Laminin induces the stable expression of surface galactosyltransferase on lamellipodia of migrating cells

We have previously shown that cell surface galactosyltransferase (GalTase) mediates cell spreading and migration on basal lamina matrices by binding N-linked oligosaccharide substrates within laminin. In this study we have examined the distribution and expression of cell surface GalTase during mesenchymal cell migration on various extracellular matrices. Antisera raised against affinity-purified beta 1,4 GalTase, as well as anti-GalTase Fab fragments, inhibited cell migration on laminin-containing matrices, whereas under identical conditions, anti-GalTase IgG had no effect on the rate of cell migration on fibronectin substrates. Cells migrating on laminin had three times the level of surface GalTase, assayed by 125I-antibody binding and by direct enzyme assay, than similar cells migrating on fibronectin. On the other hand, total cellular GalTase, assayed either enzymatically or by Northern blot analysis, was similar when cells were grown on laminin or fibronectin. The laminin-dependent increase in surface GalTase was due to its expression onto the leading and trailing edges of migrating cells in association with actin-containing microfilaments assayed by double-label indirect immunofluorescence. On stationary cells, surface GalTase levels were low, but as cells began to migrate on laminin GalTase became polarized to the growing lamellipodia. GalTase was not detectable on lamellipodia or filopodia when cells migrated on fibronectin substrates. These results show that laminin-containing matrices induce the stable expression of GalTase onto cell lamellipodia and filopodia where it mediates subsequent cell spreading and migration. Since fibronectin was unable to induce GalTase expression onto lamellipodia, these studies also suggest that the extracellular matrix can selectively influence which intracellular components are maintained on the cell surface.     

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.     

Cell surface beta-1,4-galactosyltransferase is associated with the detergent-insoluble cytoskeleton on migrating mesenchymal cells.

Cell surface beta-1,4-galactosyltransferase (GalTase) partially mediates a variety of cell interactions with laminin-containing matrices, including mesenchymal cell spreading and migration and neurite initiation, by binding to N-linked oligosaccharides within the E8 domain of laminin. Previous studies using indirect immunofluorescence have suggested that some surface GalTase colocalizes with actin-containing microfilaments in migrating cells. In this study, we present more direct biochemical evidence showing that surface GalTase is associated with the detergent-insoluble cytoskeleton and that this association is dependent upon the integrity of the cytoskeleton, valency of the anti-GalTase antibody, and migratory status of the cell. Two-thirds of the surface GalTase was associated with the detergent-insoluble cytoskeleton when assayed either by monovalent anti-GalTase Fab fragments or by extracting any detergent-soluble GalTase prior to labeling with intact anti-GalTase IgG. However, 80-100% of the surface GalTase could be induced to associate with the cytoskeleton when cross-linked with anti-GalTase IgG prior to detergent extraction. Destabilizing cytoskeleton-protein interactions with high levels of KCl, elevated pH, or cytochalasin B reduced the amount of surface GalTase retained in the detergent-insoluble cytoskeleton fraction. Finally, we have shown previously that laminin induces the expression of GalTase onto lamellipodia of migrating cells, and in this study, we show that the laminin-induced increase in surface GalTase is cytoskeletally associated. Collectively, these data suggest that cell surface GalTase participates in cell spreading and migration on laminin by virtue of its association with the cytoskeleton.     

Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin

Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin.     

Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.     

3T3 cell surface galactosyltransferase is a calcium-dependent adhesion molecule for collagen type IV.

Cell surface galactosyltransferase (GalTase) has been previously shown to mediate cell spreading or migration on laminin matrices. This work demonstrates that 3T3 cell surface GalTase also mediates cell attachment to collagen type IV. Attachment to collagen type IV was blocked by perturbations of GalTase or substrate pregalactosylation on cells possessing only calcium-dependent mechanisms of adhesion. Cells with both calcium-dependent and calcium-independent systems were not affected by GalTase perturbation. Collagen type IV was shown to possess GalTase substrates since matrices could be galactosylated by both soluble enzyme and 3T3 cells.     

Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase

A number of cell surface receptors bind to distinct laminin domains, thereby mediating laminin's diverse biological activities. Cell surface beta 1,4-galactosyltransferase (GalTase) functions as one of these laminin receptors, facilitating mesenchymal cell migration and PC12 cell neurite outgrowth on laminin. In this study, the GalTase binding site within laminin was identified as the E8 fragment by assaying purified fragments and by immunoprecipitating and immunoblotting galactosylated laminin using E8-reactive antibodies. Compared with intact laminin and other laminin fragments, E8 possessed the highest GalTase binding activity, using both membrane-bound and solubilized GalTase. More significantly, the neurite-promoting activity of fragment E8 was shown to be dependent upon its interaction with GalTase. Pregalactosylating purified E8 eliminated subsequent GalTase binding and consequently inhibited neurite initiation; parallel studies on laminin fragments E1-4 or E1 failed to affect neurite outgrowth. Furthermore, anti-GalTase IgG inhibited neurite initiation on purified E8 substrates; control IgG had no effect. These results localize the predominant GalTase binding domain in laminin to fragment E8 and demonstrate that the neurite-promoting activity of E8 is dependent upon its interaction with GalTase.     

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.     

Evidence for a novel enzymatic mechanism of neural crest cell migration on extracellular glycoconjugate matrices

Migrating embryonic cells have high levels of cell surface galactosyltransferase (GalTase) activity. It has been proposed that GalTase participates during migration by recognizing and binding to terminal N-acetylglucosamine (GlcNAc) residues on glycoconjugates within the extracellular matrix (Shur, B. D., 1982, Dev. Biol. 91:149-162). We tested this hypothesis using migrating neural crest cells as an in vitro model system. Cell surface GalTase activity was perturbed using three independent sets of reagents, and the effects on cell migration were analyzed by time-lapse microphotography. The GalTase modifier protein, alpha-lactalbumin (alpha-LA), was used to inhibit surface GalTase binding to terminal GlcNAc residues in the underlying substrate. alpha-LA inhibited neural crest cell migration on basal lamina-like matrices in a dose-dependent manner, while under identical conditions, alpha-LA had no effect on cell migration on fibronectin. Control proteins, such as lysozyme (structurally homologous to alpha-LA) and bovine serum albumin, did not effect migration on either matrix. Second, the addition of competitive GalTase substrates significantly inhibited neural crest cell migration on basal lamina-like matrices, but as above, had no effect on migration on fibronectin. Comparable concentrations of inappropriate sugars also had no effect on cell migration. Third, addition of the GalTase catalytic substrate, UDPgalactose, produced a dose-dependent increase in the rate of cell migration. Under identical conditions, the inappropriate sugar nucleotide, UDPglucose, had no effect. Quantitative enzyme assays confirmed the presence of GalTase substrates in basal lamina matrices, their absence in fibronectin matrices, and the ability of alpha-LA to inhibit GalTase activity towards basal lamina substrates. Laminin was found to be a principle GalTase substrate in the basal lamina, and when tested in vitro, alpha-LA inhibited cell migration on laminin. Together, these experiments show that neural crest cells have at least two distinct mechanisms for interacting with the substrate during migration, one that is fibronectin-dependent and one that uses GalTase recognition of basal lamina glycoconjugates.     

Dominant negative mutation in cell surface beta 1,4- galactosyltransferase inhibits cell-cell and cell-matrix interactions

In addition to its traditional location within the Golgi complex, beta 1,4-galactosyltransferase (GalTase) is also present on the cell surface, where it is thought to function as a cell adhesion molecule by binding to extracellular oligosaccharide ligands. Recent studies suggest that cells contain two forms of GalTase with distinct cytoplasmic domains. The longer form of GalTase contains a 13-amino acid cytoplasmic extension and is preferentially targeted to the plasma membrane, relative to the shorter GalTase protein that is confined primarily to the Golgi compartment. In this study, we created a dominant negative mutation that interferes with the function of cell surface GalTase by transfecting into cells cDNAs encoding truncated versions of the long form of GalTase containing the complete cytoplasmic and transmembrane domains, but devoid of the catalytic domain. In both F9 embryonal carcinoma cells and Swiss 3T3 fibroblasts, overexpressing the truncated long GalTase (TLGT) protein displaced the endogenous cell surface GalTase from its association with the cytoskeleton, resulting in a loss of intercellular adhesion and cell spreading specifically on matrices that use GalTase as a cell surface receptor. In contrast, overexpressing the analogous truncated short GalTase (TSGT) protein did not affect cell morphology or GalTase activity. In control assays, inducing the TLGT protein had no effect on cell interactions with fibronectin (which is independent of GalTase), or on the cytoskeleton attachment of another matrix receptor (beta 1 integrin), or on overall glycoprotein synthesis, thus eliminating nonspecific effects of the TLGT protein on cellular adhesion and metabolism. These results represent the first molecular manipulation of cell surface GalTase expression and confirm its function as a cell adhesion molecule. These studies further suggest that the cytoskeleton contains a defined, saturable number of binding sites for GalTase, which enables it to function as an adhesion molecule.     

Laminin carbohydrates are implicated in cell signaling

We have examined how laminin carbohydrates participate in cellular responses and have focused upon cell spreading and neurite outgrowth. Our earlier studies showed that unglycosylated laminin fully supported cell adhesion but did not promote subsequent spreading of mouse melanoma cells or neurite outgrowth of rat pheochromocytoma cells (Dean et al. (1990): J Biol Chem 265:12553-12562). In the present experiments, we determined whether those cellular responses could be restored to adherent cells. When a mixture of unglycosylated and glycosylated laminins was used as a substratum for mouse melanoma cells, some cells began to spread when 30% glycosylated laminin was present. At least 65% glycosylated laminin was required to elicit a maximal spreading response by the majority of the cells. In separate experiments, we found that cell spreading was fully restored by a pronase digest of glycosylated laminin; a similar digest of unglycosylated laminin had no effect. These results indicate that laminin carbohydrates, rather than polypeptide sequences, were responsible for cell spreading. We also conclude that substrate attachment of the carbohydrate moieties was not essential. In other experiments, laminins containing immature oligosaccharides were produced using two glycosylation pathway inhibitors, swainsonine or castanospermine. When such laminins were used to study cell spreading or neurite outgrowth, laminin containing immature oligosaccharides was as effective as laminin which contains fully processed oligosaccharides. In contrast, laminin with partially processed oligosaccharides had incomplete activity. These composite reconstitution experiments show that laminin carbohydrates provide essential information to responsive cells, enabling them to progress from an adherent state to a spread form or to extend neurite processes.     

The role of murine cell surface galactosyltransferase in trophoblast: laminin interactions in vitro

Implantation of the mouse embryo involves the invasion of the secondary trophoblast giant cells of the ectoplacental cone (EPC) into the uterine decidua. The mechanisms of this event are poorly understood. The putative substrate molecules found in the decidua which could support trophoblast invasion include laminin, fibronectin, and collagen type IV. EPCs dissected from Day 7.5 embryos were cultured on all three matrices. Galactosyltransferase (GalTase) was localized by immunolabeling on trophoblast cell surfaces when grown on laminin but not the other matrices. Perturbations of the enzyme:substrate complex with alpha-lactalbumin, uridine diphosphogalactose, anti-GalTase, and pregalactosylation of the matrix did not affect rates of EPC attachment. However, decreased rates of migration or altered morphologies of spreading cells were observed. Laminin, and not fibronectin or collagen type IV, could be galactosylated with both exogenous GalTase or EPC outgrowths. Digests of galactosylated laminin produced a glycoconjugate substrate with a molecular weight of less than 10K. The results suggest that invasive secondary trophoblast cells possess a GalTase-mediated migration system that is functional on laminin.     

Laminin alpha1 chain LG4 module promotes cell attachment through syndecans and cell spreading through integrin alpha2beta1

The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.     

A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300-1000 micrograms/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300-1000 micrograms/ml), which mimics a binding domain of LM, and alpha-lactalbumin (1-10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and alpha-lactalbumin inhibited cell migration 50%. Antibodies against integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.     

Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY- D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine- containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N- glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin- sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.     

Myocilin binding to Hep II domain of fibronectin inhibits cell spreading and incorporation of paxillin into focal adhesions

Myocilin, a novel matricellular protein found in the human eye, can modify signaling events mediated by the Heparin II domain of fibronectin. Using myocilin produced in sf9 insect cells, myocilin inhibited spreading of cycloheximide-treated human skin fibroblasts plated on substrates co-coated with myocilin and either fibronectin or its Heparin II domain. Cell spreading could be rescued by adding back either substrate adsorbed or soluble Heparin II domains. Myocilin did not inhibit cell attachment to fibronectin even in the presence of a 2400 M excess of myocilin. Myocilin impaired focal adhesion formation and specifically blocked the incorporation of paxillin, but not vinculin, into focal adhesions. The Heparin II domain mediated the incorporation of paxillin into focal adhesions, since paxillin was not assembled into focal adhesions unless the Heparin II domain was present. The effect of myocilin on focal adhesions could be overcome by treating cells with either phorbol 12-myristate (PMA) or oleoyl-L-alpha-lysophosphatidic acid (LPA). Myocilin bound to the fibroblast cell surface, but its binding could not be competed with excess fibronectin, suggesting that myocilin does not compete for cell surface binding sites of fibronectin. Myocilin therefore appears to specifically block functions mediated by the Heparin II domain possibly through direct interactions with it.     

5'-Nucleotidase is involved in chick embryo myoblast spreading on laminin

Previous studies have shown that 5'-nucleotidase, an ectoenzyme from chicken gizzard, interacts specifically with laminin and fibronectin, two glycoproteins of the extracellular matrix. Recently, we demonstrated that 5'-nucleotidase was involved in the spreading of chick embryo fibroblast on laminin. In the present communication, we report that a monoclonal antibody (CG37) raised-directed against 5'-nucleotidase inhibited the spreading of chick embryo myoblasts on laminin after their initial attachment to the substrate. Furthermore, monoclonal antibody CG37 specifically eluted 5'-nucleotidase from immobilized laminin and thus enabled its isolation from other myoblast laminin-binding proteins.     

Analysis of cell surface galactosyltransferase activity during mouse trophectodermal differentiation

The ectoplacental cone (EPC) of the Day 7.5 mouse embryo consists of a core of adhesive, proliferating trophoblast cells which transform to invasive trophoblast giant cells during implantation. Adhesive trophoblast cell types express monoclonally defined lactosaminoglycans (LAGs) at the cell surface; transformation to giant cells results in a loss of LAG cell surface expression (H. J. Hathaway and B. S. Babiarz, 1988, Cell Differ. 24, 55-66). LAGs can serve as substrates for cell surface galactosyltransferase (GalTase), providing an adhesive mechanism between a number of different cell types (B. D. Shur, 1984, Mol. Cell. Biochem. 61, 143-158). It was hypothesized that the LAGs in the EPC represented a substrate for a similar GalTase-mediated cell:cell adhesion system. Cell surface GalTase activity was demonstrated on EPC trophoblast on Day 7.5 of development by the incorporation of galactose from exogenous radiolabeled substrate. In 24- to 48-hr EPC trophoblast cultures the enzyme was localized by immunofluorescence to areas of cell:cell contact. Monolayers of differentiated trophoblast giant cells lacked this labeling pattern. The cell surface glycopeptide substrate for GalTase eluted as a single peak with an apparent molecular mass of 15,000 Da. A portion of this material was sensitive to endo-beta-galactosidase digestion, indicating that it contained a LAG structure. Perturbation of the enzyme:substrate complex in 24- to 48-hr EPC outgrowths, with alpha-lactalbumin, uridine 5'-diphosphogalactose, or anti-GalTase antibody, resulted in the disruption of cell:cell contacts. Differentiation to trophoblast giant cells resulted in a loss of sensitivity to surface GalTase perturbation. The results suggest that adhesive EPC trophoblast cells possess a GalTase-mediated cell:cell adhesion system which is downregulated upon differentiation to invasive trophoblast giant cells.     

Improved attachment and spreading in primary cell cultures of the eastern oyster, Crassostrea virginica

At present, establishment of a cell line from bivalve molluscs has been unsuccessful, and in vitro work is limited to primary cell cultures. We sought to improve attachment and spreading of cells of the eastern oyster, Crassostrea virginica, to aid primary cultures and to assist development of a bivalve cell line. Our objectives were to examine the effects of substrate on ventricle cell viability, attachment, and spreading by testing of collagen I, collagen IV, fibronectin, laminin, poly-D-lysine, and two types of uncoated tissue culture plates (Falcon and Corning). Experiments were conducted by incubating cells with the various substrates for 24 h and 5 d. An assay with a tetrazolium compound (MTS) was used to estimate cell numbers based on metabolic activity. Although differences in MTS assay values for substrate effect on cell viability were detected at 24 h and at 5 d (P > 0.0001), these were attributed to variations in metabolic activity due to different levels of attachment and spreading among treatments. Differences among treatments were detected in attachment and spreading at 24 h and 5 d (for all, P > 0.0001). At 24 h, poly-D-lysine induced the highest levels of attachment and spreading; no other factor performed better than the uncoated Falcon substrate, and collagen I performed most poorly. At 5 d, poly-D-lysine and the uncoated Corning substrate induced significantly higher levels of attachment and spreading than did the uncoated Falcons substrate, and collagen I performed most poorly. From these results, poly-D-lysine best promoted cell attachment and spreading. Fibronectin (at 24 h) and laminin (at 5 d) warrant further study. Along with improvements in medium composition, future work should involve screening of other attachment factors and combinations of factors, including those of bivalve origin.     

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.