Mass spectrometry identification of lectin-bound glycoproteins, N-glycome profiling, prediction of the glycosylation sites, and elucidation of the role of N-linked glycosylation on enzymatic activity, pharmacological properties, and lethality of Naja kaouthia venom.

The Indian monocled cobra (Naja kaouthia) venom contains many biologically active proteins and peptides. Glycosylation and other post-translational changes affect snake venom toxins. Nevertheless, our understanding of the glycoproteome profile and the impact of glycosylation on the diversity and functionality of snake venom toxins remains limited. Therefore, the glycoprotein sugars of N. kaouthia venom (NKV) that bind to different lectins, viz., concanavalin A (ConA), wheat germ agglutinin (WGA), and peanut agglutinin (PNA), were isolated and separated by SDS-PAGE, stained with glycoprotein-specific stain, and followed by mass spectrometry identification of glycoproteins. Most of the high (>90 kDa) and mid-molecular-mass (45-75 kDa) toxins bind to ConA and PNA, whereas some low-molecular-mass toxins (10-16 kDa) bind to WGA. Eighteen glycoprotein isoforms clustered into nine snake venom toxin families are identified in NKV. Most of the seventeen N-glycans found in NKV were complex-type structures, and glycoproteins contain multiple predicted N- and O-glycosylation sites within their amino acid sequences. NKV toxins with N-linked glycans removed under non-denaturing conditions demonstrated lower enzymatic activity and in vivo toxicity in mice than non-deglycosylated NKV. Deglycosylated NKV showed less cross-reactivity to commercial polyvalent antivenom than native NKV, indicating that glycosylation influences NKV toxicity and antigenicity.