Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme.
Previous studies from this laboratory using p-nitrophenyl alpha-D-mannoside (p-NPM) as substrate provided no evidence for the presence of mannosidase II in the rat epididymis [Skudlarek & Orgebin-Crist (1988) J. Reprod. Fertil. 84, 611-617]. However, rat epididymal epithelial cells cultured in the presence of swainsonine, an inhibitor of mannosidase II, produce abnormally processed N-linked glycoproteins containing hybrid-type oligosaccharides instead of complex-type [Tulsiani, Skudlarek & Orgebin-Crist (1990) Biol. Reprod. 43, 130-138], a result providing indirect evidence for the presence of mannosidase II-like enzyme in rat epididymis. In the studies described here, we present evidence for the occurrence of this processing enzyme in rat epididymal Golgi membranes. This enzyme is an integral Golgi membrane component. Like liver mannosidase II, the epididymal enzyme cleaves alpha 1,3- and alpha 1,6-linked mannosyl residues from GlcNAcMan5GlcNAc. However, unlike liver mannosidase II, the epididymal enzyme shows no activity towards the synthetic substrate, p-NPM. The epididymal mannosidase cross-reacts with liver anti-(mannosidase II) antibody, a result suggesting that the two enzymes share a common antigenic site(s). Immunoblotting studies following resolution of liver and epididymal Golgi membranes on SDS/PAGE show that, whereas the liver mannosidase II was resolved as a doublet of Mr 120,000 and 122,000, only the Mr 120,000 band was observed in the epididymal Golgi membranes. Immunoblotting of the Golgi-rich fractions, resolved under non-denaturing conditions, showed different patterns of charge and/or size isomers from the two tissues. These studies demonstrate tissue-specific differences in processing enzymes with similar function.
Skudlarek
M D
MD
Center for Reproductive Biology Research, Department of Obstetrics and Gynecology, Vanderbilt School of Medicine, Nashville, TN 37232-2633.
Orgebin-Crist
M C
MC
Tulsiani
D R
DR
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D000936:Antigen-Antibody Complex; D001216:Asparagine; D002413:Cations, Divalent; D002458:Cell Fractionation; D004822:Epididymis / Q000201:enzymology*; D006023:Glycoproteins / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D017026:Swainsonine
D000470:Alkaloids; D000936:Antigen-Antibody Complex; D002413:Cations, Divalent; D006023:Glycoproteins; D001216:Asparagine; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D017026:Swainsonine