Incompletely processed LH molecules synthesized by rat gonadotrophs treated with inhibitors of oligosaccharide processing.
Inhibitors of N-linked oligosaccharide processing are useful tools for studies on the biological function of the oligosaccharide structures in glycoprotein hormones. We have synthesized molecules of lutropin (LH) containing high-mannose- and hybrid-type oligosaccharides using rat gonadotroph-enriched primary cultures in the presence of castanospermine (a glucosidase I inhibitor) or swainsonine (a mannosidase II inhibitor), in order to compare the actions of these molecules with that of the hormone containing complex-type oligosaccharides in the activation of the receptor-adenylate cyclase system. Treatment of gonadotrophs with the above inhibitors caused an increase in the synthesis of highly basic LH molecules (pI 9.6-10.0), because addition of charged carbohydrate moieties to these molecules was prevented. Characterization of the oligosaccharide structure performed by enzymatic treatment (endoglycosidase H and neuraminidase) and the use of immobilized lectins (wheat germ agglutinin and Ricinus communis agglutinin-120) showed that these inhibitor-synthesized LH molecules contained high-mannose- and hybrid-type (asialo and sialylated) oligosaccharides. Their immunological properties were similar to that of complex-type oligosaccharide LH, but they had significantly higher receptor-binding ability in comparison with a sialylated complex-type oligosaccharide LH (about 12-fold) and an asialo complex-type oligosaccharide LH (about 3-fold). It was noted that the incompletely processed molecules were less potent than complex-type oligosaccharide LH in the activation of adenylate cyclase of Leydig cells, showing about 40-60% of the activity induced by the sialylated complex-type oligosaccharide molecule. The present data indicate that the inhibition of terminal processing of N-linked oligosaccharides by castanospermine and swainsonine impairs the full hormonal function of rat LH.
Hattori
M
M
Institute of Endocrinology, Gunma University, Maebashi, Japan.
Wakabayashi
K
K
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors*; D006031:Glycosylation; D007212:Indolizines; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D008297:Male; D009439:Neuraminidase; D009844:Oligosaccharides / Q000378:metabolism*; D009919:Orchiectomy; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D011967:Receptors, Gonadotropin / Q000187:drug effects / Q000378:metabolism; D017026:Swainsonine; D054836:Indolizidines
D000470:Alkaloids; D006063:Chorionic Gonadotropin; D000242:Cyclic AMP; D006026:Glycoside Hydrolases; D007212:Indolizines; D007986:Luteinizing Hormone; D009439:Neuraminidase; D009844:Oligosaccharides; D011967:Receptors, Gonadotropin; D017026:Swainsonine; C037806:castanospermine; D054836:Indolizidines