Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins

Thrombospondin (TSP), a major platelet-secreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum. To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP. These assays were TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSVTCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect. Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

The interaction of human platelet thrombospondin with fibrinogen. Thrombospondin purification and specificity of interaction

Human platelet thrombospondin (TSP) was purified to homogeneity by chromatography on fibrinogen coupled to cyanogen bromide-activated Sepharose. The yield of TSP was 1.3 mg or approximately 22% of that present in platelet-rich plasma as determined by radioimmunoassay. It analyzed on discontinuous sodium dodecyl sulfate gels as a single band having apparent molecular weights of 180,000 and greater than 400,000 under reducing and nonreducing conditions, respectively. Amino acid analysis gave results similar to previously published values. Antibodies raised in rabbits were monospecific as evaluated by radioimmunoassay. In double immunodiffusion tests, these antibodies gave one line of identity against TSP purified by this procedure and TSP purified by published procedures, confirming the identity of the material isolated. The protein possesses no lectin-like activity. The specificity of the TSP-fibrinogen interaction was investigated. TSP binding to fibrinogen-Sepharose occurred in the presence of EDTA, indicating that calcium and magnesium ions are not required for interaction of TSP with fibrinogen. The binding of TSP to fibrinogen-Sepharose was quantitatively blocked by pretreatment with an antibody to the cyanogen bromide cleavage fragment composed of residues 241-476 of the carboxyl-terminal end of the alpha chain of fibrinogen. Antibodies against the D and E domains of fibrinogen had no effect on the binding. Excess fibrinogen (30 mg/ml) added to platelet extract quantitatively inhibited binding of TSP to fibrinogen-Sepharose. TSP preferentially bound to uncross-linked fibrin, suggesting that the TSP-fibrinogen binding site is unavailable in cross-linked fibrin. These results indicate that TSP binds specifically to immobilized fibrinogen or uncross-linked fibrin through determinants present in the carboxyl-terminal portion of the alpha chain and that these interactions do not require calcium or magnesium ions.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.     

Purification and some properties of streptococcal protein G, a novel IgG-binding reagent

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.     

Demonstration of high affinity fibronectin receptors on rat hepatocytes in suspension

A cell-binding peptide (Mr = 85,000) which lacks the gelatin- and heparin-binding domains, was purified from trypsin-digested fibronectin. Preincubation of rat hepatocytes in suspension with the peptide, inhibited initial attachment of the cells to immobilized fibronectin while attachment to immobilized laminin and collagen was unaffected. 125I-labeled 85-kDa peptide bound to the cells in suspension at 4 degrees C in a time-dependent, saturable, and partially reversible reaction. Scatchard analysis of the binding data indicated a single class of receptors with a Kd of 1.7 X 10(-8) M. The number of binding-sites was approximately 2.8 X 10(5)/cell. Unlabeled 85-kDa peptide inhibited the binding of 125I-labeled 85-kDa peptide 30-fold more effectively than intact fibronectin. These results provide direct evidence for the presence of a domain in the fibronectin molecule which has or may acquire a high affinity for receptors involved in adhesion of hepatocytes to immobilized fibronectin.     

Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding.

To clarify the role of CD36 as a TSP receptor and to investigate the mechanisms of the TSP-CD36 interaction, transfection studies were performed using CD36-cDNA in a CDM8 plasmid. Jurkat cells transfected with CD36 cDNA express an 88kD membrane surface protein and acquire the ability to bind thrombospondin. The TSP amino acid sequence, CSVTCG, mediates the interaction of thrombospondin with CD36. CD36 transfectants but not control transfectants bind radiolabeled tyrosinated peptide (YCSVTCG). The hexapeptide inhibits thrombospondin expression on activated human platelets and results in diminished platelet aggregation. CSVTCG-albumin conjugates support CD36-dependent adhesion of tumor cells. We conclude that the CSVTCG repeat sequence is a crucial determinant of CD36 thrombospondin binding.     

A fibronectin-binding glycoprotein from human platelet membranes.

Fibronectin ('cold-insoluble globulin') has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.     

Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin

The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin- binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.     

Fibulin, a novel protein that interacts with the fibronectin receptor beta subunit cytoplasmic domain

A 100 kd protein was isolated from tissue and cell extracts by affinity chromatography on a synthetic peptide representing the cytoplasmic domain of the fibronectin receptor beta subunit. The 100 kd protein also bound to native fibronectin receptor, and this binding could be reversed with EDTA. Calcium may be the divalent cation required for the binding since the 100 kd protein was found to bind 45Ca2+. The N-terminal amino acid sequence of the 100 kd protein was not similar to any sequence in a protein data base. Immunofluorescent staining of cells cultured on fibronectin showed the 100 kd protein coinciding with the fibronectin receptor beta subunit in sites of substrate contact. Therefore this protein, which we term fibulin, interacts with the fibronectin receptor in vitro and associates with the receptor in vivo. Fibulin is a potential mediator of interactions between adhesion receptors and the cytoskeleton.     

Conformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive activities of thrombospondin-1

The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by alpha4beta1 and alpha5beta1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by alpha3beta1 integrin and required that the alpha3beta1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified alpha3beta1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by alpha3beta1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.     

Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.     

Purification of a Soluble Cytockinin-binding Protein from Etiolated Mung Bean Seedlings

A cytokinin-binding protein (CBP) was purified from a crude extract of etiolated mung bean seedlings by a protocol involving affinity chromatography on benzyladenine-linked Sepharose 4B, ion exchange chromatography on DEAE-Sephadex A50, and gel filtration on Sphacryl S-400. The molecular weight was estimatd to be about 200,000 by gel filtration. CBP appeared as two bands corresponding to molecular weights of about 45,000 and 48,000 on SDS-polyacrylamide gel electrophoresis. The dissociation constant for benzyladenine was 7.5 x 10^-7 M. ¹⁴C-Benzyladenine-binding to CBP was reversible and could be inhibited by the addition of kinetin or trans-zeatin. Adenine, AMP, and ADP had no inhibitory effect on the binding of ¹⁴C-benzyladenine to CBP but the addition of ATP to the assay mixture enhanced the binding.     

Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification

Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM.     

Identification of a 50,000 Mr protein from rabbit brush border membranes that binds Clostridium perfringens enterotoxin

A protein that binds Clostridium perfringens enterotoxin was extracted with NP-40 from rabbit intestinal brush border membranes. This protein was partially purified by affinity chromatography on enterotoxin-coupled CNBr-activated Sepharose 4B. The molecular weight of this protein was approximately 50,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity-purified samples containing this protein specifically inhibited biological activity of the enterotoxin on Vero (African green monkey kidney) cells. These studies suggest that this protein may be involved in the binding of the enterotoxin to rabbit intestinal epithelial cells.     

RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasmodium falciparum-infected erythrocytes bind to the RGD motif of fibronectin via the band 3-related adhesin

Previously it was shown that Plasmodium falciparum-infected erythrocytes bound to thrombospondin by the interaction of the peptidic sequence, HPLQKTY, of the band 3 protein of infected erythrocytes, and the RGD motif of thrombospondin. Here, we show that falciparum-parasitized erythrocytes bind to immobilized fibronectin by the RGD sequence of fibronectin. Involvement of the HPLQKTY region of band 3 in binding was demonstrated by inhibition of adhesion of parasitized erythrocytes to fibronectin by an HPLQKTY-containing peptide and the binding of the HPLQKTY peptide to the RGD sequence of immobilized fibronectin. Since fibronectin occurs on endothelial cells and platelets, this interaction may contribute to the binding of falciparum-infected erythrocytes to such host cells.