Primary Structures of Platelet Aggregation Inhibitors (Disintegrinis) Autoproteolytically Released from Snake Venom Hemorrhagic Metalloproteinases and New Flurogenic Peptide Subtrates for These Enzymes1

Abstract

A hemorrhagic protein (60 kDa), HR1B, present in the venom of Trimeresurus flavoviridis is a mosaic protein consisting of an NH2-terminal metalloproteinase-domain, a disintegrin (platelet aggregation inhibitor)-like domain, and a unique COOH-terminal Cys-rich domain. Since the gross structures of HR1B and protein precursors of disintegrins, trigramin, and rhodostomin, all of which contain the metalloproteinase domain, are similar, many disintegrins so far detected in snake venoms are assumed to be autoproteolytic fragments released from precursors. In ongoing related experiments, the newly purified hemorrhagic metalloproteinases, HR1A from T. flavoviridis venom and HT-1 from Crotalus ruber ruber venom, in addition to HR1B, were autoproteolyzed, in the absence of Ca2+, at 37 degrees C for 3-12 h. Under these conditions, HR1A, HR1B, and HT-1 each released a single major fragment of 32, 34, and 31 kDa, respectively. The entire amino acid sequences of the isolated fragments indicated the presence of disintegrin-like and Cys-rich domains in the COOH-terminal regions of HR1A, HR1B, and HT-1, respectively. It seems likely that so-called disintegrins probably originate from various metalloproteinases present in venom. On the bases of peptide sequences close to the autoproteolytic cleavage sites of these metalloproteinases and the sites of fibrinogen cleaved by these enzymes, we synthesized new intramolecularly quenched fluorogenic peptide substrates. Among the 10 peptides tested, 2-aminobenzoyl (Abz)-Ser-Pro-Met-Leu-2,4-dinitroanilinoethylamide (Dna) proved to be the best substrate for venom metalloproteinase, as deduced from kinetic analyses.

Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells

“…The disintegrin domain of atrolysin E has been isolated from C. atrox [29] ; in addition, recombinant proatrolysin E has been shown to be proteolytically processed by atrolysin A [30]. However, there is evidence that the disintegrinlike or disintegrin-like\cysteine-rich domain is autoproteolytically released from SVMPs in itro [31,32] ; the mechanisms of post-translational processing of P-II or P-III SVMPs are still unclear in snake venom. In our study, the corresponding cDNA sequence of graminelysin I precursor (N-III SVMP) was obtained from two overlapping cDNA clones.…”

What have snakes taught us about integrins?

“…The molecular masses of the large haemorrhagins range from 50 to 90 kDa. HR1B from T. fla6o6iridis venom [19], jararhagin from B. jararaca venom [21] and Ht-a from C. atrox [20] are high molecular mass haemorrhagins that have been cloned and sequenced. They are mosaic proteins with a metalloproteinase domain similar to that of the small venom proteinase at the N-terminus, a disintegrin-like domain in the middle of the molecule and a cysteine-rich domain at the C-terminus ( fig.…”

Comprehensive snake venomics of the Okinawa Habu pit viper, Protobothrops flavoviridis, by complementary mass spectrometry-guided approaches

“…This could mean, that the Habu venom contains truncated or autodigested versions of svMP P-II or P-III, which is also known from other svMPs [44]. This autolysis was firstly observed for the two svMPs HR1A and HR1B isolated from the P. flavoviridis venom [46,47].…”