N-linked oligosaccharides on free alpha interfere with its ability to combine with human chorionic gonadotropin-beta subunit.
The purpose of the study was to examine the role of N-linked oligosaccharides in preventing combination of free alpha molecules with human chorionic gonadotropin (hCG)-beta subunit to form the intact hormone, hCG. Culture media from JEG cells incubated in the presence or absence of Swainsonine were filtered on Sephadex G-100, and free alpha was identified by radioimmunoassay. Swainsonine interferes with glycosylation by inhibiting alpha-mannosidase II, resulting in formation of hybrid structures. Approximately 50% of the free alpha molecules from Swainsonine-treated cells (Swainsonine pool 2) had an apparent molecular size that was smaller than that of free alpha from control cells. The oligosaccharides of control alpha molecules were resistant to endo-beta-N-acetylglucosaminidase H treatment. In contrast, virtually all of the Swainsonine free alpha molecules contained endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides. Swainsonine free alpha and control free alpha molecules were incubated with an excess of hCG-beta subunit, followed by chromatography on Sephadex G-100. Each fraction was assayed by radioimmunoassay for intact hCG and for alpha. Less than 10% of control free alpha molecules combined with hCG-beta. In contrast, 54% of Swainsonine alpha pool 2 and 40% of Swainsonine alpha pool 1 combined with beta to form hCG. Thus, modulation of N-linked oligosaccharide processing converted free alpha molecules to forms that can combine with hCG-beta. These results indicate that the inability of a substantial portion of control free alpha molecules to combine with hCG-beta is due to the presence of N-linked oligosaccharide structures that interfere with combination.
Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
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D000470:Alkaloids / Q000494:pharmacology; D015153:Blotting, Western; D002241:Carbohydrates / Q000032:analysis; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D003208:Concanavalin A / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins; D006031:Glycosylation / Q000187:drug effects; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D046911:Macromolecular Substances; D011485:Protein Binding; D013329:Structure-Activity Relationship; D017026:Swainsonine
D000470:Alkaloids; D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D015292:Glycoprotein Hormones, alpha Subunit; D006023:Glycoproteins; D046911:Macromolecular Substances; D003208:Concanavalin A; D006596:Hexosaminidases; D017026:Swainsonine