Glycolipid linkage of a polyspermy blocking glycosidase to the ascidian egg surface.
Ascidian eggs release N-acetylglucosaminidase rapidly into the seawater following fertilization. This glycosidase is detected seconds after fertilization, and histochemical tests suggest the cell surface as the prefertilization storage site (Lambert, C. C. (1989). Development 105, 415-420). Living eggs of Ascidia ceratodes, A. callosa, and A. paratropa all cleave a fluorogenic substrate in seawater. Following cell surface biotinylation and activation of the eggs, enzyme activity binds to streptavidin further substantiating the cell surface localization. The released glycosidase has a molecular weight of 180 kDa by size exclusion chromatography and exhibits bands at 62 and 70 kDa by SDS-PAGE, suggesting a possibly multimeric enzyme. The enzyme is released by a glycophosphatidylinositol-specific phospholipase C and HNO2 deamination, both of which are specific indicators of linkage to the cell surface via phosphatidylinositol. The enzyme from unfertilized eggs is quite hydrophobic in Triton X-114 phase partition experiments but becomes hydrophyllic after release by activation or deamination. All of these observations are consistent with the glycosidase being anchored to the cell surface via a GPI anchor that is cleaved at fertilization to yield the soluble form of the enzyme which helps protect the egg against polyspermy. We discuss the possible role of a cell surface PLC in this release.
Lambert
C C
CC
Department of Biology, California State University, Fullerton 92634.
Goode
C A
CA
D000818:Animals; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D005306:Fertilization; D006026:Glycoside Hydrolases / Q000737:chemistry*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D008970:Molecular Weight; D019809:Streptavidin; D010738:Type C Phospholipases; D014561:Urochordata / Q000378:metabolism*
D001426:Bacterial Proteins; D017261:Glycosylphosphatidylinositols; D019809:Streptavidin; D010738:Type C Phospholipases; D006026:Glycoside Hydrolases