Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase.
During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.
Chayko
C A
CA
Center for Reproductive Biology Research, Department of Obstetrics & Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633, USA.
Orgebin-Crist
M C
MC
Skudlarek
M D
MD
Tulsiani
D R
DR
D000818:Animals; D004822:Epididymis / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D013087:Spermatids / Q000201:enzymology; D013094:Spermatozoa / Q000201:enzymology* / Q000648:ultrastructure*; D013737:Testis / Q000166:cytology; D001616:beta-Galactosidase / Q000032:analysis* / Q000096:biosynthesis* / Q000378:metabolism
D001616:beta-Galactosidase