Sulfated glycolipids and cell adhesion

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfatides, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The three proteins differ, however, in the inhibition of their binding to sulfatides by sulfated polysaccharides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand factor, suggesting the involvement of laminin or thrombospondin, or other unknown sulfatide-binding proteins in specific cell interactions that are also inhibited by fucoidan. Thrombospondin adsorbed on plastic promotes the attachment and spreading of some melanoma cells. Interestingly, fucoidan and an antibody against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment to thrombospondin-coated surfaces. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed on plastic also promote attachment and spreading of some cultured cell lines. Direct adhesion of melanoma cells requires high densities of adsorbed sulfatide. In the presence of laminin, however, specific adhesion of some cell types to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin is mediating adhesion by crosslinking receptors on the cell surface to sulfatide adsorbed on the plastic. Although thrombospondin also binds to sulfatides and to melanoma cells, it does not enhance but rather inhibits direct and laminin-dependent melanoma cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycolipids can participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different.     

Platelet thrombospondin mediates attachment and spreading of human melanoma cells

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.     

Sulfatide-binding domain of the laminin A chain

A sulfatide-binding site on the globular end region of the long arm of laminin has been identified. Following proteolytic digestion with thermolysin, an intact fragment of the laminin A chain carboxyl-terminal domain exhibiting sulfatide-binding activity was isolated using gel filtration and heparin affinity chromatography. This fragment is composed of two peptides that are covalently linked by at least one disulfide bond and encompass the carboxyl-terminal 394 amino acids of the A chain. The clusters of charged residues in the primary structure of these fragments are sufficient for heparin-binding activity but not sulfatide binding since reduction and alkylation of the fragments abolished sulfatide binding under conditions in which heparin binding was retained. Thus, sulfatide binding requires an intact three-dimensional structure. The iodinated fragment bound to A2058 melanoma and T47D breast carcinoma cells and could be displaced by the unlabeled fragment. Based on incorporation of [35S] sulfate, both cell lines synthesize sulfated glycolipids that bind to laminin. In agreement with previous data that indicate a synergistic interaction of the sulfatide-binding domain with other laminin-binding sites on melanoma cells during attachment, the isolated sulfatide-binding fragment significantly inhibited interaction of labeled intact laminin with melanoma and breast carcinoma cells in direct binding assays.     

Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.     

Interactions of a laminin-binding peptide from a 33-kDa protein related to the 67-kDa laminin receptor with laminin and melanoma cells are heparin-dependent.

A laminin-binding peptide (peptide G), predicted from the cDNA sequence for a 33-kDa protein related to the 67-kDa laminin receptor, specifically inhibits binding of laminin to heparin and sulfatide. Since the peptide binds directly to heparin and inhibits interaction of another heparin-binding protein with the same sulfated ligands, this inhibition is due to direct competition for binding to sulfated glycoconjugates rather than an indirect effect of interaction with the binding site on laminin for the 67-kDa receptor. Direct binding of laminin to the peptide is also inhibited by heparin. This interaction may result from contamination of the laminin with heparan sulfate, as binding is enhanced by the addition of substoichiometric amounts of heparin but inhibited by excess heparin and two heparin-binding proteins. Furthermore, laminin binds more avidly to a heparin-binding peptide derived from thrombospondin than to the putative receptor peptide. Adhesion of A2058 melanoma cells on immobilized peptide G is also heparin-dependent, whereas adhesion of the cells on laminin is not. Antibodies to the beta 1-integrin chain or laminin block adhesion of the melanoma cells to laminin but not to peptide G. Thus, the reported inhibition of melanoma cell adhesion to endothelial cells by peptide G may result from inhibition of binding of laminin or other proteins to sulfated glycoconjugate receptors rather than from specific inhibition of laminin binding to the 67-kDa receptor.     

Sialic acid-dependent adhesion of Mycoplasma pneumoniae to purified glycoproteins

Several purified glycoproteins including laminin, fetuin, and human chorionic gonadotropin promote dose-dependent and saturable adhesion of Mycoplasma pneumoniae when adsorbed on plastic. Adhesion to the proteins is energy dependent as no attachment occurs in media without glucose. Adhesion to all of the proteins requires sialic acid, and only those proteins with alpha 2-3-linked sialic acid are active. The alpha-subunit of human chorionic gonadotropin also promotes attachment, suggesting that a simple biantennary asparagine-linked oligosaccharide is sufficient for binding. Soluble laminin, asparagine-linked sialyloligosaccharides from fetuin, and 3'-sialyllactose but not 6'-sialyllactose inhibit attachment of M. pneumoniae to laminin. M. pneumoniae also bind to sulfatide adsorbed on plastic. Dextran sulfate, which inhibits M. pneumoniae binding to sulfatide, does not inhibit attachment on laminin, and 3'-sialyllactose does not inhibit binding to sulfatide, suggesting that two distinct receptor specificities mediate binding to these two carbohydrate receptors. Both 3'-sialyllactose and dextran sulfate partially inhibit M. pneumoniae adhesion to a human colon adenocarcinoma cell line (WiDr) at concentrations that completely inhibit binding to laminin or sulfatide, respectively, and in combination they inhibit binding of M. pneumoniae to these cells by 90%. Thus, both receptor specificities contribute to M. pneumoniae adhesion to cultured human cells.     

Midkine binds specifically to sulfatide the role of sulfatide in cell attachment to midkine-coated surfaces.

Midkine is a heparin-binding polypeptide which is implicated in the control of development and repair of various tissues. Recognition of sulfate groups in glycosaminoglycans is important for its function. To elucidate further its mechanism of action, the interactions of midkine with sulfated glycolipids were studied. Of various glycolipids and lipids examined, midkine bound strongly to sulfatide and cholesterol-3-sulfate (CHO-3-SO4) in a dose-dependent manner but failed to bind to other standard glycolipids and lipids. The properties of midkine binding to sulfatide and to CHO-3-SO4 differed in their sensitivity to inhibition by anionic polysaccharides, salt concentration and unlabeled midkine. Heparin inhibited midkine binding to sulfatide but weakly inhibited its binding to CHO-3-SO4. Liposomes bearing sulfatide carried out significant interactions with immobilized midkine, whereas those bearing CHO-3-SO4 did not. Incorporation of sulfatide into 32D cells and trypsinized COS cells enhanced 125I-labelled midkine binding, whereas incorporation of ganglioside or galactosylceramide had no effect. Furthermore, sulfatide-incorporated cells enhanced cell attachment to midkine-coated coverslips. These results indicate that midkine binds to sulfatide under physiological conditions and the midkine-sulfatide interaction may be important in controlling cell attachment.     

Sulfoglucuronyl Glycolipids Bind Laminin

Previous studies have shown that HNK-1 antibody reactive glycoconjugates, including the glycolipids 3-sulfoglucuronylneolactotetraosylceramide (SGGL-1) and 3-sulfoglucuronylneolactohexaosylceramide (SGGL-2), are temporally and spatially regulated antigens in the developing mammalian cortex. Extracellular matrix glycoprotein laminin is involved in cell adhesion by interacting with cell surface components and also promotes neurite outgrowth. Laminin has been shown to bind sulfatide. The interaction of sulfated glycolipids SGGL-1 and SGGL-2 with laminin was studied by employing a solid-phase radioimmunoassay and by HPTLC-immunoblotting. Laminin binding was detected with anti-laminin antibodies followed by 125I-labelled Protein A and autoradiography. Laminin binds SGGL-1 and SGGL-2, besides sulfatide, but does not bind significantly gangliosides and neutral glycolipids. The binding of SGGLs to laminin was two to three times less compared to sulfatide when compared on a molar basis. Desulfation of SGGLs and sulfatide by mild acid treatment resulted in abolition of laminin binding. On the other hand, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect. This showed that the sulfate group was essential for laminin binding. Of the various glycosaminoglycans tested, only heparin inhibited the binding of laminin to SGGLs and sulfatide in a dose-dependent manner. This indicated that SGGLs and sulfatide bind to the heparin binding site present in the laminin molecule. The availability of HNK-1 reactive glycolipids and glycoproteins such as SGGLs and several neural cell adhesion molecules to bind laminin at critical stages of neural development may serve as important physiological signals.     

Sulfatide-binding proteins

Sulfatides (galactosyl ceramide-I3-sulfate) and other sulfated glycolipids are found in many tissues. The cell adhesion proteins laminin, thrombospondin, and von Willebrand factor bind specifically to sulfated glycolipids. Methods for characterizing the specificity of these interactions using surface-adsorbed glycolipids are reviewed. The three proteins do not bind to other anionic lipids, including gangliosides, phospholipids, or cholesterol 3-sulfate. Binding to sulfatides is saturable and of relatively high affinity. Relative binding avidity depends on the oligosaccharide structure of the glycolipids. Binding to sulfatides in erythrocyte membranes can account for the hemagglutinating activities of the three proteins and may play a role in the interactions of these proteins with other cell types.     

Identification of active peptide sequences in the carboxyl-terminal cell binding domain of human thrombospondin-1.

Thrombospondin (TS) mediates attachment, spreading, and motility of several cell types through at least four cell binding domains: the amino-terminal heparin binding domain, the type I repeats containing the CSVTCG sequence, the RGDA sequence in the last of the type III calcium binding repeats and the carboxyl-terminal cell or platelet binding domain (CBD). The attachment of human melanoma cells (G361) to the COOH-terminal domain is independent of the RGDA sequence and is inhibited by the monoclonal antibody C6.7. To define the cell binding site(s) within this 212-residue COOH-terminal domain, we have synthesized eight overlapping peptides (seven 30-mers and a final 37-mer) representing the entire sequence of the CBD. Several of these peptides are insoluble in aqueous buffers at high concentration. Cell adhesion assays have been devised which employ covalent coupling of peptides in chaotropic solvents to chemically derivatized plastic 96-well plates. Three synthetic peptides, two of which are nonadjacent in the linear sequence, are potent attachment factors for G361 cells. C6.7 blocks adhesion to one of these peptides, whereas sulfated glycoconjugates inhibit adhesion of cells to all three. Polyclonal antibodies raised against the peptides inhibit cell adhesion to the peptides, the recombinant CBD, and to intact TS. The peptides GRGDSP and VTCG are not inhibitory. These sites are thus independent from the type I repeats and the RGDA sequence of TS. Each of the active peptides inhibits cell attachment to the other active peptides as well as to the CBD and to intact TS. This mutual inhibition suggests that the peptides share a common cellular receptor which may contain an associated glycoconjugate chain. These data indicate that the COOH-terminal cell binding domain of TS contains at least two peptide sequences which contribute to the attachment of a wide variety of cells.     

Binding of laminin to brain gangliosides and inhibition of laminin-neuron interaction by the gangliosides

Binding of laminin to glycolipids of neuronal membranes was studied with a thin-layer chromatography overlay assay. The major brain ganglioside GD1A was the main binding component, when chromatograms containing the same molar amount of the different brain gangliosides and the brain sulfatide were incubated with laminin at physiological ionic strength. The possible role of laminin binding to brain gangliosides in laminin-neuron interactions was studied with adhesion assays. It was found that binding of rat brain neurons to laminin is blocked by 10-40 microM brain gangliosides but not by sulfatide. The inhibition by the gangliosides is suggested to be due to competition with the cell surface interaction sites of laminin and not to binding of the gangliosides to the cells. Our findings support the idea that the adhesive and neurite-promoting effect of laminin is dependent on its interaction with gangliosides at the neuronal cell surfaces.     

The L2/HNK-1 carbohydrate of neural cell adhesion molecules is involved in cell interactions

We investigated whether the L2/HNK-1 carbohydrate epitope, expressed by two unusual glycolipids and several neural adhesion molecules, including L1, neural cell adhesion molecule, J1, and the myelin-associated glycoprotein, is involved in adhesion. Monoclonal L2 antibodies, the L2/HNK-1-reactive, sulfate-3-glucuronyl residue carrying glycolipids (L2 glycolipid) and a tetrasaccharide derived from the L2 glycolipid (L2 tetrasaccharide) were added to microexplant cultures of early postnatal mouse cerebellum, and cell migration and process extension were monitored. On the substrate poly-D-lysine, Fab fragments of L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes and migration of cell bodies, but only L2 glycolipid and L2 tetrasaccharide reduced neurite outgrowth. On laminin, L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes. Additionally, L2 glycolipid and L2 tetrasaccharide inhibited cell migration and neurite outgrowth. Several negatively charged glycolipids, lipids, and saccharides were tested for control and found to have no effect on outgrowth patterns, except for sulfatide and heparin, which modified outgrowth patterns in a similar fashion as L2 glycolipid and L2 tetrasaccharide. On astrocytes none of the tested compounds interfered with explant outgrowth. In short-term adhesion assays L2 glycolipid, sulfatide, and heparin inhibited adhesion of neural cells to laminin. L2 glycolipid and sulfatide interfered with neuron to astrocyte and astrocyte to astrocyte adhesion, but not with neuron-neuron adhesion. The most straightforward interpretation of these observations is that the L2/HNK-1 carbohydrate and the sulfated carbohydrates, sulfatide and heparin, act as ligands in cell adhesion.     

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.     

Adhesion of Mycoplasma pneumoniae to sulfated glycolipids and inhibition by dextran sulfate

A virulent strain of Mycoplasma pneumoniae was metabolically labeled with [3H]palmitate and studied for binding to glycolipids and to WiDr human colon adenocarcinoma cells. The organism binds strongly to sulfatide and other sulfated glycolipids, such as seminolipid and lactosylsulfatide which all contain terminal Gal(3SO4) beta 1-residues and weakly to some neolactoseries neutral glycolipids. M. pneumoniae do not bind gangliosides including the sialylneolacto-series and other neutral glycolipids that were tested. Only metabolically active M. pneumoniae cells bind to sulfatide, as binding is maximal in RPMI medium at 37 degrees C and almost completely abolished in nutrient-deficient medium or by keeping the cells at 4 degrees C. Dextran sulfate but not other sulfated or anionic polysaccharides at 10 micrograms/ml completely inhibits binding of M. pneumoniae to purified sulfatide. Dextran sulfate does not inhibit binding to the neolacto-series neutral glycolipids. Dextran sulfate partially inhibits adhesion of M. pneumoniae to cultured human colon adenocarcinoma cells (WiDr). The biological relevance of these data is suggested by our finding that sulfatide occurs in large amounts in human trachea, lung, and WiDr cells. Thus, there are at least two distinct receptors that mediate binding of M. pneumoniae to cells: glycolipids containing terminal Gal(3SO4) beta 1-residues as reported here, and glycoproteins containing terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc sequences (Roberts, D. D., Olson, L. D., Barile, M. F., Ginsburg, V., and Krivan, H. C. (1989) J. Biol. Chem. 264, 9289-9293).     

The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids

The human platelet glycoprotein thrombospondin (TSP) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). Binding of 125I-TSP to lipids from sheep and human erythrocytes and human platelets resolved on thin layer chromatograms indicates that sulfatides are the only lipids in the membrane which bind TSP. Binding to less than 2 ng of sulfatide could be detected. TSP failed to bind to other purified lipids including cholesterol 3-sulfate, phospholipids, neutral glycolipids, and gangliosides. Binding of 125I-TSP was inhibited by unlabeled TSP, by low pH, and by reduction of intersubunit disulfide bonds with dithiothreitol. A monoclonal antibody against TSP (A2.5), which inhibits hemagglutination and agglutination of fixed activated platelets by TSP, strongly inhibited TSP binding to sulfatides. A second monoclonal antibody (C6.7), which inhibits hemagglutination and aggregation of thrombin-activated live platelets, weakly inhibited sulfatide binding. Binding was inhibited by high ionic strength and by some monosaccharide sulfates including methyl-alpha-D-GlcNAc-3-sulfate. Neutral sugars did not inhibit. Fucoidan, a sulfated fucan, strongly inhibited binding with 50% inhibition at 0.3 micrograms/ml fucoidan. Other sulfated polysaccharides including heparin and dextran sulfates were good inhibitors, whereas hyaluronic acid and keratan sulfate were very weak.     

Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.     

Active peptides from the carboxyl-terminal globular domain of laminin α2 and Drosophila α chains

The laminin α1 chain carboxyl-terminal globular domain (G domain) contains multiple biological activities. Recently, we identified five cell binding sequences from the G domain by screening with overlapping 12-mer peptides encompassing the entire domain. The structures of these five sequences in the α1 chain are conserved in the corresponding regions of the different laminin α chains. Here we characterize the adhesion activities of the corresponding peptide segments from both the mouse laminin α2 chain and Drosophila laminin α chain using peptide-coated plastic plates and peptide-conjugated Sepharose beads. Using several cell lines, the laminin α2 chain peptides showed cell attachment and/or spreading activities with cell type specificities. Cell spreading on MG-10 was inhibited by integrin antibodies. Four of the Drosophila laminin peptides showed cell attachment activities. These results suggest that biologically active regions in the G domain are conserved in the laminin α1 and α2 chains, and that these regions in laminin play an important role in cell surface receptor interactions.     

Heparin-binding peptides from the type I repeats of thrombospondin. Structural requirements for heparin binding and promotion of melanoma cell adhesion and chemotaxis.

Synthetic peptides derived from the type I repeats of human platelet thrombospondin containing a consensus sequence Trp-Ser-Xaa-Trp bind to heparin, promote cell adhesion, and inhibit heparin-dependent interactions of melanoma cells with extracellular matrix components (Guo, N. H., Krutzsch, H. C., Nègre, E., Vogel, T., Blake, D. A., and Roberts, D. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3040-3044). In the present study, we further examined the structural requirements for activity of these peptides. The minimal active sequence for heparin or sulfatide binding based on inhibition studies is Trp-Ser-Pro-Trp, although an octapeptide is required for optimal activity. The 2 Trp residues and the Ser residue are essential. Peptides with more than 2 residues between the Trp residues are inactive. The Pro residue is essential for activity of the pentapeptide Trp-Ser-Pro-Trp-Ser, but some larger peptides with substitutions for the Pro residue are active. For direct high affinity binding to heparin, both the consensus sequence and a flanking sequence of basic amino acids are essential. Peptides containing the consensus sequence promote cell adhesion and act cooperatively with the adjacent basic amino acid sequence to promote cell spreading. Chemical modification of the Trp residues in the peptides with amino-terminal basic amino acids abolished both cell adhesion and heparin-binding. Peptides containing the consensus sequence and basic amino acids are chemotactic for A2058 human melanoma cells. The functional importance of this novel heparin and sulfatide-binding motif is suggested by its conservation in other members of the thrombospondin gene family, complement components, and in many members of the cytokine receptor and transforming growth factor beta superfamilies.     

A Mr 72K cell surface concanavalin A binding glycoprotein is specifically involved in the spreading of chick embryo fibroblasts onto laminin substrate

In the present study we have identified a 72-kDa cell surface concanavalin A binding glycoprotein (cbg 72) involved in the chick embryo fibroblast (CEF) adhesion onto laminin (LM) substrate. The cbg 72 was shown to interact specifically with immobilized laminin and to be resistant to Triton X-100 extraction when CEF were plated on laminin substrate but not on fibronectin (FN) substrate. This behavior suggested that cbg 72 could interact with cytoskeletal elements during cell spreading onto LM. This assumption is also in good agreement with the partitioning of cbg 72 in Triton X-114. Isolated cbg 72 specifically inhibited CEF spreading onto LM after their initial attachment, whereas cbg 72 did not impair the spreading of CEF onto FN. These data provide a molecular explanation to the inhibition of CEF spreading onto LM observed in the presence of the lectin concanavalin A (P. Codogno, M.-A. Doyennette-Moyne, J. Botti, and M. Aubery, 1988, J. Cell Physiol. 136, 463-470). Moreover, these results provide evidence for the role of a novel LM binding glycoprotein during the adhesion of mesenchymal derived cells. The relationship between cbg 72 and other known cell surface LM binding sites or receptors is discussed.     

Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity- purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.