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The semi-fossil and pit-less Azemiops feae is possibly the most primitive crotalid species. Here, we have cloned and sequenced cDNAs encoding four serine proteases (vSPs) from the venom glands of Chinese A. feae. Full amino-acid sequences of the major vSP (designated as AzKNa) and three minor vSPs (designated as AzKNb, AzKNc and Az-PA) were deduced. Using Protein-BLAST search, the ten most-similar vSPs for each Azemiops vSP have been selected for multiple sequence alignment, and all the homologs are crotalid vSPs. The results suggest that the A. feae vSPs are structurally most like those of eastern-Chinese Gloydius, Viridovipera, Protobothrops and North American pitvipers, and quite different from more-specialized vSPs such as Agkistrodon venom Protein-C activators. The vSPs from Chinese A. feae and those from Vietnamese A. feae show significant sequence variations. AzKNa is acidic and contains six potential N-glycosylation sites and its surface-charge distribution differs greatly from that of AzKNb, as revealed by 3D-modeling. AzKNb and AzKNc do not contain N-glycosylation sites although most of their close homologs contain one or two. Az-PA belongs to the plasminogen-activator subtype with a conserved N 20 -glycosylation site. The evolution of this subtype of vSPs in Azemiops and related pitvipers has been traced by phylogenetic analysis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acutobin, the α-fibrinogenase from Deinagkistrodon acutus venom, contains four N-glycosylation sites with disialylated complex-typed glycans. Here, we explore the functional roles of each of the N-glycan by site-directed mutagenesis. The wild-type (ATB-wt) and single glycan-knockout mutants of recombinant acutobin were prepared from HEK293T, demonstrating that mutations at Asn(77), Asn(81) and Asn(100) impaired the folding while the S79A mutant and various Asn(229)-deglycosylated mutants were correctly folded. Based on homology modeling of acutobin and multiple sequence alignment with various venom thrombin-like enzymes, the importance of a hydrophilic environment at each glycosylation site to the enzyme folding could be rationalized. Remarkably, all the mutants showed similar catalytic activities for the chromogenic substrate and similar thermal stabilities as ATB-wt, suggesting that the glycan knockout did not affect the gross conformation and stability of the active sites. Although SDS-PAGE analyses revealed that ATB-wt and the D229-mutant degraded all human fibrinogen subunits faster but less specifically in vitro as compared with other mutants that cleaved only the α-subunit, ATB-wt and D229-mutant were not able to release fibrinogen-peptide A and thus coagulated human plasma slower than the other mutants did. In the mice model, the defibrinogenating effect of ATB-wt was stronger and lasting-longer than those of all the mutants. Taken together, all the glycans contribute to the pharmacokinetics of acutobin and ATB-wt in vivo, and the microenvironment around the Asn(229)-glycan appears to regulate the fibrinogen-chain specificity of acutobin while the N-glycans at positions 77, 81 and 100 are crucial for its folding., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The crystal structure of TM-1, a P-I class snake-venom metalloproteinase (SVMP) from the Trimeresurus mucrosquamatus venom, was determined at 1.8-Å resolution. The structure exhibits the typical feature of SVMPs and is stabilized by three disulfide linkages. The active site shows a deep S1' substrate-binding pocket limited by the non-conserved Pro174 at the bottom. Further comparisons with other SVMPs suggest that the deep S1' site of TM-1 correlates with its high inhibition sensitivity to the endogenous tripeptide inhibitors. Proteolytic specificity analysis revealed that TM-1 prefers substrates having a moderate-size and hydrophobic residue at the P1' position, consistent with our structural observation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Platelet glycoprotein Ib (GPIb)-binding proteins (GPIb-BPs) from snake venoms are usually C-type lectins, which target specific sites of GPIbalpha and elicit distinct effects on platelets. In the present paper, we report a tetrameric platelet-agglutinating factor (molecular mass 121.1 kDa), termed mucrocetin, purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus ). Mucrocetin is a GPIbalpha agonist with a binding site distinct from that of flavocetin-A (a snake venom GPIbalpha antagonist) on GPIbalpha, in spite of the high sequence identity (94.6%) between the two venom lectins. The crystal structure of mucrocetin was solved and refined to 2.8 A (1 A=0.1 nm) resolution, which shows an interesting crystal packing of six-layer cylinders of doughnut-shaped molecules. The four alphabeta heterodimers are arranged in an unusual square-shaped ring stabilized by four interdimer 'head-to-tail' disulphide bridges. Detailed structural comparison between mucrocetin and flavocetin-A suggests that their disparate platelet effects are probably attributable to different charge distributions on the putative concave binding surface. A unique positively charged patch on the binding surface of mucrocetin, formed by Lys102, Lys108, Lys109 and Arg123 in the alpha-subunit coupled with Lys22, Lys102, Lys116 and Arg117 in the beta-subunit, appears to be the primary determinant of its platelet-agglutinating activity. Conceivably, this interesting venom factor may provide a useful tool to study platelet agglutination by binding to the GPIb-IX-V complex.

The crystal structure of TM-3, a small snake-venom metalloproteinase (SVMP) isolated from Taiwan habu (Trimeresurus mucrosquamatus), was determined at 1.35 A resolution with resultant R and R(free) values of 0.181 and 0.204, respectively. The overall structure of TM-3 is an oblate ellipsoid that contains three disulfide crosslinks, Cys118-Cys197, Cys159-Cys181 and Cys161-Cys164. It exhibits the typical structural features of SVMPs and is closely related to the structure of the catalytic proteinase domain of TNFalpha-converting enzyme (TACE). In the present structure, the essential catalytic zinc ion was found to be replaced by a cadmium ion during crystallization, as revealed by atomic absorption analysis and X-ray data. This cadmium ion is bound to six ligands, including three conserved histidines and three water molecules, displaying the coordination geometry of a distorted octahedron. One of the water molecules is proposed to play the role of stabilizing the tetrahedral intermediate during the catalysis of SVMPs. The putative S'(1) specificity pocket of TM-3 is relatively shallow, in contrast to the deep pockets of adamalysin II, atrolysin C and H(2)-proteinase, but is similar to those in acutolysin A and TACE. The shallow pocket is a consequence of the presence of the non-conserved disulfide bond Cys159-Cys181 and the residue Gln174 at the bottom of the S'(1) pocket. The results indicate that the active-site structure of TM-3, among the know structures of SVMPs examined thus far, is most similar to that of TACE owing to their close disulfide configurations and the S'(1) specificity pocket.

Venoms from crotalid and viperid snakes contain several peptide inhibitors which regulate the proteolytic activities of their snake-venom metalloproteinases (SVMPs) in a reversible manner under physiological conditions. In this report, we describe the high-resolution crystal structures of a SVMP, TM-3, from Taiwan habu (Trimeresurus mucrosquamatus) cocrystallized with the endogenous inhibitors pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) or pyroGlu-Lys-Trp (pEKW). The binding of inhibitors causes some of the residues around the inhibitor-binding environment of TM-3 to slightly move away from the active-site center, and displaces two metal-coordinated water molecules by the C-terminal carboxylic group of the inhibitors. This binding adopts a retro-manner principally stabilized by four possible hydrogen bonds. The Trp indole ring of the inhibitors is stacked against the imidazole of His143 in the S-1 site of the proteinase. Results from the study of synthetic inhibitor analogues showed the primary specificity of Trp residue of the inhibitors at the P-1 site, corroborating the stacking effect observed in our structures. Furthermore, we have made a detailed comparison of our structures with the binding modes of other inhibitors including batimastat, a hydroxamate inhibitor, and a barbiturate derivative. It suggests a close correlation between the inhibitory activity of an inhibitor and its ability to fill the S-1 pocket of the proteinase. Our work may provide insights into the rational design of small molecules that bind to this class of zinc-metalloproteinases.

A proteinase inhibitor (designated as TMI) was isolated and purified from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus) by using successive chromatographies which included Sephadex G-100, DEAE-Sephacel chromatographies, and C(4) reverse-phase HPLC. The purified inhibitor was shown to be a homogeneous protein with a molecular mass of about 47 or 36 kDa in the presence or absence of a reducing agent, beta-mercaptoethanol. The inhibitor decreases in molecular mass by about 23% with N-linked neuraminidase treatment, suggesting that it is a glycoprotein. Further enzymatic analyses indicated that this inhibitor possesses strong inhibitory activities toward three zinc-dependent metalloproteinases and not fibrinogenolytic serine proteases previously isolated from the venom of the same snake species with an IC(50) of about 0.2-1.1 microM. Its IC(50) value was approximately three orders of magnitude more effective than those of the tripeptide inhibitors we previously purified from the crude venom of the same snake (Biochem. Biophys. Res. Commun. 248, 562-568 (1998)). The purified inhibitor showed stronger inhibitory action against caseinolytic activities of crude venoms from closely related species of Taiwan habu than those from unrelated species. N-terminal sequence analysis showed that its sequence is distinctly different from sequences of those serum inhibitors reported for other snake species in the literature. Based on inhibition susceptibility and primary structures of various snake protease inhibitors, it is suggested that this novel inhibitor isolated from the serum of Taiwan habu may be a unique self-defense protein factor mainly for protection against envenomation from snakes of the same genus., (Copyright 1999 Academic Press.)

Three small peptide components were isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus), which show specific activity to inhibit the strong proteolytic activity of multiple metalloproteinases present in the crude venom. Using multiple chromatographies coupled with successive ultrafiltrations, three inhibitors, i.e. pyroglutamate-lysine-tryptophan (pyroGlu-Lys-Trp), pyroglutamate-asparagine-tryptophan (pyroGlu-Asn-Trp) and pyroglutamate-glutamine-tryptophan (pyroGlu-Gln-Trp) were obtained in good yields and high homogeneity. The yields of these peptide fractions were estimated to be about 0.65 mg, 0.55 mg and 0.42 mg from 250 mg total lyophilized crude venom, which corresponded to the approximate concentrations of 8.4 mM, 7.3 mM and 5.4 mM respectively in venom secretion. Detailed and unambiguous structural determination was established by amino acid analyses, mass spectrometry and microsequencing of purified peptides. Further functional characterization of these three tripeptides showed that they could weakly inhibit three metalloproteinases previously isolated from the same venom. The inhibitory activities were similar among these tripeptides and their IC50 (concentration for 50% inhibition) were estimated in a range of 0.20-0.95 mM, which is much more effective than citrate, another venom protease inhibitor of low molecular-weight component. Since these tripeptides are the endogenous peptide inhibitors present in the lumen of venom glands, it is conceivable that they may act as a self-defensive mechanism against the auto-digestive deleterious effect of the strong metalloproteinases in vivo, particularly several zinc-dependent metalloproteinases present in crotalid and viperid venoms.

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated from Trimeresurus flavoviridis and botrocetin from Bothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca2+ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated from Bothrops jararaca venom. We have therefore named this novel venom factor trimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.

Three fibrinogenolytic proteases were isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of these purified fractions with fibrinogenase activity indicated that they are single-chain proteases of approximately 24 kDa, possessing strong cleaving activity mainly on the A alpha and less on B beta and gamma chains of fibrinogen subunit chains. Enzyme activities were strongly inhibited by EDTA or 1,10-phenanthroline and not by phenylmethanesulfonyl fluoride, suggesting that these fibrinogenases belong to the family of metalloproteinases and not thrombin-like serine proteases. N-Terminal sequence analysis of these proteases failed to show any free amino-terminal residues, thus hampering the sequence determination by conventional sequencing strategy. Microsequencing on the electroblotted fragments of CNBr-treated proteases separated on SDS-PAGE was then used to determine the partial sequences. Sequence comparison of the determined partial sequences of these proteins with published sequences of the protein data bank revealed that they showed sequence homology with H2-protease. HR2a and protrigramin, which were all shown to belong to metalloproteinases present in various snake venoms. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh venom glands of the same snake species to facilitate cloning and sequencing of these proteases. Sequencing several positive clones containing amplified cDNAs revealed the existence of one fibrinogenase in the Taiwan habu, which was contained within one complete cDNA encoding the preproproteinase precursor of hemorrhagic metalloproteinases.

Several fibrinogenolytic proteases were isolated from the venom of Taiwan habu, Trimeresurus mucrosquamatus, a snake species belonging to the Crotalidae family. One protease with strong fibrinogenolytic activity was further purified to homogeneity through multiple-step chromatographies including ion-exchange chromatography, gel permeation and reversed-phase HPLC. In vitro, the purified enzyme cleaved beta-chain of fibrinogen molecules efficiently and showed relatively lower activity on alpha-chain, with almost no activity on gamma-chain even after a long period of incubation. Further characterization indicated that it is a single-chain polypeptide with molecular weight of about 28,000. Its stability at high temperatures (> 90 degrees C) distinguished it from the previously reported venom fibrinogenases. N-Terminal sequence analysis revealed that it is similar to batroxobin and ancrod, which were shown to possess either fibrinogen-clotting or antithrombotic effect. Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh venom glands of the same snake species to facilitate the cloning and sequencing of this important fibrinogenase. Sequencing several positive clones corresponding to the coding sequence of the enzyme revealed the existence of a family of novel thrombin-like fibrinogenases in the Taiwan habu, which are heat-stable and may be useful as strong antithrombotic agents.

Snake venoms, particularly those belonging to Crotalidae and Viperidae families, are known to possess fibrinogenolytic proteases which strongly affect the blood coagulation system. Three fibrinogenolytic enzymes, named as TM-1, TM-2 and TM-3, were purified from the venom of Taiwan habu using anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. These enzyme fractions were shown to be homogeneous as judged by SDS-gel electrophoresis. Further characterization of these purified fractions with fibrinogenase activity indicated that they are single-chain proteases possessing basic isoelectric points and molecular masses of approximately 27 kDa. These enzymes cleaved alpha-chain of fibrinogen first and then beta-chain, with relatively low activity on gamma-chain. Fibrinogenolytic activities of these enzymes were not significantly affected at extreme pH values, activity loss occurred only at temperatures higher than 65 degrees C. Enzyme activities were strongly inhibited by some metal chelators such as EDTA or 1,10-phenanthroline, suggesting that these fibrinogenases belong to metalloproteases. The closely similar amino-acid compositions coupled with identical cleaved products of similar molecular masses observed on the autolysis of TM-1 and TM-2 suggest that these two proteases are similar in primary structures and probably mutually related.

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.