Complete Amino Acid Sequence of Kaouthiagin, a Novel Cobra Venom Metalloproteinase with Two Disintegrin-like Sequences

Abstract

The primary structure of kaouthiagin, a metalloproteinase from the venom of the cobra snake Naja kaouthia which specifically cleaves human von Willebrand factor (VWF), was determined by amino acid sequencing. Kaouthiagin is composed of 401 amino acid residues and one Asn-linked sugar chain. The sequence is highly similar to those of high-molecular mass snake venom metalloproteinases from viperid and crotalid venoms comprised of metalloproteinase, disintegrin-like, and Cys-rich domains. The metalloproteinase domain had a zinc-binding motif (HEXXHXXGXXH), which is highly conserved in the metzincin family. Kaouthiagin had an HDCD sequence in the disintegrin-like domain and uniquely had an RGD sequence in the Cys-rich domain. Metalloproteinase-inactivated kaouthiagin had no effect on VWF-induced platelet aggregation but still had an inhibitory effect on the collagen-induced platelet aggregation with an IC(50) of 0.2 microM, suggesting the presence of disintegrin-like activity in kaouthiagin. To examine the effects of these HDCD and RGD sequences, we prepared synthetic peptides cyclized by an S-S linkage. Both the synthetic cyclized peptides from the disintegrin-like domain and from the Cys-rich domain) had an inhibitory effect on collagen-induced platelet aggregation with IC(50) values of approximately 90 and approximately 4.5 microM, respectively. The linear peptide (RAAKHDCDLPELC) and the cyclized peptide had little effect on collagen-induced platelet aggregation. These results suggest that kaouthiagin not only inhibits VWF-induced platelet aggregation by cleaving VWF but also disturbs the agonist-induced platelet aggregation by both the disintegrin-like domain and the RGD sequence in the Cys-rich domain. Furthermore, our results imply that the corresponding part of the Cys-rich domain in other snake venom metalloproteinases also has a synergistic disturbing effect on platelet aggregation, serving as a second disintegrin-like domain. This is the first report of an elapid venom metalloproteinase with two disintegrin-like sequences.

A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning

“…Berythractivase presents three potential N-glycosylation sites in its amino acid sequence ( Figure 6). Some snake-venom metalloproteinases have also been shown to be glycosylated [43,44]. Analysis of the cDNA sequence revealed that berythractivase is a member of the P-III class of the metalloproteinase protein family.…”

Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity

“…Finally, there are three PIII class SVMPs, HR1B, from the venom of Trimeresurus flavoviridis [50], BjussuMP_I, from Bothrops jararacussu [51], and kaouthiagin, from Naja kaouthia [52], that have the integrin‐binding motifs KGD or RGD in their cysteine‐rich domains (Fig. 6).…”

Snake Venom Metalloproteinase Containing a Disintegrin-like Domain, its Structure-activity Relationships at Interacting with Integrins

“…HR1B CRAAES ECD IPESC [61,99] Ht -e CR VS MVD RNDDTC [100] Jararhagi n CRASMS ECD PAEHC [60] Jararhagi n-C CRASMS ECD PAEHC [60,65] Kaouthiagin CRA AKHDCD LPELC [101] Mocarhagi n CRA AKNDCD FPELC AAM51550*…”