CHARACTERIZATION OF A CELL-SURFACE GLYCOPROTEIN ASSOCIATED WITH MATURATION OF RAT SPERMATOZOA

The principal galactose oxidase/NaB[H-3](4)-labeled membrane protein o f rat caudal epididymal spermatozoa was isolated by hydrophobic intera ction 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. Beca use 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 th e protein is blocked to Edman degradation, the majority of the protein structure was determined from a series of tryptic peptides and from l imited acid hydrolysis. Approximately 65% of the protein mass is carbo hydrate 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 pr eviously determined by gel electrophoresis. The anomalous electrophore tic behavior is undoubtedly due to the large percentage of carbohydrat e. The distribution of carbohydrate on the protein side chains suggest s the protein may form a positively charged, specialized scaffolding f or the presentation of the carbohydrate moieties. Because the appearan ce of the ability to label the protein with galactose oxidase is corre lated with sperm maturation in the epididymis, the glycoprotein struct ures may be an important component in the fertilization process. The c ombination of linkage by glycosylphosphatidylinositol and low molecula r weight mucin-like structure indicates this may be a member of a new class of membrane proteins. (C) 1994 Wiley-Liss, Inc.