Localization and characterization of blastocoelic extracellular matrix antigens in early sea urchin embryos and evidence for their proteolytic modification during gastrulation.
Previously, results were presented showing a spatiotemporal expression of matrix metalloproteases consistent with a role in remodeling the blastocoelic extracellular matrix (bECM) of the gastrulating sea urchin embryo [35]. In the present work, we provide evidence suggesting that the bECM is in fact the substrate for developmentally regulated proteolysis. Monoclonal antibody (mAb) LG11C7 was generated against testicular tissue of the sea urchin, Strongylocentrotus purpuratus, and recognizes extracellular matrix antigens overlying the perivisceral epithelium. Indirect immunofluorescence microscopy shows that mAb LG11C7 cross-reacts with components of the basal lamina lining the blastocoeles of early embryos and Western immunoblots of detergent extracts indicate that it recognizes gastrula-stage antigens with M(r)s of 158, 68, and 37 kDa. Glycosidase treatments reveal that the embryonal antigens contain multiple N-linked oligosaccharides. Developmental studies employing immunoprecipitations and Western blot analyses of staged embryonal detergent extracts show that the 68-kDa antigen appears between 18 and 24 h after fertilization and is accompanied by a substantial increase in the 37-kDa antigen. Thus, the appearances of the 68- and 37-kDa antigens occur during the blastula-gastrula transition, and their spatiotemporal expression is similar to that of the matrix metalloproteases reported previously. The appearance of the 68-kDa antigen and the increase in the 37-kDa antigen may be blocked by exposing the embryos to the metalloprotease inhibitor 1,10-phenanthroline, which also blocks gastrulation reversibly. These results suggest (1) that the 68- and 37-kDa antigens are products of developmentally regulated proteolysis of a basal laminar glycoprotein, and (2) that this proteolysis is required for the cell-cell/cell-matrix interactions and morphogenetic movements associated with normal gastrulation in the sea urchin embryo.
Vafa
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Department of Biology, Georgetown University, Washington, DC 20057, USA.
Goetzl
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Poccia
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Nishioka
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D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000941:Antigens / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins / Q000276:immunology* / Q000378:metabolism; D005260:Female; D005775:Gastrula / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D008297:Male; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012617:Sea Urchins / Q000196:embryology / Q000502:physiology*; D013737:Testis / Q000276:immunology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000941:Antigens; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D010618:Phenanthrolines; D011480:Protease Inhibitors; D006026:Glycoside Hydrolases; C025205:1,10-phenanthroline