Structural characterization of N-linked oligosaccharides of Defibrase from Agkistrodon acutus by sequential exoglycosidase digestion and MALDI-TOF mass spectrometry.
Detailed structures of N-linked oligosaccharides of Defibrase, a highly active thrombin like enzyme (TLE) purified from the venom of Agkistrodon acutus, were successfully characterized using MALDI-TOF mass spectrometry in combination with sequential exoglycosidase digestion. Monosaccharide composition analysis was performed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Galactose(Gal), mannose(Man), fucose(Fuc), N-acetylglucosamine (GlcNAc), and sialic acid (Neu5Ac) was detected and the total carbohydrate content was about 19.4% (w/w). The N-linked oligosaccharides were released by treatment with PNGase F, fluorescent labeled with 2-aminobenzamide, and fractionated by high performance liquid chromatography (HPLC). The main oligosaccharide fractions were collected and further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of five distinct oligosaccharide structures in Defibrase, which are mainly complex or hybrid type, with a small amount of oligomannosidic type. The complex type oligosaccharides are mostly tri-or bi-antennary and the hybrid oligosaccharides are all bi-antennary. Most oligosaccharides are also found to be fucosylated.
School of Pharmacy, Chinese Pharmaceutical University, Nanjing, Jiangsu, 210009, China.
Xiaoqing
X
Luo
Huaxin
H
Yang
Chenggang
C
Liang
Shaohong
S
Jin
D017836:Agkistrodon / Q000378:metabolism*; D000818:Animals; D012103:Batroxobin / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D004563:Electrochemistry; D005343:Fibrinolytic Agents / Q000737:chemistry*; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases / Q000737:chemistry; D006868:Hydrolysis; D008745:Methylation; D009005:Monosaccharides / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry*; D010084:Oxidation-Reduction; D012794:Sialic Acids / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D005456:Fluorescent Dyes; D009005:Monosaccharides; D009844:Oligosaccharides; D012794:Sialic Acids; D006026:Glycoside Hydrolases; D012103:Batroxobin