In vitro biological activities of echinonectin.
Echinonectin (EN) is a 230-kDa extracellular matrix glycoprotein found in the hyaline layer of sea urchin embryos. Dissociated embryonic cells attached strongly to EN-coated microtiter wells in a centrifugal-based in vitro adhesion assay, suggesting that EN is one of the hyaline layer proteins to which cells adhere in vivo (Alliegro et al., 1988). The present study examines the molecular properties of that adhesion using monoclonal antibodies as probes to block cell attachment, and also demonstrates that EN possesses lectin activity. EN binds tenaciously to agarose-based chromatography resins, such as Sepharose. The sugar-binding activity is associated with the polypeptide component of EN, and not with the carbohydrate moiety. Binding is inhibited with galactose and fucoidan, but not with glucose or locust bean gum. Although functional sites both for polysaccharide binding and for cell attachment are present on each subunit of the EN molecule, the sites appear to be functionally distinct because galactose and fucoidan are completely without effect on cell attachment in vitro. Proteolytic digestion of EN yields a highly limited set of immunoreactive peptides. Digestion with trypsin yields a 20-kDa fragment, chymotrypsin, a doublet at 20 kDa, and 20- and 23-kDa fragments with thermolysin. McAb's directed against these peptides block cell adhesion in vitro, suggesting that they possess the cell attachment domain of EN. This is supported by the observations that trypsin-digested EN is an effective substrate in adhesion assays and that adhesion to the tryptic fragments is also blocked by McAb's to the 20-kDa domain.
Alliegro
M C
MC
Department of Zoology, Duke University, Durham, North Carolina 27706.
Burdsal
C A
CA
McClay
D R
DR
D000818:Animals; D000911:Antibodies, Monoclonal; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins / Q000378:metabolism*; D037102:Lectins; D011134:Polysaccharides / Q000378:metabolism; D011489:Protein Denaturation; D012617:Sea Urchins
D000911:Antibodies, Monoclonal; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D037102:Lectins; D011134:Polysaccharides; C057966:echinonectin protein, Lytechinus variegatus