Enzymatic removal of asparagine-linked carbohydrate chains from heterodimer human chorionic gonadotrophin and effect on bioactivity
Craig A. H. Richard A C D , Mitchell D. Creinin A , Carolyn J. Kubik B and Julie A. DeLoia AA Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine and Magee-Women’s Research Institute, Pittsburgh, PA 15213, USA.
B Reproductive Health Specialists Inc., 665 Rodi Road, Pittsburgh, PA 15235, USA.
C Present address: Department of Biopharmaceutical Sciences, Shenandoah University, School of Pharmacy, 1775 North Sector Court, Winchester, VA 22601, USA.
D Corresponding author. Email: crichard@su.edu
Reproduction, Fertility and Development 19(8) 933-946 https://doi.org/10.1071/RD07077
Submitted: 9 May 2007 Accepted: 6 August 2007 Published: 13 September 2007
Abstract
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.
Additional keywords: deglycosylation, glycosylation, MA-10 cells.
Acknowledgements
The authors thank Dr Kaoru Takegawa (Kagawa University, Miki-cho, Kagawa, Japan) for kindly supplying endoglycosidase A and Mario Ascoli (University of Iowa, Iowa City, IA, USA) for the generous provision of MA-10 cells. This work was supported by The Magee-Women’s Research Institute Fellowship, University of Pittsburgh (Pittsburgh, PA, USA), The Lalor Foundation (Boston, MA, USA) and Family Health International (Research Triangle Park, NC, USA).
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