Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry

Chad Borges, Douglas S. Rehder

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein—leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide—precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

Original languageEnglish (US)
Pages (from-to)167-179
Number of pages13
JournalArchives of Biochemistry and Biophysics
StatePublished - Sep 15 2016


  • Disaccharide
  • ESI-MS
  • GcMAF
  • Linear trisaccharide
  • Linkage analysis
  • Vitamin D binding protein

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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