Abstract
Background N-linked glycosylation is one of most important post-translational modification for several physiological processes in all eukaryote cells. There is growing interest in understanding the roles of the attached N-glycans on the structures and stability of the investigated proteins be-cause recent studies have shown that N-linked glycans are not only play a role in the quality control process during protein synthesis, but also affect significantly on their biological func-tions. Snake venom toxins also consist of glycoproteins, but the function of glycosylation re-mains illusive. Methods In this study, we investigate two P-III snake venom metalloproteinases (SVMPs), for example, Atragin and K-like from Naja atra, and correlate its enzymatic activities before and after cleaving the N-glycan complexes with Endoglycosydase. We express Endoglycosidase F3 and use it to remove N-glycan complexes of Atragin and K-like SVMPs to understand whether the truncated sugar molecule with one N-acetylglucosamine and a fuccose residue remaining on the asparagine could still maintain their structure and activity. Three different substrates from extracellular matrix, for instance, fibrinogen, fibronectin, collagen type I, and azocasein were tested and the protease activity of Atragin and K-like SVMPs were compared. Finally, their structures were examined by Circular Dichroism and fluorescence spectroscopy. Results The P-III SVMPs and its mammalian homologues of ADAMs, and ADAMTSs all consist of at least one N-linked protein glycosylation, but the exact location of the N-glycans appear to vary significantly among different SVMPs and its homologues. Comparison of the enzymatic activities between K-like and Atragin SVMPs indicate that the effect of deglycosylation is substrate dependent. For instance, while there was not a significant change in the enzymatic activity of N-deglycosylated Atragin and – K-like with fibronectin and vitronectin substrates, the fibrinogenolytic and collagenolytic activity of Atragin and K-like, however, were signifi-cantly perturbed after deglycosylating. Moreover, the azocaseinolytic activity with sulfonila-mide-azocasein, a nonspecific substrate, showed a significantial fall off in the N-deglycosylated K-like activity, whereas Atragin retain the same activity. Finally, we also dis-covered that the N-deglycosylated K-like bound to heparin column much more stronger than native K-like, and the observed heparin binding could significantly enhance its enzymatic ac-tivity. In order to understand the observed change in its enzymatic activity, we examine the possible effect of glycosylation on its structural stability. The N-glycans contributed to a change in negatively charged surface and shifting the pH value of protein toward acidic pI of Atragin and K-like. Since there is no detected conformational change based on Circular Di-chroism result, the N-glycan appears to retain the stability of protein avoided to aggregation and precipitation, likely leading to an decrease in protein stability with high temperatures and denaturant.