Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation

Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC₅₀) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a better treatment for viper bites.     

Molecular cloning of novel serine proteases and phospholipases A2 from green pit viper (Trimeresurus albolabris) venom gland cDNA library.

Green pit viper (Trimeresurus albolabris) is the most common venomous snake responsible for bites in Bangkok. It causes local edema and systemic hypofibrinogenemia resulted from the thrombin-like, as well as the fibrinolytic effects of the venom. However, the amino acid sequences of these venom proteins have never been reported. In this study, we have cloned five novel serine proteases from the Thai T. albolabris venom gland cDNA library. They were all closely homologous to the corresponding serine proteases from Chinese green viper (Trimeresurus stejnegeri), suggesting the evolutionary proximity of the two species. In addition, their functional activities could be deduced. There were predicted to be two thrombin-like enzymes (GPV-TL1 and GPV-TL-2), two isoforms of a fibrinogenolytic enzyme (albofibrase) and a plasminogen activator (GPV-PA), suggesting that defibrination syndrome in patients is a combination of these enzymatic effects. By multiple sequence alignment, no conserved residue or motif responsible for distinct functions of snake venom serine proteases could be observed. Moreover, one Lys 49 and one Asn 49 phospholipase A2 (PLA2) genes were cloned. Lys 49 PLA2 was predicted to devoid of catalytic activity, but showed a carboxy terminal cytotoxic region. No Asp 49 PLA2 was found in 150 clones screened. This explains the marked limb edema but no hemolysis in patients. These novel serine proteases have potentials to be therapeutic anti-thrombotic and thrombolytic agents in the future.     

Molecular cloning and sequence analysis of alboaggregin B

Green pit viper (Trimeresurus albolabris) venom contains a variety of C-type lectin-like proteins (CLPs) causing platelet aggregation and consumptive thrombocytopenia in biting victims. Alboaggregin B (AL-B), a heterodimeric glycoprotein (Gp) Ib-binding protein, was purified from the venom, but there is no reported cDNA sequence and the platelet agglutination mechanism is poorly understood. The full-length AL-B beta clone was obtained from T. albolabris venom gland cDNA library. AL-B alpha was, later, derived using 3'-RACE based on the conserved sequence. In this study, purified AL-B dimer agglutinated human platelets with the EC(50) of 180 nM and was completely inhibited by anti GpIb antibody. MALDI ToF mass spectroscopy found no glycosylation. The peptide mass fingerprints were matched with deduced amino acid sequences of cloned genes. AL-B alpha and beta contained 156 and 146 amino acid, respectively, including 23-residue signal peptides. AL-B beta showed the conserved hydrophilic patches, putative sites for GpIb binding. Furthermore, there was another conserved motif (SRTY) exclusively in platelet-agglutinating AL-B, TSV-GPIb-BP and Mamushigin. We propose that these three CLPs may function as bivalent adhesive proteins linking two GpIb molecules on adjacent platelets.     

Expression and characterization of a recombinant fibrinogenolytic serine protease from green pit viper (Trimeresurus albolabris) venom

Viper venom serine proteases (SPs) display several effects on hemostatic system. Molecular cloning showed that Trimeresurus albolabris venom comprised a mixture of five SPs with thrombin-like (2), fibrinogenase (2) and plasminogen-activating (1) activities. Because only few fibrinogenolytic SP sequences were reported, we decided to express albofibrase, a novel fibrinogenase from T. albolabris using Pichia pastoris system. The recombinant active form of enzyme was 30 kDa including 2.2 kDa of glycosylation. Albofibrase showed an fibrinogenase activity. In addition, a plasminogen activating and clotting effect were detectable. Albofibrase prolonged APTT and PT in a time-dependent manner. The effect was neutralized by pre-incubation with equine antivenom to T. albolabris. Therefore, the protein is potentially useful as a new anticoagulant as the antidote is clinically available. Sequence analysis compared with other snake venom fibrinogenases and SPs could not find any unique residues responsible for their various effects. Structure–function relationship should be further studied using mutagenesis in order to explore the mechanisms of venom protease functional diversity.