The sea urchin egg receptor for sperm: isolation and characterization of the intact, biologically active receptor.
The species-specific binding of sea urchin sperm to the egg is mediated by an egg cell surface receptor. Although earlier studies have resulted in the cloning and sequencing of the receptor, structure/function studies require knowledge of the structure of the mature cell surface protein. In this study, we report the purification of this glycoprotein to homogeneity from a cell surface complex of Strongylocentrotus purpuratus eggs using lectin and ion exchange chromatography. Based on the yield of receptor it can be calculated that each egg contains approximately 1.25 x 10(6) receptor molecules on its surface. The receptor, which has an apparent M(r) of 350 kD, is a highly glycosylated transmembrane protein composed of approximately 70% carbohydrate. Because earlier studies on the partially purified receptor and on a pure, extracellular fragment of the receptor indicated that the carbohydrate chains were important in sperm binding, we undertook compositional analysis of the carbohydrate in the intact receptor. These analyses and lectin binding studies revealed that the oligosaccharide chains of the receptor are sulfated and that both N- and O-linked chains are present. Functional analyses revealed that the purified receptor retained biological activity; it inhibited fertilization in a species-specific and dose-dependent manner, and polystyrene beads coated with it bound to acrosome-reacted sperm in a species-specific manner. The availability of biochemical quantities of this novel cell recognition molecule opens new avenues to studying the interaction of complementary cell surface ligands in fertilization.
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794-5215.
K
K
Ohlendieck
S T
ST
Dhume
J S
JS
Partin
W J
WJ
Lennarz
D000177:Acrosome / Q000378:metabolism; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D005260:Female; D005306:Fertilization; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification*; D012617:Sea Urchins; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism* / Q000648:ultrastructure
D002241:Carbohydrates; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C035027:egg surface sperm receptor