The effect of human C-reactive protein on the cell-attachment activity of fibronectin and laminin

We have previously reported that purified human C-reactive protein (CRP) specifically binds to the cell-binding region of plasma fibronectin (Fn) in a Ca2+-dependent reaction that is saturable at a molar ratio of CRP/Fn of approximately 9. In this study, the binding of CRP to Fn was found to interfere with the cell-attachment promoting activity of Fn. The inhibition of cell attachment was dependent on the concentration of the CRP and involved the phosphorylcholine (PC) binding site of CRP since inhibition was prevented by allowing the CRP to react with either PC (or closely related monophosphate compounds) or a mAb specific for the PC-binding site of CRP. Binding of CRP to laminin was also Ca2+-dependent; however, this binding did not alter the cell-attachment promoting activity of laminin. CRP by itself does not mediate cell attachment. Since CRP is selectively deposited at sites of tissue damage along with plasma Fn and has the ability to bind to Fn and alter its cell-binding activity, CRP may modulate early events in tissue repair.     

Cell attachment peptide of C-reactive protein: critical amino acids and minimum length

Human C-reactive protein (CRP) is an acute phase blood component that accumulates at sites of tissue damage and necrosis and is degraded by neutrophils to biologically active peptides. A dodecapeptide composed of amino acids 27–38 of CRP mediates cell attachment in vitro. This peptide was designated the cell-binding peptide (CB-Pep) of CRP. Characterization of the interaction between fibroblasts and modified synthetic peptides with sequential deletions from either the N-terminus or C-terminus revealed that the minimal sequence for cell attachment or inhibition of cell attachment to the CB-Pep was Phe-Thr-Val-Cys-Leu , which corresponds to residues 33–37 within each of the five 206 amino acid subunits of CRP. The pentapeptide by itself mediated cell attachment. Substitutions for each residue within the CB-Pep indicated that the critical residues for activity were Phe-33 and Thr-34. This cell-binding pentapeptide represents a recognition motif for cell adhesion not found in other proteins.     

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.     

Inhibition of fibronectin binding to platelets by proteolytic fragments and synthetic peptides which support fibroblast adhesion

To define regions within fibronectin (Fn) recognized by platelet binding sites, inhibition of Fn binding by an Fn fragment and synthetic peptides has been analyzed. A highly purified 120-kDa chymotryptic fragment, which has cell attachment activity but did not bind to insolubilized heparin or gelatin, inhibited Fn binding to platelets with an ID50 approximately 3 microM. Previous work indicates that fibroblasts attach to an 11.5-kDa subfragment of this 120-kDa fragment, and that one of four 30-residue synthetic peptides containing sequences from this region supports cell attachment. Only the peptide containing the COOH terminus of the 11.5-kDa fragment inhibited Fn binding to platelets, with an ID50 approximately 10 microM and is the peptide which supports fibroblast attachment. Of the smaller peptides studied from this sequence, all peptides containing the Arg-Gly-Asp-Ser sequence, including the tetrapeptide itself, were active in inhibiting Fn binding to platelets (ID50 values approximately 10-20 microM). The same peptides support fibroblast attachment. Those which lacked this sequence including Gly-Asp-Ser-Pro and Thr-Gly-Arg-Gly (immediately adjacent tetrapeptides) lacked both activities. Further evidence for specificity of inhibition was provided by structurally modified peptides in which substitution of a Glu for Asp abolished inhibitory activity and substitution of Lys for Arg or Ala for Gly reduced activity 6- and 8-fold, respectively. In addition, Arg-Gly-Asp-Ser-containing peptides inhibited the rate and extent of thrombin-induced platelet aggregation. These data suggest that the Arg-Gly-Asp-Ser tetrapeptide contains a recognition specificity involved in the binding of Fn to platelets and that platelets share features of this recognition specificity with fibroblasts.     

Binding site on human C-reactive Protein (CRP) recognized by the Leukocyte CRP-receptor

C-reactive protein (CRP), the prototypical inflammatory acute phase reactant in humans, interacts with monocytes and neutrophils via a specific receptor. To map the site on CRP recognized by the CRP receptor (CRP-R), synthetic peptides corresponding to the surface region on each of the five identical subunits were tested as competitors vs. [¹²⁵l]-CRP for cell binding. A peptide of residues 27–38 (TKPLKAFTVCLH) efficiently inhibited CRP binding when compared to other nonoverlapping peptides. This peptide was termed the cell-binding peptide (CB-Pep). The F(ab′)₂ of an IgG Ab to the CB-Pep specifically inhibited CRP binding upon reacting with the ligand. Competitive binding studies with synthetic peptides truncated from either the NH₂- or COOH-terminus of the CB-Pep revealed that the minimum length recognized by the CRP-R consisted of residues 31–36: KAFTVC. Conservative substitutions of residues within the CB-Pep indicated that the four residues AFTV were critical for CRP-R binding. The CB-Pep also inhibited induced superoxide generation by HL-60 granulocytes. The minimum length required for the inhibition was also KAFTVC; however, only Phe-33 and Leu-37 were critical residues in this assay. Anti-CB-Pep IgG Ab reacted more extensively with heat-modified CRP, suggesting that an altered conformation of CRP is preferentially recognized by the CRP-R. The results suggest that this contiguous sequence on a β-strand on one face of each of five subunits of the CRP pentamer serves as a unique recognition motif for inflammatory leukocytes. J. Cell. Biochem. 64:140–151. © 1997 Wiley-Liss, Inc.     

Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding

We have probed for active sites in the B1 chain of laminin using synthetic peptides comprising certain regions of its amino acid sequence as deduced from cDNA clones. An antibody to a 19-mer from domain III inhibited attachment of HT-1080 and CHO cells to laminin, while the peptide itself was inactive. A nearby peptide (CDPGYIGSR) from domain III with homology to epidermal growth factor was synthesized and found to be one of the principle sites in laminin mediating cell attachment, migration, and receptor binding.     

Identification of active sequences in human laminin α5 G domain

Human laminin‐511 (α5β1γ1) and its truncated protein, laminin‐511 E8 fragment, bind to integrin α6β1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder‐free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin‐511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1‐hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide‐coated plate and peptide‐chitosan matrix (peptide‐ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26‐coated plates and hA5G74‐ChtMs promoted integrin β1‐mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin‐511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.     

Cyclic peptides from the loop region of the laminin alpha 4 chain LG4 module show enhanced biological activity over linear peptides

Laminins, heterotrimeric glycoproteins in the basement membrane, are involved in diverse biological activities. So far, five alpha, three beta, and three gamma chains have been identified, and at least 15 laminin isoforms exist composed of various combinations of the different three chains. The major cell-surface receptors for laminins are integrins and proteoglycans, such as dystroglycans and syndecans. Previously, we reported that synthetic peptide A4G82 (TLFLAHGRLVFM, mouse laminin alpha4 chain residues 1514-1525) showed strong cell attachment and syndecan binding activities. On the basis of the crystal structure of the LG module and sequence alignment, A4G82 is located in the connecting loop region between beta-strands E and F in the laminin alpha4 chain LG4 module. Here, we have focused on the structural importance of this E-F loop region for the biological activity of the alpha4 chain LG4 module. To determine the importance of the loop structure, we synthesized peptide A4G82X (cyclo-A4G82X, Cys-TLFLAHGRLVFX-Cys, X= norleucine), which was cyclized via disulfide bridges at both the N- and C-termini. The cyclic peptides derived from A4G82X inhibited the heparin binding activity of the alpha4 chain G domain and promoted HT-1080 cell attachment better than the corresponding linear peptides. We determined FLAHGRLVFX as a minimal sequence of cyclo-A4G82X important for cell adhesion and heparin binding using a series of truncated peptides. Moreover, HT-1080 cell attachment to the cyclic peptides was more efficiently blocked by heparin than cell attachment to the linear peptides. Furthermore, the cyclic peptides showed significantly enhanced syndecan-2-mediated cell attachment activity. These results indicate that the activity of A4G82 is highly conformation-dependent, suggesting that the E-F loop structure is crucial for its biological activity.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.     

A laminin-pepsin fragment with cell attachment and neurite outgrowth activity at distinct sites

Laminin is a large basement membrane glycoprotein which influences the behavior and morphology of a variety of cells. We have found that laminin and a pepsin fragment of laminin (P-lam) contain distinct sites for HT-1080 human fibrosarcoma cell attachment and for neurite outgrowth activity of PC12 and NG108-15 cell lines. Reduction and alkylation of laminin and P-lam fragment disulfide bonds, in the absence of denaturing agents, markedly reduced the cell attachment activity without reducing the neurite outgrowth response. The P-lam fragment (approximately 375 kDa) was found to contain part of the cross region of laminin and a portion of the long arm, on the basis of recognition by antisera against laminin synthetic peptides and fusion proteins. Modification of arginine residues by cyclohexanedione also had no effect on neurite outgrowth but reduced HT-1080 cell adhesion. Modification of lysine residues by succinic and citraconic anhydride, however, abolished laminin neurite outgrowth but not cell attachment activity. Neurite outgrowth activity was recovered by reversing the lysine modification. These data support the existence on laminin of separate sites for cell attachment and for neurite outgrowth.     

Cell adhesive peptide screening of the mouse laminin α1 chain G domain

Cell adhesive peptides have been widely applied for therapeutic drugs, drug delivery systems, and biomaterials. Previously, we identified various cell adhesive sequences in the G domains of four laminin α chains (α2-α5) by the systematic soluble peptide screening. We also identified five cell-binding sequences in the laminin α1 chain G domain using synthetic peptide-polystyrene beads. Here, we re-screened cell adhesive peptides in the laminin α1 chain G domain by the systematic soluble peptides screening. The 110 soluble peptides were evaluated for their cell adhesive activities using human fibrosarcoma HT1080 cells and human dermal fibroblasts. Fourteen peptides were newly identified as a cell adhesive. Additionally, four peptides (AG22: SSFHFDGSGYAM, AG42: TFDLLRNSYGVRK, AG76: HQNQMDYATLQLQ, AG86: LGGLPSHYRARNI) promoted integrin-mediated cell adhesion. Further, neurite outgrowth activity with rat pheochromocytoma PC12 cells was evaluated and two peptides (AG20: SIGLWNYIEREGK, AG26: SPNGLLFYLASNG) were newly identified for neurite outgrowth activity. These results suggested that the systematic soluble peptides screening approach is an accurate and powerful strategy for finding biologically active sequences. The active sequences newly identified here could be involved in the biological functions of this domain. The active peptides are useful for evaluating molecular mechanisms of laminin-receptor interactions and for developing cell adhesive biomaterials.     

B133 (DSITKYFQMSLE), a laminin β1-derived peptide, contains distinct core sequences for both integrin α2β1-mediated cell adhesion and amyloid-like fibril formation

The B133 peptide (DSITKYFQMSLE, mouse laminin β1 chain 1319-1330) promotes cell attachment, and forms amyloid-like fibrils. Here, we evaluated the active core sequences using B133 deletion peptides. B133a, lacking the N-terminal Asp residue, promoted cell spreading via integrin α2β1, whereas B133g, lacking the C-terminal Glu residue, lost the activity. Congo red analysis using the truncated peptides determined that B133g forms amyloid-like fibrils but B133a did not. These results suggest that the N- and C-terminal amino acids contribute to integrin α2β1 binding and to fibril formation, respectively. Further analyses using the truncated peptides showed that the C-terminal eight residues (B133d: KYFQMSLE) are a minimum active sequence for integrin α2β1-mediated cell attachment and the N-terminal nine residues (B133i: DSITKYFQM) are critical for amyloid-like fibril formation. These results suggest that peptide B133 is multifunctional with two different active core sequences: integrin α2β1-mediated cell attachment and amyloid-like fibril formation. Moreover, alanine substitution analysis of B133a indicated that six amino acids, Ile, Thr, Tyr, Phe, Met, and Glu, are important for cell attachment activity. When the Ser residue at the 9th position of B133a was replaced with Ala, the cell attachment activity was enhanced. Further mutation analysis at the 9th position of B133a using various amino acids suggests that hydrophobic amino acids are effective for the integrin α2β1-mediated cell attachment activity. These findings define multifunctional and overlapping sites on the B133 peptide and are useful for designing multifunctional synthetic molecules.     

层粘连蛋白及其肽段对小鼠胚泡粘附和扩展的作用

作为细胞外基质的主要成分之一的层粘连蛋白（ＬＮ）,对小鼠胚泡的粘附、扩展有显著促进作用。ＬＮ分子上的一些活性位点对胚泡的粘附和扩展也具有一定的作用,含ＲＧＤ位点序列的合成肽段ＲＧＤＳ对胚泡的粘附有促进作用;含ＹＩＧＳＲ位点序列的合成肽段ｃＹＩＧＳＲ对胚泡的粘附和扩展均有促进作用;且ＲＧＤＳ和ｃＹＩＧＳＲ可以竞争性抑制ＬＮ对胚泡粘附和扩展的促进作用。以上结果表明ＬＮ对胚泡的作用是通过胚泡上不同的ＬＮ     

A substrate of the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser) is sufficient to promote transition of arterial smooth muscle cells from a contractile to a synthetic phenotype

Extracellular matrix components strongly influence the differentiated properties of isolated rat arterial smooth muscle cells during in vitro cultivation. The attachment and spreading of the cells on a substrate of fibronectin or a 105-kDa cell-binding fragment of fibronectin are accompanied by a structural and functional transformation, referred to as a transition or modulation from a contractile to a synthetic phenotype. Here, the ability of the cell-attachment sequence of fibronectin, Arg-Gly-Asp-Ser (RGDS), to promote this process was studied. The results demonstrate that freshly isolated smooth muscle cells attached to a substrate of the synthetic peptide Gly-Arg-Gly-Asp-Ser-Cys (GRGDSC) in a specific manner and as well as to substrates of fibronectin and the 105-kDa fragment. Subsequent spreading of the cells on the peptide substrate followed the same kinetics and was as extensive as on fibronectin, even if protein synthesis was blocked by treatment of the cultures with cycloheximide. Like fibronectin, the peptide substrate induced formation of actin filament bundles, again without ongoing protein synthesis. Moreover, it was as efficient as fibronectin in supporting the transition of the cells from a contractile to a synthetic phenotype as analyzed by electron microscopy. Antibodies against the beta subunit of the fibronectin receptor interfered with the attachment, spreading, and fine structural reorganization of the cells in a similar manner on substrates of fibronectin, the 105-kDa fragment, and GRGDSC. Taken together, the findings indicate that the cell-attachment sequence (RGDS) mimics intact fibronectin in promoting a change in the differentiated properties of arterial smooth muscle cells and does so by interacting with a cell surface receptor for fibronectin.     

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin.

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.     

Development of laminin receptor agonists: identification of important functional residues by alanine scanning

An antagonist of cellular adhesion and motility, acetyl-C-[S-Acm]-VIGYSGDRC-[S-Acm]-NH(2) (mEGF(33-42)), shares homology with the agonist sequence CDPGYIGSR-NH(2). It has been proposed that the latter peptide binds to the high affinity 67 kDa laminin receptor. Both peptides have equal affinities for the receptor and similar conformations have been derived for both. We have examined the importance of individual non-homologous residues with respect to receptor binding and antagonistic properties of mEGF(33-42). Alanine scanning of non-conserved residues in the N-terminal half of mEGF(33-42) caused loss of biological activity with respect to cell attachment, receptor binding and migratory response. Substitution of alanine for serine (position 6) caused loss of laminin-specific cell attachment and receptor binding activities. However, the peptide did stimulate migration suggesting that this peptide may be a non-specific stimulator of migration. In contrast, alanine substitution for the C-terminal Cys-S-Acm had no apparent effect on the attachment or receptor binding activities of the peptide but generated an agonist from the antagonist parent. Comparison of the modelled folds of the alanine containing peptides revealed the presence of significant helical content in those peptides capable of stimulating migration and suggests that a reduction in bulk in the N-terminal residues is not conducive to adopting a productive binding conformation.     

Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: unique and shared features

Synthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly-Arg-Gly-Asp-Ser was considerably more active than Arg-Gly-Asp-Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser-Asp-Gly-Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly-Arg-Gly-Asp-Ser was only weakly active, but the inverted peptide Ser-Asp-Gly-Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser-Asp-Gly-Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.     

Syndecan binding sites in the laminin alpha1 chain G domain

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.