Abstract
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 language | English (US) |
---|---|
Pages (from-to) | 167-179 |
Number of pages | 13 |
Journal | Archives of Biochemistry and Biophysics |
Volume | 606 |
DOIs | |
State | Published - Sep 15 2016 |
Keywords
- Disaccharide
- ESI-MS
- GcMAF
- Linear trisaccharide
- Linkage analysis
- Vitamin D binding protein
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology