The polypeptide part of human chorionic gonadotrophin affects the kinetics of alpha 6-sialylation of its N-linked glycans but does not alter the branch specificity of CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase.
Human chorionic gonadotrophin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha- and a beta-subunit, both containing two N-linked, complex-type glycans. Using this hormone as a model glycoprotein, the influence of its polypeptide part on the activity and specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase (alpha 6-sialyltransferase) was investigated. Initial rates of sialic acid incorporation into the desialylated glycans of hCG alpha and hCG beta in the heterodimer were higher with the alpha-subunit. This appeared to be due to a higher V which, together with a slightly lowered affinity (higher Km), resulted in a higher kinetic efficiency of the sialyltransferase for the glycans of this subunit. By contrast, the kinetic parameters did not differ significantly when the subunits were in the free form, indicating that the differences in the kinetics of sialylation found for the subunits in the heterodimeric state were not caused by the differences in N-linked carbohydrate structures of the subunits. It is proposed that these effects are due to conformational constraints which the polypeptide moieties put on the glycan chains upon dimerization. Furthermore, it was investigated whether the polypeptide of hCG would interfere with the sialyltransferase so as to alter the branch specificity of the enzyme. 1H-NMR spectroscopy (400 MHz) of the glycan chains, alpha 6-sialylated in vitro, showed that the enzyme highly prefers the galactosyl residue at the Gal beta 1----4GlcNAc beta 1----2-Man alpha 1----3Man branch for attachment of the first mol of sialic acid into the diantennary glycans of desialylated hCG.(ABSTRACT TRUNCATED AT 250 WORDS)
Nemansky
M
M
Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Edzes
H T
HT
Wijnands
R A
RA
Van den Eijnden
D H
DH
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D010455:Peptides / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011487:Protein Conformation; D012794:Sialic Acids / Q000737:chemistry*; D012799:Sialyltransferases / Q000737:chemistry*; D013379:Substrate Specificity; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D006063:Chorionic Gonadotropin; D010455:Peptides; D011134:Polysaccharides; D012794:Sialic Acids; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase