Enzymatic removal of asparagine-linked carbohydrate chains from heterodimer human chorionic gonadotrophin and effect on bioactivity.
The native form of human chorionic gonadotropin (hCG) is a heterodimer protein with two asparagine (Asn)-linked carbohydrate chains on each subunit. Removal of the Asn-linked carbohydrate chains from hCG has resulted in hCG variants with consistent antagonistic properties on isolated murine cells. Specific and direct enzymatic removal of these carbohydrate chains from native hCG with resultant antagonistic properties has not been reported. An antagonist to the hCG/luteinising hormone (LH) receptor could be used as an anticancer therapy, emergency contraceptive or for therapeutic resolution of ectopic pregnancies. Therefore, our aim was to use enzymes to specifically remove Asn-linked carbohydrate chains from hCG in the heterodimer form and analyse the resultant bioactivity. Native hCG was treated with endoglycosidases, carbohydrate removal was analysed with electrophoresis and the hCG variants were tested for altered bioactivity with human and murine cells. Endoglycosidases were able to cleave most of the Asn-linked carbohydrate chains from the native hCG. The deglycosylated hCG demonstrated a 75% reduction in bioactivity on a murine Leydig cell line and a 65% reduction in bioactivity on human granulosa cells. These results exemplify a simple and efficient method for creating deglycosylated hCG and provide the most direct evidence for the importance of Asn-linked carbohydrate chains in maintaining hCG bioactivity.
Richard
Craig A H
CA
Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine and Magee-Women's Research Institute, Pittsburgh, PA 15213, USA. crichard@su.edu
Creinin
Mitchell D
MD
Kubik
Carolyn J
CJ
DeLoia
Julie A
JA
D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D021122:Protein Subunits; D013379:Substrate Specificity
D006063:Chorionic Gonadotropin; C034923:HCG, deglycosylated; D021122:Protein Subunits; D011374:Progesterone; D001216:Asparagine; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase