دانلود رایگان مقاله ساختار گلیکان فاکتور فعال ماکروفاژ مشتق شده از پروتئین GC

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.

4. Conclusions

The primary O-glycan of GcMAF derived from the 1F allele product is a Gal-b(1-3)-GalNAc-Thr disaccharide and not a GalNAcThr monosaccharide as has long been believed based on inference from activity studies. This disaccharide is derived from the common sialylated core 1 O-glycanda linear trisaccharide in which the sialic acid residue is attached to galactose at its 3-position. Indeed, based on glycosidase digestion studies followed by analysis of the intact protein by ESI-MS as well as glycan linkage analysis data presented here, both the Gc1F and Gc1S trisaccharides are linear, sialylated core 1 O-glycans (i.e., NeuNAc-(2-3)-Gal-(1-3)-GalNAc-Thr) and neither contains mannose. Moreover, a-N-Acetylgalactosamindase (nagalase) lacks endoglycosidase activity and is only capable of removing the DBP O-glycan GalNAc residue once the glycan has been trimmed down to just the single reducing-end GalNAc residueda result that corroborates previous findings demonstrating that DBP is not deglycosylated in cancer patients.