Characterization of a cell surface glycoprotein associated with maturation of rat spermatozoa.
The principal galactose oxidase/NaB[3H]4-labeled membrane protein of rat caudal epididymal spermatozoa was isolated by hydrophobic interaction chromatography. The protein is released from the membrane by the action of phosphatidylinositol specific phospholipase C, and thereby its properties are transformed from those of a protein anchored to the hydrophobic membrane to those of a hydrophilic solution protein. Because it is the only membrane-associated protein released by the enzyme which did not absorb to a propylaspartate resin, a simple, single step purification procedure was devised. Although the amino terminus of the protein is blocked to Edman degradation, the majority of the protein structure was determined from a series of tryptic peptides and from limited acid hydrolysis. Approximately 65% of the protein mass is carbohydrate which is primarily attached through O-glycosidic bonds to the 18 threonines. The molecular weight of the glycoprotein was estimated to be 16,600, considerably smaller than the M(r) = 26,000 to 37,000 previously determined by gel electrophoresis. The anomalous electrophoretic behavior is undoubtedly due to the large percentage of carbohydrate. The distribution of carbohydrate on the protein side chains suggests the protein may form a positively charged, specialized scaffolding for the presentation of the carbohydrate moieties. Because the appearance of the ability to label the protein with galactose oxidase is correlated with sperm maturation in the epididymis, the glycoprotein structures may be an important component in the fertilization process. The combination of linkage by glycosylphosphatidylinositol and low molecular weight mucin-like structure indicates this may be a member of a new class of membrane proteins.
Eccleston
E D
ED
Department of Biochemistry, University of Minnesota, Minneapolis.
White
T W
TW
Howard
J B
JB
Hamilton
D W
DW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D016601:RNA-Binding Proteins; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013079:Sperm Maturation; D013094:Spermatozoa / Q000378:metabolism / Q000502:physiology*; D000097796:Nucleolin
D000596:Amino Acids; D002241:Carbohydrates; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C086330:SMemG protein, rat