Deglycosylation of gonadotropins with an endoglycosidase.
A commercially available endoglycosidase (N-glycanase, Genzyme, Boston, Mass.) purified from Flavobacterium meningosepticum with a specificity for cleaving asparagine-linked carbohydrate moieties in glycoproteins was tested on several pituitary and chorionic gonadotropins as substrates. All intact hormones tested were resistant to the action of the enzyme as were all beta subunits from the respective gonadotropins. All alpha subunits, however, were susceptible to the enzyme as evidenced by a decrease in molecular size when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparative experiments with ovine luteinizing hormone subunit (oLH alpha) indicated that only 35-40% of the carbohydrate was removed after N-glycanase treatment, suggesting that perhaps only one of the two carbohydrate moieties was cleavable under the conditions employed. The enzyme-modified subunit (DG-oLH alpha) was able to recombine with untreated oLH beta. An in vitro steroidogenic bioassay (rat Leydig cell) showed that the recombinant (DG-oLH alpha-oLH beta) was about 22% as potent as the native oLH, but in a testicular membrane binding assay for LH, it was equal in potency to the native hormone in competing with the radioligand.
Swedlow
J R
JR
Matteri
R L
RL
Papkoff
H
H
D000818:Animals; D001681:Biological Assay; D002241:Carbohydrates; D006063:Chorionic Gonadotropin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000494:pharmacology*; D006062:Gonadotropins / Q000378:metabolism*; D006064:Gonadotropins, Equine; D006801:Humans; D007986:Luteinizing Hormone / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D013552:Swine
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D006062:Gonadotropins; D006064:Gonadotropins, Equine; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase