O-Glycosylated 24 kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60

Juan J. Bustamante, Leticia Gonzalez, Christopher A. Carroll, Susan T. Weintraub, Roberto M. Aguilar, Jesus Muñoz, Andrew O. Martinez, Luis S. Haro

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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 (NeuAc2, N-acetyl galactosamine1, 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.

Original languageEnglish (US)
Pages (from-to)3474-3488
Number of pages15
JournalProteomics
Volume9
Issue number13
DOIs
StatePublished - Jul 2009

Keywords

  • Glycosylated
  • Human growth hormone
  • MS
  • O-linked oligosaccharide
  • Structural analysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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