MUTANTS OF HUMAN CHORIOGONADOTROPIN LACKING N-GLYCOSYL CHAINS IN THE ALPHA-SUBUNIT .1. MECHANISM FOR THE DIFFERENTIAL ACTION OF THE N-LINKED CARBOHYDRATES

Analogs of human choriogonadotropin (hCG) lacking N-glycosyl chains at alpha 52Asn and alpha 78Asn were purified from the culture media of i nsect cells by immunoaffinity chromatography using a monoclonal antibo dy column. As previously reported, while analogs lacking carbohydrate at alpha 52Asn and alpha 78Asn had similar receptor binding activities compared with the wild type recombinant hCG (hCGwt), they differed in their signal transduction properties. The mutant lacking carbohydrate at alpha 78Asn had 20% less cAMP-stimulating activity than hCGwt, but the absence of glycosylation at alpha 52Asn resulted in the reduction of cAMP accumulation by 90-95%. A similar effect of the mutations was observed on the stimulation of steroidogenesis. Circular dichroism sp ectra of the two mutants showed significant differences. The mutant la cking carbohydrate at alpha 52Asn had a much higher negative mean resi due ellipticity (MRE) at 200 nm and a lower negative MRE at 220 nm tha n that lacking carbohydrate at alpha 78Asn and hCGwt. The dissociation rates of the alpha 52Asn and alpha 78Asn carbohydrate deficient mutan ts at pH 3 and room temperature, measured by using 1-anilino-8-naphtha lenesulfonate, were 9.4 x 10(-5) and 3.8 x 10(-5) s(-1), respectively, as compared with 1.5 x 10(-5) s(-1) for hCGwt. The results of both CD measurements and dissociation studies strongly suggest that the absen ce of carbohydrate at alpha 52Asn results in conformational changes in the mutant which might explain the loss in its signal transduction fu nction. This is further supported by indirect evidence from two other lines of experimentation. Unlike the mutant lacking carbohydrate at al pha 78Asn, the one lacking carbohydrate at alpha 52Asn cross-reacted w ith the two subunit specific monoclonal antibodies, anti-hCG alpha and anti-hCG beta, which normally did not cross-react With the native or the hCGwt. Also, polyclonal anti-hCG beta but not anti-hCG alpha was a ble to restore the cAMP-producing activity of the alpha 52Asn carbohyd rate deficient mutant. From all the data taken together, it appears th at the loss of second messenger-producing activity of hCG with the abs ence of the glycosyl chain at alpha 52Asn was probably due to a confor mational change in the heterodimer rather than due to the loss of the alpha 52Asn-carbohydrate-receptor interaction.