin binding proteins often contain consensus sequences consisting of clusters of basic amino acid residues. Lysine, arginine, asparagine, histidine and glutamine are frequently involved in electrostatic contacts with the sulphate and carboxyl groups on the GAG. The spacing of the basic amino acids, sometimes determined by the proteins 3-D structure, are assumed to control the GAG binding specificity and affinity. The biological activity of the ligand can also be affected by the kinetics of HS-protein interaction. Reducing the dimension of growth factor diffusion is one of the suggested HSPG functions for which the long repetitive character of the GAG chains as well as their relatively fast on and off rates of protein binding are ideally suited. In some cases, kinetics rather than thermodynamics drives the physiological function of HS-protein binding. Most HS ligands require GAG sequences of well-defined length and structure. Heparin, which is produced by mast cells, is structurally very similar to heparan sulphate but is characterised by higher levels of post-polymerisation modifications resulting in a uniformly high degree of sulphation with a relatively small degree of structural diversity. Thus, the highly modified blocks in heparan sulphate are sometimes referred to as “heparin-like”. For this reason, heparin can be used as a perfect HS analogue for protein biophysical studies as it is, in addition, available in larger quantities and therefore less expensive than HS. Different cell types have been shown to synthesise proteoglycans with different glycosaminoglycan structure which changes during pathogenesis, during development or in response to extracellular signals such as growth factors. This structural diversity of HSPGs leads to a high binding versatility emphasising the great importance of proteoglycans.
  • Since the demonstration that heparan sulphate proteoglycans are critical for FGF 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 targetted 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.
  • Figure shows graphics of results from fluorescence anisotropy tests of two mutants.
  • Figure shows the graphic of results from isothermal fluorescence titrations.
  • Figure shows the graphic of results from unfolding experiments of various mutants.
  • Figure shows chemotaxis index of IL-8 mutants.
  • Figure 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: Kd GAG(mutant)<10 μM.
  • Examples of said engineered proteins with new GAG binding sites are for example the Fc part of IgG as well as the complement factors C3 and C4 modified as follows:
  • Fc: (439)KSLSLS(444) −> KSKKLS (SEQ ID NOS 1 & 2)
    C3: (1297)WIASHT(1302) −> WKAKHK (SEQ ID NOS 3 & 4)
    C4: (1)MLDAERLK(8) −> MKKAKRLK (SEQ ID NOS 5 & 6)
  • A further aspect of the present invention is a protein obtainable by the inventive method as described above. The inventive protein therefore comprises a—compared to the wild-type protein—new GAG binding site as defined above and will therefore act as competitor with natural GAG binding proteins, in particular since the GAG binding affinity of the inventive protein is very high, e.g. 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 syntesis of GAG regions are described; Butcher et al., (FEBS Letters 4009 (1997), 183-187) (relates to artificial peptides, not proteins); Jinno-Oue et al, (J. Virol. 75 (2001), 12439-12445) de novo synthesis)).
  • 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, 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 17.2% to 45.2% out of the total structure. Nevertheless, this fact suggests that the overall structure of IL-8 wt was maintained despite many changes within the proteins sequence. This could not have been previously predicted. Having already found that heparan sulphate oligosaccharides only, and not heparin, were able to affect IL-8 wt secondary structure, attention was focused on the effects induced by unfractionated heparan sulphate. All examined mutants showed structural changes upon HS binding which can be seen as evidence of complex formation.
  • 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 nM), which therefore dimerised worst, was Δ6 F17RF21R E70RN71K. Previous studies identified the β-sheets to be mainly involved in the dimerisation process, a fact, which correlates with the results for this mutants' secondary structure, which showed a very low share of β-sheet of only 11.4%. The mutant with the lowest 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 Ed. 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 Δ6 F17RN71R E70K a Kd of 107 nM was found, for Δ6 F17RF21R N71K the Kd was 95 nM and the mutant Δ6 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 2Mio/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) 60TVVEKFLKR68 (residues 60-68 of SEQ ID NO: 16)
    (SEQ ID NO: 16)
    SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS
    MGSA/GROα: 19HPK21, 45KNGR48 (residues 45-48 of SEQ ID NO: 17),
    60KKIIEK66 (residues 60-66 of SEQ ID NO: 17)
    (SEQ ID NO: 17)
    ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN
    MIP-2α/GROβ: 19HLK21, K45, 60KKIIEKMLK68 (residues 60-68 of SEQ ID NO: 18)
    (SEQ ID NO: 18)
    APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIEKMLKNGKSN
    NAP-2: 15HPK18, 42KDGR45 (residues 42-45 of SEQ ID NO: 19), 57KKIVQK62
    (residues 57-62 of SEQ ID NO: 19)
    (SEQ ID NO: 19)
    AELRCLCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDESAD
    PF-4: 20RPRH23 (residues 20-23 of SEQ ID NO: 20), 46KNGR49 (residues 46-49
    of SEQ ID NO: 20), 61KKIIKK66 (residues 61-66 of SEQ ID NO: 20)
    (SEQ ID NO: 20)
    EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES
    SDF-1α: K1, 24KHLK27 (residues 24-27 of SEQ ID NO: 21), 41RLK43
    (SEQ ID NO: 21)
    KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN
                                        CC-chemokines
    RANTES: (17RPLPRAH23 (residues 17-23 of SEQ ID NO: 22)) 44RKNR47 (residues
    44-47 of SEQ ID NO: 22)
    (SEQ ID NO: 22)
    SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS
    MCP-2: 18RKIPIQR24 (residues 18-24 of SEQ ID NO: 23), 46KRGK49 (residues 46-49
    of SEQ ID NO: 23)
    (SEQ ID NO: 23)
    QPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLKP
    MCP-3: 22KQR24, 47KLDK50 (residues 47-50 of SEQ ID NO: 24),
    66KHLDKK71 (residues 66-71 of SEQ ID NO: 24)
    (SEQ ID NO: 24)
    QPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL
    MIP-1α: R17, 44KRSR47 (residues 44-47 of SEQ ID NO: 25)
    (SEQ ID NO: 25)
    SLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVCADPSEEWVQKYVSDLELSA
    MIP-1β: R18, 45KRSK48 (residues 45-48 of SEQ ID NO: 26)
    (SEQ ID NO: 26)
    APMGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYDLELN
    MPIF-1: R18, 45KKGR48 (residues 45-48 of SEQ ID NO: 27)
    (SEQ ID NO: 27)
    MDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANPSDKQVQVCMRMLKLDTRIKTRKN
    MIP-5/HCC-2: 40KKGR43 (residues 40-43 of SEQ ID NO: 28)
    (SEQ ID NO: 28)
    HFAADCCTSYISQSIPCSLMKSYFETSSECSKPGVIFLTKKGRQVCAKPSGPGVQDCMKKLKPYSI
  • TABLE 3
    SEQ ID NO:
    Peroxisome biogenesis factor 1 29 181 TRRAKE 186
    30 367 QKKIRS 372
    31 1263 PKRRKN 1268
    32 181 TRRAKE 186
    33 367 QKKIRS 372
    34 1263 PKRRKN 1268
    MLTK-beta 35 415 SKRRGKKV 422
    36 312 ERRLKM 317
    37 416 KRRGKK 421
    38 312 ERRLKM 317
    39 416 KRRGKK 421
    BHLH factor Hes4 40 43 EKRRRARI 50
    41 43 EKRRRA 48
    42 43 EKRRRA 48
    Protocadherin 11 43 867 MKKKKKKK 874
    44 867 MKKKKK 872
    45 867 MKKKKK 872
    46 899 MKKKKKKK 906
    47 899 MKKKKK 904
    48 899 MKKKKK 904
    catenin (cadherin-associated protein), 49 315 RRRLRS 320
    delta 1 50 404 VRKLKG 409
    51 460 LRKARD 465
    52 545 RRKLRE 550
    53 621 AKKGKG 626
    54 787 AKKLRE 792
    55 315 RRRLRS 320
    56 404 VRKLKG 409
    57 460 LRKARD 465
    58 545 RRKLRE 550
    59 621 AKKGKG 626
    60 787 AKKLRE 792
    Muscarinic acetylcholine receptor M5 61 221 EKRTKD 226
    62 427 TKRKRV 432
    63 514 WKKKKV 519
    64 221 EKRTKD 226
    65 427 TKRKRV 432
    66 514 WKKKKV 519
    Alpha-2A adrenergic receptor 67 147 PRRIKA 152
    68 224 KRRTRV 229
    69 147 PRRIKA 152
    70 224 KRRTRV 229
    IL-5 promoter REII-region-binding protein 71 440 TKKKTRRR 447
    72 569 GKRRRRRG 576
    73 38 ARKGKR 43
    74 437 GKKTKK 442
    75 444 TRRRRA 449
    76 569 GKRRRR 574
    77 38 ARKGKR 43
    78 437 GKKTKK 442
    79 444 TRRRRA 449
    80 569 GKRRRR 574
    Mitofusin 1 81 291 ARKQKA 296
    82 395 KKKIKE 400
    83 291 ARKQKA 296
    84 395 KKKIKE 400
    N-cym protein 85 71 VRRCKI 76
    86 71 VRRCKI 76
    Smad ubiquitination regulatory factor 1 87 672 ERRARL 677
    88 672 ERRARL 677
    CUG-BP and ETR-3 like factor 5 89 468 MKRLKV 473
    90 475 LKRPKD 480
    91 468 MKRLKV 473
    92 475 LKRPKD 480
    Ewings sarcoma EWS-Flil 93 347 QRKSKP 352
    94 347 QRKSKP 352
    NUF2R 95 455 LKRKMFKM 462
    96 331 LKKLKT 336
    97 347 VKKEKL 352
    98 331 LKKLKT 336
    99 347 VKKEKL 352
    Kruppel-like zinc finger protein 100 22 EKRERT 27
    GLIS2 101 22 EKRERT 27
    FKSG32 202 15 LKRVRE 20
    103 431 VRRGRI 436
    104 15 LKRVRE 20
    105 431 VRRGRI 436
    BARH-LIKE 1 PROTEIN 106 175 LKKPRK 180
    107 228 NRRTKW 233
    108 175 LKKPRK 180
    109 228 NRRTKW 233
    Nucleolar GTP-binding protein 1 110 393 SRKKRERD 400
    111 624 GKRKAGKK 631
    112 48 MRKVKF 53
    113 141 IKRQKQ 146
    114 383 ARRKRM 388
    115 393 SRKKRE 398
    116 490 KKKLKI 495
    117 543 ARRSRS 548
    118 550 TRKRKR 555
    119 586 VKKAKT 591
    120 629 GKKDRR 634
    121 48 MRKVKF 53
    122 141 IKRQKQ 146
    123 383 ARRKRM 388
    124 393 SRKKRE 398
    125 490 KKKLKI 495
    126 543 ARRSRS 548
    127 550 TRKRKR 555
    128 586 VKKAKT 591
    129 629 GKKDRR 634
    EVG1 130 17 RRRPKT 22
    131 138 ERKRKA 143
    132 17 RRRPKT 22
    133 138 ERKRKA 143
    ASPL 134 282 PKKSKS 287
    135 282 PKKSKS 287
    Zinc transporter 1 136 477 EKKPRR 482
    137 477 EKKPRR 482
    Uveal autoantigen 138 603 EKKGRK 608
    139 995 ERKFKA 1000
    140 1023 VKKNKQ 1028
    141 603 EKKGRK 608
    142 995 ERKFKA 1000
    143 1023 VKKNKQ 1028
    RAB39 144 7 VRRDRV 12
    145 7 VRRDRV 12
    Down syndrome cell adhesion molecule 146 320 PRKVKS 325
    147 387 VRKDKL 392
    148 320 PRKVKS 325
    149 387 VRKDKL 392
    Protein-tyrosine phosphatase, non-receptor 150 139 GRKKCERY 146
    type 12 151 59 VKKNRY 64
    152 59 VKKNRY 64
    WD-repeat protein 11 153 752 VRKIRF 757
    154 752 VRKIRF 757
    Gastric cancer-related protein 155 20 SRKRQTRR 27
    VRG107 156 25 TRRRRN 30
    157 25 TRRRRN 30
    Early growth response protein 4 158 356 ARRKGRRG 363
    159 452 EKKRHSKV 459
    160 357 RRKGRR 362
    161 357 RRKGRR 362
    Vesicle transport-related protein 162 309 PKRKNKKS 316
    163 226 DKKLRE 231
    164 310 KRKNKK 315
    165 355 VKRLKS 360
    166 226 DKKLRE 231
    167 310 KRKNKK 315
    168 355 VKRLKS 360
    UPF3X 169 140 AKKKTKKR 147
    170 141 KKKTKK 146
    171 217 ERRRRE 222
    172 225 RKRQRE 230
    173 233 RRKWKE 238
    174 240 EKRKRK 245
    175 296 DKREKA 301
    176 373 RRRQKE 378
    177 393 MKKEKD 398
    178 426 VKRDRI 431
    179 140 AKKKTKKRD 148
    180 141 KKKTKK 146
    181 217 ERRRRE 222
    182 225 RKRQRE 230
    183 233 RRKWKE 238
    184 240 EKRKRK 245
    185 296 DKREKA 301
    186 373 RRRQKE 378
    187 393 MKKEKD 398
    188 426 VKRDRI 431
    CGI-201 protein, type IV 189 49 ARRTRS 54
    190 49 ARRTRS 54
    RING finger protein 23 191 98 KRKIRD 103
    192 98 KRKIRD 103
    FKSG17 193 72 EKKARK 77
    194 95 IRKSKN 100
    195 72 EKKARK 77
    196 95 IRKSKN 100
    P83 197 681 ARKERE 686
    198 681 ARKERE 686
    Ovarian cancer-related protein 1 199 62 LKRDRF 67
    200 62 LKRDRF 67
    MHC class II transactivator CIITA 201 407 HRRPRE 412
    202 741 PRKKRP 746
    203 783 DRKQKV 788
    204 407 HRRPRE 412
    205 741 PRKKRP 746
    206 783 DRKQKV 788
    Platelet glycoprotein VI-2 207 275 SRRKRLRH 282
    208 275 SRRKRL 280
    209 275 SRRKRL 280
    Ubiquitin-like 5 protein 210 11 GKKVRV 16
    211 11 GKKVRV 16
    Protein kinase D2 212 191 ARKRRL 196
    213 191 ARKRRL 196
    Homeobox protein GSH-2 214 202 GKRMRT 207
    215 252 NRRVKH 257
    216 202 GKRMRT 207
    217 252 NRRVKH 257
    ULBP3 protein 218 166 ARRMKE 171
    219 201 HRKKRL 206
    220 166 ARRMKE 171
    221 201 HRKKRL 206
    Type II iodothyronine deiodinase 222 87 SKKEKV 92
    223 87 SKKEKV 92
    224 299 SKRCKK 304
    225 299 SKRCKK 304
    Sperm antigen 226 160 LKKYKE 165
    227 478 IKRLKE 483
    228 160 LKKYKEKRT 168
    229 160 LKKYKE 165
    230 478 IKRLKE 483
    UDP-GalNAc: polypeptide N- 231 4 ARKIRT 9
    acetylgalactosaminyltransferase 232 44 DRRVRS 49
    233 138 PRKCRQ 143
    234 4 ARKIRT 9
    235 44 DRRVRS 49
    236 138 PRKCRQ 143
    NCBE 237 62 HRRHRH 67
    238 73 RKRDRE 78
    239 1012 SKKKKL 1017
    240 62 HRRHRH 67
    241 73 RKRDRE 78
    242 1012 SKKKKL 1017
    WD repeat protein 243 372 LKKKEERL 379
    244 384 EKKQRR 389
    245 400 AKKMRP 405
    246 384 EKKQRR 389
    247 400 AKKMRP 405
    Phosphodiesterase 11A 248 27 MRKGKQ 32
    249 27 MRKGKQ 32
    Probable cation-transporting ATPase 2 250 891 ERRRRPRD 898
    251 306 SRKWRP 311
    252 891 ERRRRP 896
    253 306 SRKWRP 311
    254 891 ERRRRP 896
    HMG-box transcription factor TCF-3 255 420 GKKKKRKR 427
    256 399 ARKERQ 404
    257 420 GKKKKR 425
    258 420 GKKKKRKRE 428
    259 399 ARKERQ 404
    260 420 GKKKKR 425
    HVPS11 261 793 VRRYRE 798
    262 793 VRRYRE 798
    PIST 263 165 NKKEKM 170
    264 165 NKKEKM 170
    FYN-binding protein 265 473 KKREKE 478
    266 501 KKKFKL 506
    267 682 LKKLKK 687
    268 696 RKKFKY 701
    269 473 KKREKE 478
    270 501 KKKFKL 506
    271 682 LKKLKK 687
    272 696 RKKFKY 701
    C1orf25 273 620 GKKQKT 625
    274 620 GKKQKT 625
    C1orf14 275 441 LRRRKGKR 448
    276 70 LRRWRR 75
    277 441 LRRRKG 446
    278 70 LRRWRR 75
    279 441 LRRRKG 446
    T-box transcription factor TBX3 280 144 DKKAKY 149
    281 309 GRREKR 314
    282 144 DKKAKY 149
    283 309 GRREKR 314
    Mitochondrial 39S ribosomal protein L47 284 121 AKRQRL 126
    285 216 EKRARI 221
    286 230 RKKAKI 235
    287 121 AKRQRL 126
    288 216 EKRARI 221
    289 230 RKKAKI 235
    CGI-203 290 33 VRRIRD 38
    291 33 VRRIRD 38
    Jagged1 292 1093 LRKRRK 1098
    293 1093 LRKRRK 1098
    Secretory carrier-associated 294 102 DRRERE 107
    membrane protein 1 295 102 DRRERE 107
    Vitamin D receptor-interacting 296 673 KKKKSSRL 680
    protein complex component DRIP205 297 672 TKKKKS 677
    298 954 QKRVKE 959
    299 978 GKRSRT 983
    300 995 PKRKKA 1000
    301 1338 GKREKS 1343
    302 1482 HKKHKK 1487
    303 1489 KKKVKD 1494
    304 672 TKKKKS 677
    305 954 QKRVKE 959
    306 978 GKRSRT 983
    307 995 PKRKKA 1000
    308 1338 GKREKS 1343
    309 1482 HKKHKK 1487
    310 1489 KKKVKD 1494
    Secretory carrier-associated 311 100 ERKERE 105
    membrane protein 2 312 100 ERKERE 105
    Nogo receptor 313 420 SRKNRT 425
    314 420 SRKNRT 425
    FLAMINGO 1 315 169 GRRKRN 174
    316 2231 ARRQRR 2236
    317 169 GRRKRN 174
    318 2231 ARRQRR 2236
    CC-chemokine receptor 319 58 CKRLKS 63
    320 58 CKRLKS 63
    Prolactin regulatory element-binding protein 321 271 HKRLRQ 276
    322 271 HKRLRQ 276
    Kappa B and V(D)J recombination 323 17 PRKRLTKG 24
    signal sequences binding protein 324 713 RKRRKEKS 720
    325 903 PKKKRLRL 910
    326 180 HKKERK 185
    327 629 TKKTKK 634
    328 712 LRKRRK 717
    329 903 PKKKRL 908
    330 1447 QKRVKE 1452
    331 1680 SRKPRM 1685
    332 180 HKKERK 185
    333 629 TKKTKK 634
    334 712 LRKRRK 717
    335 903 PKKKRL 908
    336 1447 QKRVKE 1452
    337 1680 SRKPRM 1685
    Breast cancer metastasis-suppressor 1 338 200 SKRKKA 205
    339 229 IKKARA 234
    340 200 SKRKKA 205
    341 229 IKKARA 234
    Forkhead box protein P3 342 414 RKKRSQRP 421
    343 413 FRKKRS 418
    344 413 FRKKRS 418
    FAS BINDING PROTEIN 345 228 LKRKLIRL 235
    346 391 RKKRRARL 398
    347 358 ARRLRE 363
    348 390 ERKKRR 395
    349 629 CKKSRK 634
    350 358 ARRLRE 363
    351 390 ERKKRR 395
    352 629 CKKSRK 634
    Ubiquitin carboxyl-terminal hydrolase 12 353 228 HKRMKV 233
    354 244 LKRFKY 249
    355 228 HKRMKV 233
    356 244 LKRFKY 249
    KIAA0472 protein 357 110 HRKPKL 115
    358 110 HRKPKL 115
    PNAS-101 359 68 LKRSRP 73
    360 106 PRKSRR 111
    361 68 LKRSRP 73
    362 106 PRKSRR 111
    PNAS-26 363 118 DRRTRL 123
    364 118 DRRTRL 123
    Myelin transcription factor 2 365 176 GRRKSERQ 183
    Sodium/potassium-transporting ATPase 366 47 SRRFRC 52
    gamma chain 367 55 NKKRRQ 60
    368 47 SRRFRC 52
    369 55 NKKRRQ 60
    Mdm4 protein 370 441 EKRPRD 446
    371 464 ARRLKK 469
    372 441 EKRPRD 446
    373 464 ARRLKK 469
    G antigen family D 2 protein 374 87 QKKIRI 92
    375 87 QKKIRI 92
    NipSnap2 protein 376 153 FRKARS 158
    377 153 FRKARS 158
    Stannin 378 73 ERKAKL 78
    379 73 ERKAKL 78
    Sodium bicarbonate cotransporter 380 973 EKKKKKKK 980
    381 165 LRKHRH 170
    382 666 LKKFKT 671
    383 966 DKKKKE 971
    384 973 EKKKKK 978
    385 165 LRKHRH 170
    386 666 LKKFKT 671
    387 966 DKKKKE 971
    388 973 EKKKKK 978
    Myosin X 389 683 YKRYKV 688
    390 828 EKKKRE 833
    391 1653 LKRIRE 1658
    392 1676 LKKTKC 1681
    393 683 YKRYKV 688
    394 828 EKKKRE 833
    395 1653 LKRIRE 1658
    396 1676 LKKTKC 1681
    PNAS-20 397 21 RKRKSVRG 28
    398 20 ERKRKS 25
    399 20 ERKRKS 25
    Pellino 400 36 RRKSRF 41
    401 44 FKRPKA 49
    402 36 RRKSRF 41
    403 44 FKRPKA 49
    Hyaluronan mediated motility receptor 404 66 ARKVKS 71
    405 66 ARKVKS 71
    Short transient receptor potential channel 7 406 753 FKKTRY 758
    407 753 FKKTRY 758
    Liprin-alpha2 408 825 PKKKGIKS 832
    409 575 IRRPRR 580
    410 748 LRKHRR 753
    411 839 GKKEKA 844
    412 875 DRRLKK 880
    413 575 IRRPRR 580
    414 748 LRKHRR 753
    415 839 GKKEKA 844
    416 875 DRRLKK 880
    Transcription intermediary factor 1-alpha 417 904 DKRKCERL 911
    418 1035 PRKKRLKS 1042
    419 321 NKKGKA 326
    420 1035 PRKKRL 1040
    421 321 NKKGKA 326
    422 1035 PRKKRL 1040
    CARTILAGE INTERMEDIATE LAYER PROTEIN 423 719 QRRNKR 724
    424 719 QRRNKR 724
    UBX domain-containing protein 1 425 194 YRKIKL 199
    426 194 YRKIKL 199
    Arachidonate 12-lipoxygenase, 12R type 427 166 VRRHRN 171
    428 233 WKRLKD 238
    429 166 VRRHRN 171
    430 233 WKRLKD 238
    Hematopoietic PBX-interacting protein 431 159 LRRRRGRE 166
    432 698 LKKRSGKK 705
    433 159 LRRRRG 164
    434 703 GKKDKH 708
    435 159 LRRRRG 164
    436 703 GKKDKH 708
    NAG18 437 28 LKKKKK 33
    438 28 LKKKKK 33
    POU 5 domain protein 439 222 ARKRKR 227
    440 222 ARKRKR 227
    NRCAM PROTEIN 441 2 PKKKRL 7
    442 887 SKRNRR 892
    443 1185 IRRNKG 1190
    444 1273 GKKEKE 1278
    445 2 PKKKRL 7
    446 887 SKRNRR 892
    447 1185 IRRNKG 1190
    448 1273 GKKEKE 1278
    protocadherin gamma cluster 449 11 TRRSRA 16
    450 11 TRRSRA 16
    SKD1 protein 451 288 IRRRFEKR 295
    452 251 ARRIKT 256
    453 362 FKKVRG 367
    454 251 ARRIKT 256
    455 362 FKKVRG 367
    ANTI-DEATH PROTEIN 456 58 HRKRSRRV 65
    457 59 RKRSRR 64
    458 59 RKRSRR 64
    Centrin 3 459 14 TKRKKRRE 21
    460 14 TKRKKR 19
    461 14 TKRKKR 19
    Ectonucleoside triphosphate 462 512 TRRKRH 517
    diphosphohydrolase 3 463 512 TRRKRH 517
    Homeobox protein prophet of PIT-1 464 12 PKKGRV 17
    465 69 RRRHRT 74
    466 119 NRRAKQ 124
    467 12 PKKGRV 17
    468 69 RRRHRT 74
    469 119 NRRAKQ 124
    PROSTAGLANDIN EP3 RECEPTOR 470 77 YRRRESKR 84
    471 389 MRKRRLRE 396
    472 82 SKRKKS 87
    473 389 MRKRRL 394
    474 82 SKRKKS 87
    475 389 MRKRRL 394
    Pituitary homeobox 3 476 58 LKKKQRRQ 65
    477 59 KKKQRR 64
    478 112 NRRAKW 117
    479 118 RKRERS 123
    480 59 KKKQRR 64
    481 112 NRRAKW 117
    482 118 RKRERS 123
    HPRL-3 483 136 KRRGRI 141
    484 136 KRRGRI 141
    Advillin 485 812 MKKEKG 817
    486 812 MKKEKG 817
    Nuclear LIM interactor-interacting factor 1 487 32 GRRARP 37
    488 109 LKKQRS 114
    489 32 GRRARP 37
    490 109 LKKQRS 114
    Core histone macro-H2A.1 491 5 GKKKSTKT 12
    492 114 AKKRGSKG 121
    493 70 NKKGRV 75
    494 132 AKKAKS 137
    495 154 ARKSKK 159
    496 302 DKKLKS 307
    497 70 NKKGRV 75
    498 132 AKKAKS 137
    499 154 ARKSKK 159
    500 302 DKKLKS 307
    Villin-like protein 501 180 KRRRNQKL 187
    502 179 EKRRRN 184
    503 179 EKRRRN 184
    BETA-FILAMIN 504 254 PKKARA 259
    505 2002 ARRAKV 2007
    506 254 PKKARA 259
    507 2002 ARRAKV 2007
    Tripartite motif protein TRIM31 508 290 LKKFKD 295
    alpha 509 290 LKKFKD 295
    Nuclear receptor co-repressor 1 510 106 SKRPRL 111
    511 299 ARKQRE 304
    512 330 RRKAKE 335
    513 349 IRKQRE 354
    514 412 QRRVKF 417
    515 497 KRRGRN 502
    516 580 RRKGRI 585
    517 687 SRKPRE 692
    518 2332 SRKSKS 2337
    519 106 SKRPRL 111
    520 299 ARKQRE 304
    521 330 RRKAKE 335
    522 349 IRKQRE 354
    523 412 QRRVKF 417
    524 497 KRRGRN 502
    525 580 RRKGRI 585
    526 687 SRKPRE 692
    527 2332 SRKSKS 2337
    BRAIN EXPRESSED RING FINGER PROTEIN 528 432 KRRVKS 437
    529 432 KRRVKS 437
    PB39 530 231 TKKIKL 236
    531 231 TKKIKL 236
    Sperm acrosomal protein 532 48 FRKRMEKE 55
    533 24 RRKARE 29
    534 135 KRKLKE 140
    535 213 KKRLRQ 218
    536 24 RRKARE 29
    537 135 KRKLKE 140
    538 213 KKRLRQ 218
    VESICLE TRAFFICKING PROTEIN SEC22B 539 177 SKKYRQ 182
    540 177 SKKYRQ 182
    Nucleolar transcription factor 1 541 79 VRKFRT 84
    542 102 GKKLKK 107
    543 125 EKRAKY 130
    544 147 SKKYKE 152
    545 156 KKKMKY 161
    546 240 KKRLKW 245
    547 451 KKKAKY 456
    548 523 EKKEKL 528
    549 558 SKKMKF 563
    550 79 VRKFRT 84
    551 102 GKKLKK 107
    552 125 EKRAKY 130
    553 147 SKKYKE 152
    554 156 KKKMKY 161
    555 240 KKRLKW 245
    556 451 KKKAKY 456
    557 523 EKKEKL 528
    558 558 SKKMKF 563
    Plexin-B3 559 248 FRRRGARA 255
    Junctophilin type3 560 626 QKRRYSKG 633
    Plaucible mixed-lineage kinase protein 561 773 YRKKPHRP 780
    562 312 ERRLKM 317
    563 312 ERRLKM 317
    fatty acid binding protein 4, adipocyte 564 78 DRKVKS 83
    565 105 IKRKRE 110
    566 78 DRKVKS 83
    567 105 IKRKRE 110
    exostoses (multiple) 1 568 78 SKKGRK 83
    569 78 SKKGRK 83
    DHHC-domain-containing cysteine-rich protein 570 64 HRRPRG 69
    571 64 HRRPRG 69
    Myb proto-oncogene protein 572 2 ARRPRH 7
    573 292 EKRIKE 297
    574 523 LKKIKQ 528
    575 2 ARRPRH 7
    576 292 EKRIKE 297
    577 523 LKKIKQ 528
    Long-chain-fatty-acid--CoA ligase 2 578 259 RRKPKP 264
    579 259 RRKPKP 264
    syntaxinlB2 580 260 ARRKKI 265
    581 260 ARRKKI 265
    Dachshund 2 582 162 ARRKRQ 167
    583 516 QKRLKK 521
    584 522 EKKTKR 527
    585 162 ARRKRQ 167
    586 516 QKRLKK 521
    587 522 EKKTKR 527
    DEAD/DEXH helicase DDX31 588 344 EKRKSEKA 351
    589 760 TRKKRK 765
    590 760 TRKKRK 765
    Androgen receptor 591 628 ARKLKK 633
    592 628 ARKLKK 633
    Retinoic acid receptor alpha 593 364 RKRRPSRP 371
    594 163 NKKKKE 168
    595 363 VRKRRP 368
    596 163 NKKKKE 168
    597 363 VRKRRP 368
    Kinesin heavy chain 598 340 WKKKYEKE 347
    599 605 VKRCKQ 610
    600 864 EKRLRA 869
    601 605 VKRCKQ 610
    602 864 EKRLRA 869
    DIUBIQUITIN 603 30 VKKIKE 35
    604 30 VKKIKE 35
    BING1 PROTEIN 605 519 NKKFKM 524
    606 564 ERRHRL 569
    607 519 NKKFKM 524
    608 564 ERRHRL 569
    Focal adhesion kinase 1 609 664 SRRPRF 669
    610 664 SRRPRF 669
    EBN2 PROTEIN 611 20 TKRKKPRR 27
    612 13 PKKDKL 18
    613 20 TKRKKP 25
    614 47 NKKNRE 52
    615 64 LKKSRI 69
    616 76 PKKPRE 81
    617 493 SRKQRQ 498
    618 566 VKRKRK 571
    619 13 PKKDKL 18
    620 20 TKRKKP 25
    621 47 NKKNRE 52
    622 64 LKKSRI 69
    623 76 PKKPRE 81
    624 493 SRKQRQ 498
    625 566 VKRKRK 571
    CO16 PROTEIN 626 33 ARRLRR 38
    627 115 PRRCKW 120
    628 33 ARRLRR 38
    629 115 PRRCKW 120
    KYNURENINE 3-MONOOXYGENASE 630 178 MKKPRF 183
    631 178 MKKPRF 183
    MLN 51 protein 632 4 RRRQRA 9
    633 255 PRRIRK 260
    634 407 ARRTRT 412
    635 4 RRRQRA 9
    636 255 PRRIRK 260
    637 407 ARRTRT 412
    MHC class II antigen 638 99 QKRGRV 104
    MHC class II antigen 639 99 QKRGRV 104
    Transforming acidic coiled-coil-containing 640 225 SRRSKL 230
    protein 1 641 455 PKKAKS 460
    642 225 SRRSKL 230
    643 455 PKKAKS 460
    Neuro-endocrine specific protein VGF 644 479 EKRNRK 484
    645 479 EKRNRK 484
    Organic cation transporter 646 230 GRRYRR 235
    647 535 PRKNKE 540
    648 230 GRRYRR 235
    649 535 PRKNKE 540
    DNA polymerase theta 650 215 KRRKHLKR 222
    651 214 WKRRKH 219
    652 220 LKRSRD 225
    653 1340 GRKLRL 1345
    654 1689 SRKRKL 1694
    655 214 WKRRKH 219
    656 220 LKRSRD 225
    657 1340 GRKLRL 1345
    658 1689 SRKRKL 1694
    CDC45-related protein 659 169 MRRRQRRE 176
    660 155 EKRTRL 160
    661 170 RRRQRR 175
    662 483 NRRCKL 488
    663 155 EKRTRL 160
    664 170 RRRQRR 175
    665 483 NRRCKL 488
    Chloride intracellular channel protein 2 666 197 AKKYRD 202
    667 197 AKKYRD 202
    Methyl-CpG binding protein 668 85 KRKKPSRP 92
    669 83 SKKRKK 88
    670 318 QKRQKC 323
    671 354 YRRRKR 359
    672 83 SKKRKK 88
    673 318 QKRQKC 323
    674 354 YRRRKR 359
    Protein kinase C, eta type 675 155 RKRQRA 160
    676 155 RKRQRA 160
    Heterogeneous nuclear 677 71 LKKDRE 76
    ribonucleoprotein H 678 169 LKKHKE 174
    679 71 LKKDRE 76
    680 169 LKKHKE 174
    ORF2 681 11 SRRTRW 16
    682 155 ERRRKF 160
    683 185 LRRCRA 190
    684 530 SRRSRS 535
    685 537 GRRRKS 542
    686 742 ERRAKQ 747
    687 11 SRRTRW 16
    688 155 ERRRKF 160
    689 185 LRRCRA 190
    690 530 SRRSRS 535
    691 537 GRRRKS 542
    692 742 ERRAKQ 747
    F-box only protein 24 693 9 LRRRRVKR 16
    694 9 LRRRRV 14
    695 29 EKRGKG 34
    696 9 LRRRRV 14
    697 29 EKRGKG 34
    Leucin rich neuronal protein 698 51 NRRLKH 56
    699 51 NRRLKH 56
    RER1 protein 700 181 KRRYRG 186
    701 181 KRRYRG 186
    Nephrocystin 702 3 ARRQRD 8
    703 430 PKKPKT 435
    704 557 NRRSRN 562
    705 641 EKRDKE 646
    706 3 ARRQRD 8
    707 430 PKKPKT 435
    708 557 NRRSRN 562
    709 641 EKRDKE 646
    Adenylate kinase isoenzyme 2, mitochondrial 710 60 GKKLKA 65
    711 116 KRKEKL 121
    712 60 GKKLKA 65
    713 116 KRKEKL 121
    Chlordecone reductase 714 245 AKKHKR 250
    715 245 AKKHKR 250
    Metaxin 2 716 166 KRKMKA 171
    717 166 KRKMKA 171
    Paired mesoderm homeobox protein1 718 89 KKKRKQRR 96
    719 88 EKKKRK 93
    720 94 QRRNRT 99
    721 144 NRRAKF 149
    722 88 EKKKRK 93
    723 94 QRRNRT 99
    724 144 NRRAKF 149
    Ring finger protein 725 174 LKRKWIRC 181
    726 8 TRKIKL 13
    727 95 MRKQRE 100
    728 8 TRKIKL 13
    729 95 MRKQRE 100
    Ataxin 7 730 55 PRRTRP 60
    731 377 GRRKRF 382
    732 704 GKKRKN 709
    733 834 GKKRKC 839
    734 55 PRRTRP 60
    735 377 GRRKRF 382
    736 704 GKKRKN 709
    737 834 GKKRKC 839
    Growth-arrest-specific protein 1 738 169 ARRRCDRD 176
    SKAP55 protein 739 115 EKKSKD 120
    740 115 EKKSKD 120
    Serine palmitoyltransferase 1 741 232 PRKARV 237
    742 232 PRKARV 237
    Serine palmitoyltransferase 2 743 334 KKKYKA 339
    744 450 RRRLKE 455
    745 334 KKKYKA 339
    746 450 RRRLKE 455
    Synaptopodin 747 405 KRRQRD 410
    748 405 KRRQRD 410
    Alpha-tectorin 749 1446 TRRCRC 1451
    750 2080 IRRKRL 2085
    751 1446 TRRCRC 1451
    752 2080 IRRKRL 2085
    LONG FORM TRANSCRIPTION FACTOR C-MAF 753 291 QKRRTLKN 298
    Usher syndrome type IIa protein 754 1285 MRRLRS 1290
    755 1285 MRRLRS 1290
    MSin3A associated polypeptide p30 756 95 QKKVKI 100
    757 124 NRRKRK 129
    758 158 LRRYKR 163
    759 95 QKKVKI 100
    760 124 NRRKRK 129
    761 158 LRRYKR 163
    Ig delta chain C region 762 142 KKKEKE 147
    763 142 KKKEKE 147
    THYROID HORMONE RECEPTOR-ASSOCIATED 764 383 AKRKADRE 390
    PROTEIN COMPLEX COMPONENT TRAP100 765 833 KKRHRE 838
    766 833 KKRHRE 838
    P60 katanin 767 369 LRRRLEKR 376
    768 326 SRRVKA 331
    769 326 SRRVKA 331
    Transcription factor jun-D 770 286 RKRKLERI 293
    771 273 RKRLRN 278
    772 285 CRKRKL 290
    773 273 RKRLRN 278
    774 285 CRKRKL 290
    Sterol/retinol dehydrogenase 775 152 VRKARG 157
    776 152 VRKARG 157
    Glycogen [starch] synthase, liver 777 554 DRRFRS 559
    778 578 SRRQRI 583
    779 554 DRRFRS 559
    780 578 SRRQRI 583
    Estrogen-related receptor gamma 781 173 TKRRRK 178
    782 353 VKKYKS 358
    783 173 TKRRRK 178
    784 353 VKKYKS 358
    Neural retina-specific leucine zipper protein 785 162 QRRRTLKN 169
    Cytosolic phospholipase A2-gamma 786 514 NKKKILRE 521
    787 31 LKKLRI 36
    788 218 FKKGRL 223
    789 428 CRRHKI 433
    790 31 LKKLRI 36
    Cytosolic phospholipase A2-gamma 791 218 FKKGRL 223
    792 428 CRRHKI 433
    GLE1 793 415 AKKIKM 420
    794 415 AKKIKM 420
    Multiple exostoses type II protein 795 296 VRKRCHKH 303
    EXT2.I 796 659 RKKFKC 664
    797 659 RKKFKC 664
    Cyclic-AMP-dependent transcription 798 86 EKKARS 91
    factor ATF-7 799 332 GRRRRT 337
    800 344 ERRQRF 349
    801 86 EKKARS 91
    802 332 GRRRRT 337
    803 344 ERRQRF 349
    Protein kinase/endoribonulcease 804 886 LRKFRT 891
    805 886 LRKFRT 891
    Transcription factor E2F6 806 23 RRRCRD 28
    807 59 VKRPRF 64
    808 98 VRKRRV 103
    809 117 EKKSKN 122
    810 23 RRRCRD 28
    811 59 VKRPRF 64
    812 98 VRKRRV 103
    813 117 EKKSKN 122
    MAP kinase-activating death domain protein 814 1333 IRKKVRRL 1340
    815 160 KRRAKA 165
    816 943 MKKVRR 948
    817 1034 DKRKRS 1039
    818 1334 RKKVRR 1339
    819 1453 TKKCRE 1458
    820 160 KRRAKA 165
    821 943 MKKVRR 948
    822 1034 DKRKRS 1039
    823 1334 RKKVRR 1339
    824 1453 TKKCRE 1458
    Orphan nuclear receptor PXR 825 126 KRKKSERT 133
    826 87 TRKTRR 92
    827 125 IKRKKS 130
    828 87 TRKTRR 92
    829 125 IKRKKS 130
    LENS EPITHELIUM-DERIVED GROWTH FACTOR 830 149 RKRKAEKQ 156
    831 286 KKRKGGRN 293
    832 145 ARRGRK 150
    833 178 PKRGRP 183
    834 285 EKKRKG 290
    835 313 DRKRKQ 318
    836 400 LKKIRR 405
    837 337 VKKVEKKRE 345
    838 145 ARRGRK 150
    839 178 PKRGRP 183
    840 285 EKKRKG 290
    841 313 DRKRKQ 318
    842 400 LKKIRR 405
    LIM homeobox protein cofactor 843 255 TKRRKRKN 262
    844 255 TKRRKR 260
    845 255 TKRRKR 260
    MULTIPLE MEMBRANE SPANNING RECEPTOR TRC8 846 229 WKRIRF 234
    847 229 WKRIRF 234
    Transcription factor SUPT3H 848 172 DKKKLRRL 179
    849 169 MRKDKK 174
    850 213 NKRQKI 218
    851 169 MRKDKK 174
    852 213 NKRQKI 218
    GEMININ 853 50 KRKHRN 55
    854 104 EKRRKA 109
    855 50 KRKHRN 55
    856 104 EKRRKA 109
    Cell cycle-regulated factor p78 857 165 EKKKVSKA 172
    858 124 IKRKKF 129
    859 188 TKRVKK 193
    860 381 DRRQKR 386
    861 124 IKRKKF 129
    862 188 TKRVKK 193
    863 381 DRRQKR 386
    lymphocyte antigen 6 complex, locus D 864 61 QRKGRK 66
    865 85 ARRLRA 90
    866 61 QRKGRK 66
    867 85 ARRLRA 90
    Delta 1-pyrroline-5-carboxylate synthetase 868 455 LRRTRI 460
    869 455 LRRTRI 460
    B CELL LINKER PROTEIN BLNK 870 36 IKKLKV 41
    871 36 IKKLKV 41
    B CELL LINKER PROTEIN BLNK-S 872 36 IKKLKV 41
    873 36 IKKLKV 41
    fetal Alzheimer antigen 874 5 ARRRRKRR 12
    875 16 PRRRRRRT 23
    876 93 WKKKTSRP 100
    877 5 ARRRRK 10
    878 16 PRRRRR 21
    879 26 PRRPRI 31
    880 35 TRRMRW 40
    881 5 ARRRRK 10
    882 16 PRRRRR 21
    883 26 PRRPRI 31
    884 35 TRRMRW 40
    Transient receptor potential channel 885
    4 zeta splice variant 505 CKKKMRRK 512
    886 506 KKKMRR 511
    887 676 HRRSKQ 681
    888 506 KKKMRR 511
    889 676 HRRSKQ 681
    Myofibrillogenesis regulator MR-2 890 65 RKRGKN 70
    891 65 RKRGKN 70
    SH2 domain-containing phosphatase 892 269 IKKRSLRS 276
    anchor protein 2c 893 394 SKRPKN 399
    immunoglobulin superfamily, member 3 894 394 SKRPKN 399
    Meis (mouse) homolog 3 895 112 PRRSRR 117
    896 120 WRRTRG 125
    897 112 PRRSRR 117
    898 120 WRRTRG 125
    Deleted in azoospermia 2 899 105 GKKLKL 110
    900 114 IRKQKL 119
    901 105 GKKLKL 110
    902 114 IRKQKL 119
    Centaurin gamma3 903 543 NRKKHRRK 550
    904 544 RKKHRR 549
    905 544 RKKHRR 549
    Pre-B-cell leukemia transcription factor-1 906 233 ARRKRR 238
    907 286 NKRIRY 291
    908 233 ARRKRR 238
    909 286 NKRIRY 291
    60S ribosomal protein L13a 910 112 DKKKRM 117
    911 158 KRKEKA 163
    912 167 YRKKKQ 172
    913 112 DKKKRM 117
    914 158 KRKEKA 163
    915 167 YRKKKQ 172
    WD40-and FYVE-domain containing protein 3 916 388 IKRLKI 393
    917 388 IKRLKI 393
    LENG1 protein 918 34 RKRRGLRS 41
    919 84 SRKKTRRM 91
    920 1 MRRSRA 6
    921 33 ERKRRG 38
    922 85 RKKTRR 90
    923 1 MRRSRA 6
    924 33 ERKRRG 38
    925 85 RKKTRR 90
    MRIP2 926 375 NKKKHLKK 382
    G protein-coupled receptor 927 430 EKKKLKRH 437
    928 290 WKKKRA 295
    929 395 RKKAKF 400
    930 431 KKKLKR 436
    931 290 WKKKRA 295
    932 395 RKKAKF 400
    933 431 KKKLKR 436
    934 143 LKKFRQ 148
    935 228 LRKIRT 233
    936 143 LKKFRQ 148
    937 228 LRKIRT 233
    938 232 QKRRRHRA 239
    939 232 QKRRRH 237
    940 232 QKRRRH 237
    Sperm ion channel 941 402 QKRKTGRL 409
    A-kinase anchoring protein 942 2232 KRKKLVRD 2239
    943 2601 EKRRRERE 2608
    944 2788 EKKKKNKT 2795
    945 370 RKKNKG 375
    946 1763 SKKSKE 1768
    947 2200 EKKVRL 2205
    948 2231 LKRKKL 2236
    949 2601 EKRRRE 2606
    950 2785 EKKEKK 2790
    951 1992 QKKDVVKRQ 2000
    952 370 RKKNKG 375
    953 1763 SKKSKE 1768
    954 2200 EKKVRL 2205
    955 2231 LKRKKL 2236
    956 2601 EKRRRE 2606
    957 2785 EKKEKK 2790
    Lymphocyte-specific protein LSP1 958 315 GKRYKF 320
    959 315 GKRYKF 320
    similar to signaling lymphocytic 960 261 RRRGKT 266
    activation molecule (H. sapiens) 961 261 RRRGKT 266
    Dermatan-4-sulfotransferase-1 962 242 VRRYRA 247
    963 242 VRRYRA 247
    Moesin 964 291 MRRRKP 296
    965 325 EKKKRE 330
    966 291 MRRRKP 296
    967 325 EKKKRE 330
    A-Raf proto-oncogene 968 288 KKKVKN 293
    serine/threonine-protein kinase 969 358 LRKTRH 363
    970 288 KKKVKN 293
    971 358 LRKTRH 363
    Cytochrome P450 2C18 972 117 GKRWKE 122
    973 117 GKRWKE 122
    974 117 GKRWKE 122
    975 156 LRKTKA 161
    976 117 GKRWKE 122
    977 156 LRKTKA 161
    Protein tyrosine phosphatase, non-receptor 978 594 IRRRAVRS 601
    type 3 979 263 FKRKKF 268
    980 388 IRKPRH 393
    981 874 VRKMRD 879
    982 263 FKRKKF 268
    983 388 IRKPRH 393
    984 874 VRKMRD 879
    similar to kallikrein 7 985
    (chymotryptic, stratum corneum) 15 VKKVRL 20
    986 15 VKKVRL 20
    Hormone sensitive lipase 987 703 ARRLRN 708
    988 703 ARRLRN 708
    40S ribosomal protein S30 989 25 KKKKTGRA 32
    990 23 EKKKKK 28
    991 23 EKKKKK 28
    Zinc finger protein 91 992 617 LRRHKR 622
    993 617 LRRHKR 622
    NNP-1 protein 994 320 NRKRLYKV 327
    995 387 ERKRSRRR 394
    996 432 QRRRTPRP 439
    997 454 EKKKKRRE 461
    998 29 VRKLRK 34
    999 355 GRRQKK 360
    1000 361 TKKQKR 366
    1001 388 RKRSRR 393
    1002 454 EKKKKR 459
    1003 29 VRKLRK 34
    1004 355 GRRQKK 360
    1005 361 TKKQKR 366
    1006 388 RKRSRR 393
    1007 454 EKKKKR 459
    Methionyl-tRNA synthetase 1008 725 WKRIKG 730
    1009 725 WKRIKG 730
    ELMO2 1010 560 NRRRQERF 567
    Meningioma-expressed antigen 6/11 1011 432 RKRAKD 437
    1012 432 RKRAKD 437
    Inositol polyphosphate 4-phosphatase 1013
    type I-beta 375 LRKKLHKF 382
    1014 829 ARKNKN 834
    1015 829 ARKNKN 834
    1016 815 SKKRKN 820
    1017 815 SKKRKN 820
    C7ORF12 1018 40 SRRYRG 45
    1019 338 HRKNKP 343
    1020 40 SRRYRG 45
    1021 338 HRKNKP 343
    Rap guanine nucleotide exchange 1022 138 SRRRFRKI 145
    factor 1023 1071 QRKKRWRS 1078
    1024 1099 HKKRARRS 1106
    1025 139 RRRFRK 144
    1026 661 SKKVKA 666
    1027 930 LKRMKI 935
    1028 1071 QRKKRW 1076
    1029 1100 KKRARR 1105
    1030 1121 ARKVKQ 1126
    1031 139 RRRFRK 144
    1032 661 SKKVKA 666
    1033 930 LKRMKI 935
    1034 1071 QRKKRW 1076
    1035 1100 KKRARR 1105
    1036 1121 ARKVKQ 1126
    Sigma 1C adaptin 1037 27 ERKKITRE 34
    Alsin 1038 883 GRKRKE 888
    1039 883 GRKRKE 888
    NOPAR2 1040 14 LKRPRL 19
    1041 720 VKREKP 725
    1042 14 LKRPRL 19
    1043 720 VKREKP 725
    AT-binding transcription factor 1 1044 294 SKRPKT 299
    1045 961 EKKNKL 966
    1046 1231 NKRPRT 1236
    1047 1727 DKRLRT 1732
    1048 2032 QKRFRT 2037
    1049 2087 EKKSKL 2092
    1050 2317 QRKDKD 2322
    1051 2343 PKKEKG 2348
    1052 294 SKRPKT 299
    1053 961 EKKNKL 966
    1054 1231 NKRPRT 1236
    1055 1727 DKRLRT 1732
    1056 2032 QKRFRT 2037
    1057 2087 EKKSKL 2092
    1058 2317 QRKDKD 2322
    1059 2343 PKKEKG 2348
    Suppressin 1060 232 YKRRKK 237
    1061 232 YKRRKK 237
    Midline 1 protein 1062 100 TRRERA 105
    1063 494 HRKLKV 499
    1064 100 TRRERA 105
    1065 494 HRKLKV 499
    High mobility group protein 2a 1066 6PKKPKG11
    1067 84GKKKKD89
    1068 6PKKPKG11
    1069 84GKKKKD89
  • This application claims priority to A 1952/2003 filed on Dec. 4, 2003, the entirety of which is hereby incorporated by reference.

Claims (15)

1. A method of making a modified GAG binding protein by modifying a GAG binding site of the GAG binding protein, wherein the GAG binding site is modified by a method comprising the steps of:
a) identifying a region in the protein which is not essential for structure maintenance; and
b) introducing at least one basic amino acid into the site and/or deleting at least one bulky and/or acidic amino acid in the site;
whereby the GAG binding site has a GAG binding affinity of Kd≦10 μM.
2. A method according to claim 1, wherein the GAG binding site has a GAG binding affinity of ≦1 μM.
3. A method according to claim 1, wherein the GAG binding site has a GAG binding affinity of ≦0.1 μM.
4. A method according to claim 1, wherein the GAG binding affinity is higher by a factor of minimum 5 compared with wild-type GAG binding protein.
5. A method according to claim 1, wherein at least one basic amino acid selected from the group consisting of Arg, Lys, and His is inserted into the GAG binding region.
6. A method according to claim 1, wherein the protein is a chemokine, preferably IL-8, RANTES or MCP-1.
7. A method according to claim 6, wherein the GAG binding region is a C terminal a-helix.
8. A method according to claim 7, wherein positions 17, 21, 70, and/or 71 are substituted by Arg, Lys, His, Asn, and/or Gln.
9. A method according to claim 1, wherein the increased GAG binding affinity is an increased binding affinity to heparan sulfate and/or heparin.
10. A method according to claim 1, wherein a further biologically active region is modified thereby inhibiting or down-regulating a further biological activity of the protein.
11. A method according to claim 10, wherein the further biologically active region is modified by deletion, insertion, and/or substitution, preferably with alanine, a sterically and/or electrostatically similar residue.
12. A method according to claim 10, wherein the protein is a chemokine and the further biological activity is leukocyte activation.
13. A method according to claim 12, wherein the protein is IL-8 and the further biologically active region is located within the first 10 N-terminal amino acids.
14. A method according to claim 13, wherein the protein is a mutant with the first 6 N-terminal amino acids deleted.
15. A method according to claim 14, wherein the protein is a mutant, and wherein the protein is modified such that
Arg is at position 17, Lys is at position 70, and Arg is at position 71;
Arg is at position 17, Arg is at position 70, and Lys is at position 71;
or
Lys is at positions 70 and 71.
US12/437,121 2003-12-04 2009-05-07 Gag binding protein Abandoned US20110144305A1 (en)

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