title: Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.
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.
authors: School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA; The Biodesign Institute at Arizona State University, Tempe, AZ 85287, USA. Electronic address: chad.borges@asu.edu.
authors: Chad R
authors: CR
authors: Borges
authors: The Biodesign Institute at Arizona State University, Tempe, AZ 85287, USA. Electronic address: doug.rehder@gilead.com.
authors: Douglas S
authors: DS
authors: Rehder
mesh_terms: D000483:Alleles; D004187:Disaccharides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008262:Macrophage Activation; D016215:Macrophage-Activating Factors / Q000737:chemistry*; D008358:Mannose / Q000737:chemistry; D009439:Neuraminidase / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014312:Trisaccharides / Q000737:chemistry; D014809:Vitamin D-Binding Protein / Q000737:chemistry; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: D004187:Disaccharides; D006027:Glycosides; D016215:Macrophage-Activating Factors; D011134:Polysaccharides; D014312:Trisaccharides; D014809:Vitamin D-Binding Protein; C101908:vitamin D-binding protein-macrophage activating factor; D014269:Trifluoroacetic Acid; D006026:Glycoside Hydrolases; D009439:Neuraminidase; D001616:beta-Galactosidase; D048809:alpha-N-Acetylgalactosaminidase; D008358:Mannose
keywords: Disaccharide; ESI-MS; GcMAF; Linear trisaccharide; Linkage analysis; Vitamin D binding protein