Oligosaccharide mapping reveals hormone-specific glycosylation patterns on equine gonadotropin alpha-subunit Asn56.
Equine gonadotropin alpha-subunit glycosylation was examined by releasing oligosaccharides using a sequential enzymatic deglycosylation protocol and comparing the released oligosaccharide populations using a high resolution oligosaccharide mapping technique. Digestion of native alpha-subunit preparations with peptide-N-glycosidase altered their mobilities during SDS-PAGE under reducing conditions to positions intermediate between the corresponding native alpha-subunit and completely deglycosylated alpha-subunit bands. Complete alpha-subunit deglycosylation required reduction of disulfide bonds. Results of solid-phase Edman degradation demonstrated that partial deglycosylation occurred exclusively at Asn56. Oligosaccharide mapping of total oligosaccharides obtained by enzymatic deglycosylation of reduced, carboxymethylated alpha-subunit preparations revealed hormone-specific patterns of glycosylation in eLH alpha and eCG alpha. Oligosaccharide mapping of individual glycosylation sites revealed that hormone-specific glycosylation was primarily restricted to Asn56 of both subunit preparations and revealed a hormone-specific pattern of Asn56 glycosylation in eFSH alpha that was obscured in the total oligosaccharide map. eLH alpha Asn56 oligosaccharides appeared to be primarily seven variants of a monoantennary structure. eCG alpha Asn56 oligosaccharides consisted of one of two forms, either a sialylated biantennary oligosaccharide that appeared identical to a commercial carbohydrate standard or a lactosamine variant of that structure.
Gotschall
R R
RR
Department of Biological Sciences, Wichita State University, Kansas 67260-0026, USA.
Bousfield
G R
GR
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D003488:Cyanogen Bromide; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006736:Horses; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
D004220:Disulfides; D015292:Glycoprotein Hormones, alpha Subunit; D009844:Oligosaccharides; D001216:Asparagine; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D003488:Cyanogen Bromide