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

Arch Biochem Biophys. 2016 Sep 15;606:167-79. doi: 10.1016/ Epub 2016 Aug 5.


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.

Keywords: Disaccharide; ESI-MS; GcMAF; Linear trisaccharide; Linkage analysis; Vitamin D binding protein.

MeSH terms

  • Alleles
  • Disaccharides / chemistry
  • Escherichia coli / enzymology
  • Glycoside Hydrolases / chemistry
  • Glycosides / chemistry
  • Glycosylation
  • Humans
  • Macrophage Activation
  • Macrophage-Activating Factors / chemistry*
  • Mannose / chemistry
  • Neuraminidase / chemistry
  • Polysaccharides / chemistry*
  • Spectrometry, Mass, Electrospray Ionization
  • Trifluoroacetic Acid / chemistry
  • Trisaccharides / chemistry
  • Vitamin D-Binding Protein / chemistry
  • alpha-N-Acetylgalactosaminidase / chemistry
  • beta-Galactosidase / chemistry


  • Disaccharides
  • Glycosides
  • Macrophage-Activating Factors
  • Polysaccharides
  • Trisaccharides
  • Vitamin D-Binding Protein
  • vitamin D-binding protein-macrophage activating factor
  • Trifluoroacetic Acid
  • Glycoside Hydrolases
  • Neuraminidase
  • beta-Galactosidase
  • alpha-N-Acetylgalactosaminidase
  • Mannose