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O ‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60
Author(s) -
Bustamante Juan J.,
Gonzalez Leticia,
Carroll Christopher A.,
Weintraub Susan T.,
Aguilar Roberto M.,
Muñoz Jesus,
Martinez Andrew O.,
Haro Luis S.
Publication year - 2009
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800989
Subject(s) - oligosaccharide , chemistry , glycoprotein , biochemistry , tetrasaccharide , glycopeptide , mucin , polysaccharide , antibiotics
MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O ‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N ‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc 2 , N ‐acetyl galactosamine 1 , Gal 1 ). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O ‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O ‐oligosaccharide sequence linked to Thr‐60 begins with N ‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.