Effects of N-linked glycans on the activity and stability of Atragin and K-like from Naja atra.

Atragin and K-like are N-linked glycoproteins that belong to the P-III class of snake venom metalloproteinases, which hydrolyze components of the extracellular matrix and plasma proteins, thus contributing to vascular damage and coagulation disorders. In this study, we examined the effects of N-linked glycans on the proteolytic activity and structural stability of Atragin and K-like, which were purified from Naja atra cobra venom. Deglycosylation was performed using recombinant endo F3 under nondenaturing conditions. The removal of N-linked glycans significantly altered the proteolytic activity of both proteins against extracellular matrix substrates. In addition, neuraminidase-mediated desialylation markedly reduced the fibrinogenolytic activity of K-like. Free sialic acid binds to fibrinogen and inhibits its cleavage by K-like. Deglycosylation enhanced the heparin-binding affinity of Atragin and K-like. Heparin efficiently enhanced the proteolytic activity of both proteins irrespective of their glycosylation state. Deglycosylation reduced resistance to guanidine hydrochloride-induced unfolding, and K-like was more sensitive to thermal denaturation. These findings highlight the critical role of N-linked glycans in maintaining the enzymatic activity and structural integrity of venom metalloproteinases, and reveal how specific carbohydrate moieties, including terminal sialic acid residues, modulate their interactions with substrates. This study improves our understanding of venom proteins and may inform therapeutic strategies for snakebite treatment.