Glycoconjugates and cell adhesion: the adhesive proteins laminin, thrombospondin and von Willebrand's factor bind specifically to sulfated glycolipids

The adhesive glycoproteins laminin, thrombospondin and von Willebrand's factor bind specifically and with high affinity to sulfated glycolipids, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The 3 proteins differ, however, in the effect of sulfated polysaccharides on their binding to sulfatides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand's 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 onto plastic promotes the attachment and spreading of G361 melanoma cells. Interestingly, fucoidan and an antibody directed against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed onto plastic also promote attachment and spreading of G361 melanoma cells. Direct adhesion of G361 cells requires high densities of sulfatide. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin mediates adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed onto the plastic. Although thrombospondin binds to sulfatide and to G361 cells, it does not enhance but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycoconjugates participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different.     

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.     

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 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.     

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.     

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.     

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.     

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.     

von Willebrand factor binds specifically to sulfated glycolipids

The human plasma glycoprotein Factor VIII/von Willebrand factor (vWF) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). vWF does not bind to gangliosides, neutral glycolipids, phospholipids, or cholesterol 3-sulfate. Although the largest oligomers of vWF bind preferentially to sulfatides, vWF monomers and dimers also bind but with reduced affinity. vWF binding is inhibited at high ionic strength or low pH, by some sulfated polysaccharides and by antibodies to vWF. Binding of vWF to sulfatides is probably responsible for its agglutination of aldehyde-fixed erythrocytes and may play a role in vWF-induced platelet adhesion or platelet aggregation.     

Gangliosides indirectly inhibit the binding of laminin to sulfatides

Laminin, a glycoprotein of basement membranes, agglutinates aldehyde-fixed erythrocytes. Laminin-mediated hemagglutination is strongly inhibited by some gangliosides and anionic phospholipids. Laminin, however, binds only to sulfatides among the lipids extracted from erythrocytes. We now report that gangliosides are remarkably potent inhibitors of laminin binding to sulfatides when both lipids are adsorbed on plastic. A 50% inhibition of laminin binding to 100 ng of sulfatides is obtained with 10 ng of GM3 and 8 ng of GM1, respectively. Mixing of sulfatides with neutral glycolipids, phosphatidyl choline, or cholesterol does not inhibit laminin binding, whereas mixing with sulfatide-depleted erythrocyte lipids enhances binding. Inhibition of binding by gangliosides is not due to competition for adsorption to the plastic, as preincubation of the adsorbed lipids with neuraminidase reverses inhibition by GM3, but not by GM1 which is not a substrate for the enzyme. These results are consistent with the observations that treatment of fixed erythrocytes with neuraminidase increases their agglutinability by laminin and that pretreatment of erythrocytes with gangliosides followed by washing gives similar inhibition as seen when gangliosides are present as competitive inhibitors. Thus, inhibition of laminin-mediated agglutination by gangliosides probably results from masking of erythrocyte sulfatides due to adsorption of gangliosides onto the membrane rather than from a direct competition for laminin binding sites.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts

Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.     

Cell attachment to thrombospondin: the role of ARG-GLY-ASP,calcium, and integrin receptors

Thrombospondin is a 420,000-D glycoprotein that has recently been shown to have several properties in common with the members of a class of adhesive proteins. To characterize further the adhesive properties of thrombospondin, we have studied its ability to support cell attachment. Thrombospondin adsorbed to plastic dishes supports the attachment of human endothelial and smooth muscle cells and the monocyte-like cell line (U937) as well as normal rat kidney cells. The majority of attached cells do not spread on the solid-phase thrombospondin. The attachment of all four cell types to thrombospondin is abolished if the assay is performed in the presence of EGTA, although the cells still attach to fibronectin. If thrombospondin is adsorbed to the dishes in the presence of EGTA and then washed with buffer containing calcium before addition of the cells, attachment is still markedly inhibited, indicating that calcium affects the conformation and function of thrombospondin. Attachment of all four cell types is also markedly inhibited by the synthetic peptides gly-arg-gly-asp-ser-pro (GRG-DSP) and gly-arg-gly-asp-ala-cys (GRGDAC) but not by the control peptide gly- arg-gly-glu-ser-pro (GRG-ESP). Affinity chromatography of n- octylglucoside extracts of surface-labeled endothelial cells or smooth muscle cells on thrombospondin-Sepharose and GRG-DSP-Affigel columns was used to identify an integrin complex related to glycoprotein IIb- IIIa as an RGD-dependent receptor for thrombospondin. In addition, a monoclonal antibody (LM609) that blocks attachment of endothelial cells to vitronectin, fibrinogen, and von Willebrand factor also inhibits attachment of endothelial cells to thrombospondin. These data indicate that the attachment of cells to thrombospondin is mediated by RGD and calcium-dependent mechanisms and is consistent with the hypothesis that the GRGDAC sequence in thrombospondin is a site for interaction with an integrin receptor of the beta 3 subclass.     

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).