Carbohydrate Structure Analysis of Batroxobin, a Thrombin-Like Serine Protease from Bothrops moojeni Venom

Abstract

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.

Correlation between the Glycan Variations and Defibrinogenating Activities of Acutobin and Its Recombinant Glycoforms

“…Most of the four N-glycosylation sites of acutobin carry the disialylated glycans, and the full range of 1 to 4 disialyl LacNAc antenna [8] is distinct from the sugars in ancrod and batroxobin [9], [10]. It has been well known that sialylation of otherwise exposed termini may extend the circulatory half-life of many serum glycoproteins by reducing their clearance through hepatic asialoglycoprotein receptor [23].…”

“…Acutobin contains four glycosylation sites at N77, N81, N100 and N231 (the acutobin numbering [8]); among them only the N231 is conserved among most of the SVTLEs which release only FpA [9], [10], [27]. The role of each N-glycan site in acutobin remains to be explored by site directed mutagenesis.…”

“…For past decades, SVTLEs have been used as therapeutics to prevent or treat stroke and other cardiovascular diseases [4]–[6]. The amino acid sequences and glycan structures of SVTLE glycoproteins from different species are not similar [7] and the number and positions of their N-glycosylation sites are not conserved [8]–[10].…”

Glycan labeling strategies and their use in identification and quantification

“…Typically, volatile buffers such as acetic acid/triethylamine are applied at concentrations of up to 200 mM [156]. The use of amine-bonded phases at lower ionic strength results in a mixed-mode HILIC/anion-exchange separation, which is discussed later.…”

Novel pathways in complex-type oligosaccharide synthesis: new vistas opened by studies in invertebrates

“…For example, as snail P4-GalNAc-T efficiently acts on GlcNAcP( 1-4)GlcNAc [ 131, the concerted action of the two glycosyltransferases might lead to the formation of a GalNAcP( 1-4)GlcNAcP( 1-4)GlcNAc structural element on certain snail glycans. Interestingly, this element has been reported as a minor constituent on the glycans of the snake venom serine protease batroxobin [29].…”