NEUROENDOCRINOLOGY AND BIOLOGY OF THE PITUITARY GONADOTROPINS
7:30 AM-10:00 AM
(514) ENZYMATIC REMOVAL OF ASPARAGINE-LINKED CARBOHYDRATES FROM DIMER hCG AND RESULTANT BIOACTIVITY.
Richard, Craig1, DeLoia, J1, 1 Dept of Obstetrics, Gynecology and Reproductive Sciences, Pittsburgh, PA
ABSTRACT- Human chorionic gonadotropin (hCG) is best known for its critical role in the establishment and maintenance of early pregnancy. The bioactivity of hCG has been attributed to the asparagine-linked carbohydrates on each subunit of hCG, especially the carbohydrate at asparagine 52 on the alpha subunit (asp52). Specific removal of the asparagine-linked carbohydrates from dimer hCG has only been done by site-directed mutagenesis. We therefore utilized endoglycosidases to specifically remove asparagine-linked carbohydrates and examine the resultant bioactivity of the dimer. Different sources of dimer hCG were incubated with various endoglycosidases. HCG was subsequently run on an SDS PAGE gel, proteins were stained and the extent of deglycosylation was evaluated. HCG bioactivity was evaluated on human granulosa-luteal cells in culture. Endoglycosidases were able to cleave asparagine-linked carbohydrates from dimer hCG. The specific removal of asp52 did not alter the bioactivity of dimer hCG. The removal of 70% of all the N-linked carbohydrates from dimer hCG also did not alter the bioactivity of the dimer. The results of these studies demonstrate that the asparagine-linked carbohydrates may not be involved directly in the receptor signaling of hCG.
KEY WORDS: human chorionic gonadotropin, bioactivity, carbohydrates, endoglycosidases