ABSTRACT
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.
Subject(s)
Cell Adhesion/physiology , Fibroblasts/physiology , Laminin/physiology , Membrane Glycoproteins/physiology , Receptors, Concanavalin A/physiology , Animals , Binding, Competitive , Chick Embryo , Fibroblasts/chemistry , Fibronectins/physiology , In Vitro Techniques , Membrane Glycoproteins/isolation & purification , Membrane Glycoproteins/metabolism , Molecular Weight , Receptors, Concanavalin A/isolation & purification , Receptors, Concanavalin A/metabolismABSTRACT
In vitro assays for the beta-N-acetylglucosaminyltransferases (GlcNAcTase) were performed on crude microsomal fractions prepared from 8-day chick embryo fibroblasts (8-day-CEF) and 16-day chick embryo fibroblasts (16-day-CEF) using [3H]mannose-labeled GlcNAc beta 1----2 Man alpha 1----6 (GlcNAc beta 1----2 Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (Fuc alpha 1----6) GlcNAc-Asn and UDP-GlcNAc as substrates. 8-day-CEF synthesize preferentially triantennary complex type chains, whereas 16-day-CEF produce essentially tetraantennary complex type chains. Furthermore oligosaccharides containing the GlcNAc beta 1----4 Man alpha 1----3 Man sequence represent 90% of the structures found in 16-day-CEF versus 30% in 8-day-CEF, indicating an increase in beta-N-acetylglucosaminyltransferase IV activity during embryo development.
Subject(s)
Chick Embryo/growth & development , Glucosyltransferases/metabolism , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Cells, Cultured , Chick Embryo/enzymology , Microsomes/enzymology , Molecular Sequence Data , Time FactorsABSTRACT
Concanavalin A (Con A), a tetravalent lectin, was shown to impair 8 chick embryo fibroblast (8 d CEF) spreading on a laminin (LM) substrate but not on a fibronectin substrate (FN), suggesting that cell surface Con A binding proteins could be involved in 8 d CEF spreading on a LM substrate. The interaction of Con A-binding proteins with Con A is dependent upon the carbohydrate moieties of the isolated glycoproteins; since they interact strongly with Con A-Sepharose and are eluted with 0.3 Mol/l alpha-methylmannopyranoside, the isolated Con A binding-proteins inhibit 8 d CEF adhesion to a Con A substrate to the same extent as alpha-methylmannopyranoside. Furthermore, the isolated Con A binding proteins specifically inhibit in a dose-dependent manner 8 d CEF spreading on LM but not on FN.
Subject(s)
Fibroblasts/cytology , Laminin , Receptors, Concanavalin A/physiology , Animals , Binding Sites , Cell Adhesion/drug effects , Chick Embryo , Concanavalin A/pharmacology , Fibroblasts/drug effects , Glycoproteins/physiology , Methylmannosides/pharmacology , Receptors, Concanavalin A/pharmacologyABSTRACT
Eight d (8d) and 16d (16d) chick embryo fibroblasts (CEF) exhibited marked differences in their adhesive capacity on plastic support, but not on fibronectin substratum. This suggests differences in fibronectin (FN) expression and/or FN receptor expression. Both 8d and 16d CEF expressed an identical number of membrane receptors for FN with similar affinity. In contrast, the newly synthesized FN appeared de novo in 30 min in 8d CEF versus 60 min in 16d CEF. This difference is not due to a modification of the polypeptide chain biosynthetic rate. The FN synthesized in 8d CEF became insensitive to endo beta-N-acetyl-glucosaminidase H (endo H) treatment after 20 min, whereas it remained sensitive to endo H until 60 min in 16d CEF. Post-translational modifications of N-linked mannose-rich chains to complex type chain may account for the difference in the expression of cell surface FN and thus for the difference in cell adhesion capacity to plastic.
