Differential effects of alpha subunit Asparagine56 oligosaccharide structure on equine lutropin and follitropin hybrid conformation and receptor-binding activity

Abstract

The gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG), are cysteine-knot growth-factor superfamily glycoproteins composed of a common alpha subunit noncovalently associated with a hormone-specific beta subunit. The cysteine-knot motifs in both subunits create two hairpin loops, designated L1 and L3, on one side of the knot, with the intervening long loop, L2, on the opposite side. As the average alpha-subunit loop 2 oligosaccharide mass increased from 1482 to 2327, LH and FSH receptor-binding affinities of the dual-specificity eLH declined significantly, while the decrease in FSH receptor-binding affinity for eFSH was not significant. In the present study, we characterized hormone-specific glycosylation of alphaL2 oligosaccharides in eLHalpha, eFSHalpha, and eCGalpha preparations. MALDI mass spectrometry revealed 28-57 structures, including high mannose, hybrid, bi-, and triantennary oligosaccharides. The same intact subunit preparations and their alphaL2 loop-deglycosylated derivatives were combined with either eLHbeta or eFSHbeta, and the circular dichroism (CD) spectrum for each preparation was determined. We predicted that hybrid hormone preparations obtained by combining intact eLHalpha, eFSHalpha, and eCGalpha preparations with eLHbeta might exhibit differences in conformation that would disappear when the alphaL2 oligosaccharide attached to alphaAsn(56) was removed by selective peptide-N-glycanase digestion (N(56)dg-alpha). CD data supported the first prediction; however, elimination of alphaL2 oligosaccharide actually increased the conformational differences. The intact alpha subunit:eFSHbeta hybrids had virtually identical CD spectra, as expected. However, the N(56)dg-alpha:eFSHbeta hybrid spectra differed from each other. Oligosaccharide removal altered the conformation of most hybrids, suggesting that alphaAsn(82) oligosaccharide (located in alphaL3) also influenced gonadotropin conformation.