Carbohydrate moieties of small placental hCG: requirement of mannose structure for biological activity.
The 43 kDa human chorionic gonadotropin (hCG) (SP-hCG) was purified from human placenta and analyzed for sugar moieties. The low hexosamine content suggests that SP-hCG probably lacks O-linked sugar chains in the beta-subunit and incompletely formed N-linked sugar chains in the alpha- and beta-subunits. In the present study SP-hCG was hydrolyzed with various glycosidases. Treatment of hCG or SP-hCG with O-glycan peptide hydrolase increased the mobility of asialo-hCG beta in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) while that of SP-hCG beta was unaffected, indicating that SP-hCG beta does not contain NeuNAc-Gal-GalNAc unit. Alpha-Mannosidase and endoglycosidase H hydrolyzed mannose and the high mannose-GlcNAc moieties, respectively, from alpha- and beta-subunits of SP-hCG, but not from the subunits of authentic hCG. Glycopeptidase F hydrolyzed completely the N-linked sugar chains from SP-hCG subunits, producing alpha- and beta-subunits with estimated Mr of 15,000 and 18,500, respectively. The biological activity of purified SP-hCG is about 50-80% of highly purified authentic hCG. In an in vitro system SP-hCG increased cAMP accumulation and testosterone production by rat Leydig cells to the same levels as that induced by hCG. However, the biological activity of SP-hCG was markedly reduced, following treatment with endoglycosidase H or alpha-mannosidase. To attain the level of testosterone production equivalent to that induced with untreated SP-hCG, 10-20 times higher dose of treated SP-hCG was required. On the other hand, cAMP accumulation induced with treated SP-hCG even at a very high concentration was substantially lower than that attained with untreated SP-hCG. In conclusion, the mannose moieties are essential structural components of the hormone in stimulating cAMP accumulation and steroidogenesis by rat Leydig cells.
Wang
H Y
HY
Center for Biomedical Research, Population Council, New York, NY 10021.
Segal
S J
SJ
Koide
S S
SS
D000818:Animals; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis*; D006063:Chorionic Gonadotropin / Q000032:analysis* / Q000378:metabolism; D005260:Female; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D008297:Male; D008358:Mannose / Q000032:analysis* / Q000378:metabolism; D010920:Placenta / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D006026:Glycoside Hydrolases; D008358:Mannose