Carbohydrate structure analysis of batroxobin, a thrombin-like serine protease from Bothrops moojeni venom.
The carbohydrate side chains of batroxobin were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, pyridylaminated and separated by two-dimensional HPLC. Neutral oligosaccharide derivatives obtained after desialylation were characterized by methylation analysis, liquid secondary-ion mass spectrometry, digestion with exoglycosidases and endoglycosidases and, in part, by acetolysis, whereas sialic acid constituents were identified by reverse-phase HPLC after conjugation with 1,2-diamino-4,5-methylene-dioxybenzene. The overall glycosylation status of the protein was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results revealed that batroxobin is heterogeneously glycosylated carrying predominantly diantennary, partially incomplete complex-type glycans in addition to hybrid-type species. Most glycans were core-fucosylated at C6 of the innermost GlcNAc. As a characteristic feature, galactose was completely replaced by GalNAc beta 4-substituents in complex-type antennae, the GlcNAc-residues of which were, in part, fucosylated at C3. Furthermore, evidence was obtained that suggested the presence of a novel type of glycoprotein-N-glycan comprising two GalNAc beta 4GlcNAc beta 4GlcNAc beta 2Man-antennae. Sialic acid residues represented a mixture of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2), which were exclusively linked to C3 of subterminal GalNAc. A precise assignment of these sialic acid derivatives to distinct oligosaccharide structures or antennae, however, was not carried out. Finally, MALDI-TOF-MS demonstrated that both potential N-glycosylation sites of batroxobin are substituted by carbohydrate chains. In conclusion, our studies revealed that this snake venom glycoprotein is characterized by a unique oligosaccharide pattern partly comprising novel structural elements.
Lochnit
G
G
Biochemisches Institut am Klinikum, Universität, Giessen, Germany.
Geyer
R
R
D000818:Animals; D012103:Batroxobin / Q000737:chemistry*; D017837:Bothrops; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006031:Glycosylation; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D011134:Polysaccharides / Q000737:chemistry; D012794:Sialic Acids / Q000737:chemistry
D009844:Oligosaccharides; D011134:Polysaccharides; D012794:Sialic Acids; D012103:Batroxobin; D019158:N-Acetylneuraminic Acid