The existence of at least six mammalian GalNAc-transferases makes it unlikely that O-glycosylation can be predicted with the same level of confidence as N-glycosylation. Unlike N-glycosylation, which occurs at the consensus motif Asn-Xaa-Thr/Ser (Xaa is any amino acid other than proline), several O-glycosylation motifs have been suggested. It is not known whether this resulted from glycosylation of more of the five threonine (Thr) or 11 serine (Ser) residues located in the C-terminal extension, a greater percentage of carbohydrate present at the same sites, larger oligosaccharides attached to the same sites, or all three factors. In terms of O-glycosylation, the eCGβ C-terminal extension possessed a greater carbohydrate content than that of eLHβ. Because these structural differences were unlikely to be responsible for the reduced activity of eCG in vitro and its extended survival in vivo, we began to characterize the O-glycosylation of both eLH and eCG. The βAsn 13 oligosaccharides have been reported to consist of largely biantennary structures that were terminated with GalNAc sulfate in eLHβ and sialylated galactose in eCGβ. The α subunit contribution was largely due to the addition of a Man(α1-6)Man branch to asparagine (Asn) 56 oligosaccharides and its extension by addition of lactosamine repeats. Studies involving hybrid hormone preparations composed of all possible combinations of equine gonadotropin subunits have demonstrated that both α and β subunits contributed to the difference in receptor-binding affinity between eLH and eCG. The most heavily O-glycosylated eCG isoform was significantly less active than the other two eCG isoforms in terms of LH receptor-binding activity. Recently, however, a series of eCG glycosylation isoforms were described that exhibited the same patterns of N-glycosylation but differed in their β subunit O-glycosylation. The potential role of the C-terminal extension in extending circulatory survival was demonstrated by adding it to recombinant bovine LHβ and recombinant human FSH subunits and noting the decreased metabolic clearance rates of the resulting LH and FSH derivatives. Deletion of the C-terminus from eLHβ and hCGβ by mild acid hydrolysis or by mutation of recombinant hCGβ produced no significant effect on LH receptor binding or in vitro biological activity. Comparison of the equid LH/CGβ sequences with those available for the primate CGβ subunits indicated a greater conservation of glycosylation patterns in the former.Ī unique structural feature of primate chorionic gonadotropins and equid LH (eLH)/chorionic gonadotropins is an O-glycosylated C-terminal extension of the β subunit that appears to extend survival of these hormones in the circulation but seems to have little to do with cellular activation. Thus, O-glycosylation appears to be responsible for the β subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLHβ or des(121-149)eCGβ were combined with the same α subunit preparation. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHβ or eCGβ, hybrid hormones could be obtained by reassociating eLHα,eFSHα, or eCGα with the truncated β subunit derivatives. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGβ and from 10% to 100% for eLHβ. Both subunits were O-glycosylated at the same 12 positions, rather than the 4–6 sites anticipated. The O-glycosylation sites for equine LHβ (eLHβ) and eCGβ were identified by solid-phase Edman degradation of four glycopeptides derived from the C-terminal region.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |