Identification of twelve O-glycosylation sites in equine chorionic gonadotropin β and equine luteinizing hormone β by solid-phase Edman degradation

The O-glycosylation sites for equine LH beta (eLH beta) and eCG beta were identified by solid-phase Edman degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4-6 sites anticipated, These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCG beta and from 10% to 100% for eLH beta. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLH beta or eCG beta, hybrid hormones could be obtained by reassociating eLH alpha, eFSH alpha, or eCG alpha with the truncated beta subunit derivatives. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLH beta or des(121-149)eCG beta were combined with the same alpha subunit preparation, Thus, O-glycosylation appears to be responsible for the beta subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG, Comparison of the equid LH/CG beta sequences with those available for the primate CG beta subunits indicated a greater conservation of glycosylation patterns in the former.