Agkistrodon Acutus Research Articles

Purification and Immunoprotection Evaluation of AaHIV from Complex Venom Metalloproteinases of Deinagkistrodon acutus.

The aim of this study was to investigate the immunoprotective effects of AaHIV in mice. After purification, a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Bicinchoninic acid was used to determine the molecular weight and concentration of AaHIV. AaHIV, venom complex (VC), and phosphate buffered saline (PBS) were subsequently used to immunize the mice three times, and the blood was sampled 1 week after the third immunization to determine the serum immunoglobulin G (IgG) antibody titer. A skin-bleeding inhibition assay and toxin-eliminating assay were performed on the immunized mice. The purity and concentration of AaHIV were 86.6% and 1.20 mg/mL, respectively. The AaHIV group exhibited higher antibody titers than the VC group. The survival rate of the AaHIV group (7/10) was significantly higher than that of the PBS group (0/10) (P = 0.0031). The high titer of antibodies induced by AaHIV partially neutralized the bleeding activity of the Deinagkistrodon acutus venom complex.

High-resolution hyaluronidase inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of anti-necrosis constituents in Clausena excavata

On average 100,000 persons are bitten by venomous snakes in China each year, with a mortality rate of 5 – 10% [1]. Gloydius blomhoffi brevicaudus, Deinagkistrodon acutus, Naja naja atra and Trimeresurus stejnegeri bites are most common. Necrotizing enzymes phospholipase A2, proteases, and hyaluronidase in the snake venom cause local tissue damage. Clausena excavata is a common plant used against snakebite in China. The aim of our study was to explore the anti-necrosis potential of plant C. excavata and identify non-tannin anti-necrosis constituents.

Homogeneous fluorescent specific PCR for the authentication of medicinal snakes using cationic conjugated polymers.

A label-free, homogenous and sensitive one-step method for the molecular authentication of medicinal snakes has been developed by combining a rapid PCR technique with water-soluble cationic conjugated polyelectrolytes (CCPs). Three medicinal snake materials (Deinagkistrodon acutus, Zaocys dhumnades and Bungarus multicinctus; a total of 35 specimens) and 48 snake specimens with similar morphologies and textures were clearly distinguished by the naked eye by utilizing a CCP-based assay in a high-throughput manner. The identification of medicinal snakes in patented Chinese drugs was successfully performed using this detection system. In contrast to previous fluorescence-labeled oligonucleotide detection and direct DNA stain hybridization assays, this method does not require designing dye-labeled primers, and unfavorable dimer fluorescence is avoided in this homogenous method.

Antibodies against Venom of the Snake Deinagkistrodon acutus.

Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10(8) and 5.28 × 10(8) transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future.

A novel fibrinogenase from Agkistrodon acutus venom protects against LPS-induced endotoxemia via regulating NF-κB pathway

Context: Endotoxins including lipopolysaccharide (LPS) could cause endotoxemia which often results in excessive inflammation, organ dysfunction, sepsis, disseminated intravascular coagulation (DIC) or even death. Previously, a novel fibrinogenase (FII) showed protective effects on LPS-induced DIC via activating protein C and suppressing inflammatory cytokines.Objective: To evaluate whether FII has protective effect on LPS-induced endotoxemia in mice and learn about the role of NF-κB pathway in TNF-α producing process.Methods: BALB/C mice were intraperitoneally injected (i.p.) with (a) 30 mg/kg LPS, (b) LPS + 0.3 mg/kg FII, (c) LPS + 1.0 mg/kg FII, (d) LPS + 3.0 mg/kg FII or (e) saline. Both survival rate and organ function were tested, including alanine aminotransferase (ALT), blood urine nitrogen (BUN) and tissue section, and TNF-α was examined by ELISA. RAW 264.7 macrophage was administered with (a) LPS, (b) LPS + FII, (c) FII alone or (d) saline, and TNF-α and phosphorylation (P)-NF-κB (P65) were determined by Western blot.Results: The administration of LPS led to 65% mortality rate, a rise of serum TNF-α, BUN and ALT levels, and both liver and renal tissue damage were observed. While FII treatment significantly increased the survival rate of LPS-induced endotoxemia mice model, histopathology and protein analysis results also revealed that FII remarkably protected liver and renal from LPS damage as well as decreasing TNF-α level. In vitro, FII significantly decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65).Conclusions: Our findings suggested that FII had protective effect on LPS-induced endotoxemia and organ injuries by suppressing the activation of NF-κB which decreased TNF-α level.

Design, synthesis of novel tryptophan derivatives for antiplatelet aggregation activity based on tripeptide pENW (pGlu-Asn-Trp)

pENW, a three mer peptide derived from Agkistrodon acutus Guenther venom, has been found to be an antagonist of the GPIIb/IIIa receptor and shows antiplatelet aggregation activity. Based on pENW and a GPIIb/IIIa inhibitor Tirofiban, a series of tryptophan derivatives were designed, synthesized and evaluated for their antiplatelet aggregation activity induced by ADP. The most potent compound 87 was also tested for the bleeding time and antithrombotic activity in vivo in comparison with Tirofiban. The results indicated that 87 shows similar antiplatelet aggregation activity as Tirofiban to the aggregation of platelet induced by all of the four agonists, but has lower bleeding risk than Tirofiban, representing a promising lead compound for further study.

Balancing the Expression and Production of a Heterodimeric Protein: Recombinant Agkisacutacin as a Novel Antithrombotic Drug Candidate

Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and β-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and β-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris.

Population-based study of venomous snakebite in Taiwan

The epidemiological features of snakebite are fragmented and inadequate in most countries. The true impact of snakebite is also very likely to be underestimated, and reliable information on its incidence, morbidity, and mortality is limited worldwide. We perform a nationwide epidemiological study of snakebite by extracting claim record data from Taiwan's National Health Insurance database to explore the epidemiology of venomous snakebite from years 2005 to 2009. A total of 4647 snakebites were reported in 2005–2009. The nationwide annual incidence of snakebite was 40.49 per million persons. Hemorrhagic-type snakebites (Viridovipera stejnegeri and Protobothrops mucrosquamatus) accounted for 71.78% of the cases, while neurotoxic-type snakebites (Naja atra and Bungarus multicinctus) accounted for 19.21%. Only a few cases of snakebites were caused by Deinagkistrodon acutus (0.73%). Although the east part of Taiwan accounted for only one-sixth of the total number of cases, the same area had the highest incidence of snakebite, about seven times the national incidence. Fifty-nine percent (n = 2747) of victims were between the age of 41 years and 70 years, and mostly in the age group of 51–60 years (n = 1026, 22%). The highest incidence (130.4 per million persons) was in the age group of 71–80 years. In general, snakebite victims suffered minor injuries. Overall, hospital admission was 35.8%, and only about 3.6% patients were needed to be admitted to the intensive care unit. During this study period, only two cases of mortality were documented. In this population-based study, the annual incidence of venomous snakebite was 40 per million people. Cases of venomous snakebite are geographically unevenly distributed in Taiwan. Due to effective antivenom therapy, the outcome of the snakebite patients was favorable.

Isolation and Characterization of a Novel Antithrombotic Peptide from Enzymatic Hydrolysate of Agkistrodon acutus Venom

Snake venoms contain a large variety of proteins and peptides that affect the hemostasis and thrombosis. Numerous antithrombotic peptides have been found in snake venom. However, few studies have been performed on the proteolysis of snake venom for obtaining bioactive peptides. In this study, the Agkistrodon acutus venom was hydrolyzed using four commercial proteases (pepsin, papain, neutrase, and alcalase) and the hydrolysate was tested for antiplatelet aggregation activity. The pepsin hydrolysate, exhibiting the highest activity, was further purified by gel filtration and reversed-phase high performance liquid chromatography. A novel antithrombotic peptide SP-14 was obtained, and its sequence was identified as SHIHGDYSSPSGAP using tandem electrospray mass spectrometry. SP-14 inhibited U46619-induced rabbit platelet aggregation in a dose-dependent manner. It reduced the mortality of mice in acute pulmonary thrombosis model and decreased thrombus weight in rat arteriovenous shunt model, while with lower bleeding risk than aspirin. Therefore, SP-14 may be beneficial for new antithrombotic drug design and development.

Effects of single N-glycosylation site knockout on folding and defibrinogenating activities of acutobin recombinants from HEK293T.

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 invitro 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 invivo, 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.

Application of rapid PCR to authenticate medicinal snakes

To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

Correlation between the glycan variations and defibrinogenating activities of acutobin and its recombinant glycoforms.

Acutobin isolated from Deinagkistrodon acutus venom has been used to prevent or treat stroke in patients. This defibrinogenating serine protease is a 39 kDa glycoprotein containing terminal disialyl-capped N-glycans. After sialidase treatment, the enzyme showed similar catalytic activities toward chromogenic substrate, and cleaved the Aα chain of fibrinogen as efficiently as the native acutobin did. However, the level of fibrinogen degradation products in mice after i.p.-injection of desialylated-acutobin was significantly lower than the level after acutobin injection, suggesting that the disialyl moieties may improve or prolong the half-life of acutobin. Two recombinant enzymes with identical protein structures and similar amidolytic activities to those of native acutobin were expressed from HEK293T and SW1353 cells and designated as HKATB and SWATB, respectively. Mass spectrometric profiling showed that their glycans differed from those of acutobin. In contrast to acutobin, HKATB cleaved not only the Aα chain but also the Bβ and γ chains of human fibrinogens, while SWATB showed a reduced α-fibrinogenase activity. Non-denaturing deglycosylation of these proteases by peptide N-glycosidase F significantly reduced their fibrinogenolytic activities and thermal stabilities. The in vivo defibrinogenating effect of HKATB was inferior to that of acutobin in mice. Taken together, our results suggest that the conjugated glycans of acutobin are involved in its interaction with fibrinogen, and that the selection of cells optimally expressing efficient glycoforms and further glycosylation engineering are desirable before a recombinant product can replace the native enzyme for clinical use.

Age-related Variation in Snake Venom: Evidence from Two Snakes (Naja atra and Deinagkistrodon acutus) in Southeastern China

Age-related Variation in Snake Venom: Evidence from Two Snakes (Naja atra and Deinagkistrodon acutus) in Southeastern China

Purification and partial characterization of a novel fibrinogenase from the venom of Deinagkistrodon acutus: Inhibition of platelet aggregation

A novel fibrinogenase, DAnase, was purified from the venom of Deinagkistrodon acutus by a combination of anion and cation exchange chromatography. Unlike other fibrinogenases which are usually single polypeptide chain proteins, the enzyme was a disulfide-linked dimer with an isoelectric point of 6.03 and an apparent molecular weight of 25kDa on SDS–polyacrylamide gel electrophoresis. DAnase showed α-fibrinogenase activity devoid of fibrinolytic activity. It hydrolyzed rapidly the Aα-chain of fibrinogen and followed by the Bβ-chain and did not cleave the γ-chain. It also exhibited arginine esterase activity. The fibrinogenolytic and arginine esterase activities were completely inhibited by phenylmethanesulfonyl fluoride or tris-(2-carboxyethyl)phosphine hydrochloride, but not by EDTA, indicating that DAnase is a serine protease requiring disulfide bridge(s) for its activity. The protease strongly inhibited ADP-induced platelet aggregation in human platelet-rich plasma but was lack of ADPase activity, indicating that its fibrinogenolytic activity is involved in its inhibition of ADP-induced platelet aggregation. DAnase was devoid of hemorrhagic activity and Factor XIII activation activity. DAnase may have a potential clinical application for the therapy of thrombosis disease.

A critical role for the regulation of Syk from agglutination to aggregation in human platelets

Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to integrin αIIbβ3-dependent aggregation in human platelets.

The First In Vitro and In Vivo Assessment Of Anfibatide, a Novel Glycoprotein Ib Antagonist, In Mice and In a Phase I Human Clinical Trial

BackgroundPlatelet adhesion and subsequent aggregation at the site of vascular injury are critical for hemostasis and thrombosis. It has been well accepted that interaction between the GPIb complex and von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear. Platelet surface integrin αIIbβ3, through interaction with fibrinogen or other ligands, then mediates platelet aggregation to form a stable hemostatic plug or thrombus. Recently, the indispensable role of the GPIb-VWF interaction in platelet aggregation at extremely high shear (e.g. > 10,000s-1; areas of stenosis following arteriosclerosis and/or thrombus growth) has been highlighted. Therefore, both the GPIb complex and αIIbβ3 are considered major targets for antithrombotic therapies. Interestingly, although several inhibitors of αIIbβ3 have been developed for antithrombotic therapies, no drug has been developed to target the GPIb complex even though there are limitations for anti-αIIbβ3 therapies. The GPIb complex is, therefore, an attractive target for anti-thrombotic therapy. Here, we evaluated the efficacy and safety in vitro and in vivo of Anfibatide, a novel GPIb antagonist, in mice and in a phase I clinical trial. MethodsAnfibatide was purified from venom of the Agkistrodon acutus snake and its purity was analyzed by mass spectrometry. The effect of Anfibatide on murine platelet function was assessed by in vitro platelet aggregometry, ex vivo perfusion chamber, and two complementary in vivo intravital microscopy models. The effects of Anfibatide on human platelet aggregation and thrombus formation were studied in vitro, and thrombealastography (TEG) was also performed. Most importantly, we evaluated the safety and efficacy of Anfibatide on platelet function and coagulation in a total of 94 healthy human volunteers in a phase I clinical trial. ResultsMALDI-TOF mass spectrometry of Anfibatide showed only one peak and the mass to charge ratio is 29799.7. Anfibatide specifically inhibited ristocetin-induced human platelet aggregation. Interestingly, Anfibatide was not able to inhibit botrocetin-induced murine platelet aggregation in plate-rich plasma (PRP), suggesting that its binding site may differ from other snake venom-derived GPIb antagonists. We found Anfibatide did not affect ADP-, TRAP- or collagen-induced aggregation in PRP, suggesting its specificity to GPIb. In ex vivo perfusion, Anfibatide strongly inhibited murine and human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface at both high and low shear flow conditions although it is far more sensitive at high shear. Importantly, Anfibatide effectively dissolved the preformed thrombi when we continuously perfused Anfibatide-treated whole blood through perfusion chambers, demonstrating its potential as an anti-thrombotic therapy. In the mesenteric arteriole thrombosis model, Anfibatide strongly inhibited platelet adhesion, thrombus formation, and prevented vessel occlusion in response to FeCl3 injury (P<0.05). At sites of laser-injured cremaster arterioles, Anfibatide also dramatically inhibited platelet accumulation and thrombus growth. Anfibatide did not cause significant murine platelet activation in vitro and had no significant change in coagulation parameters in TEG when we treated human whole blood with Anfibatide, suggesting it had minimal side effects. In the phase I clinical trial, results showed that Anfibatide can occupy approximately 95% of GPIb and inhibit up to 90% of ristocetin specific platelet aggregation. The inhibitory effect was undetectable four hours after Anfibatide was withdrawn. There were no serious adverse events, or deaths that occurred during the study. Anfibatide did not significantly prolong bleeding time, activated partial thromboplastin time (APTT), prothrombin time (PT), or thrombin time (TT). There was also no spontaneous bleeding or bleeding from blood collection sites. Anfibatide did not significantly affect platelet count and no anti-Anfibatide antibodies were detected in the subjects, suggesting that Anfibatide is well-tolerated in healthy individuals. ConclusionThese comprehensive studies in mice and human subjects and in the first clinical trial clearly demonstrated that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy. Disclosures:Hou:Lee's pharmaceutical holdings limited: Research Funding. Lei:Lee's pharmaceutical holdings limited: Research Funding. Zhao:Lee's pharmaceutical holdings limited: Research Funding. Shen:Lee's pharmaceutical holdings limited: Research Funding. Zhou:Lee's pharmaceutical holdings limited: Research Funding. Wang:Lee's pharmaceutical holdings limited: Research Funding. Marshall:Lee's pharmaceutical holdings limited: Research Funding. Ni:Lee's pharmaceutical holdings limited: Research Funding.

Extraction, purification and identification of type Ⅱ collagen from Agkistrodon acutus

The object of the research was to extract, purify and identify the type II collagen of Agkistrodon acutus. Type II collagen of A. acutus was extracted by enzyme decomposition method, and purified by ion exchange column chromatography. It was characterized by SDS-PAGE gel electrophoresis, ultraviolet spectrophotometry, infrared absorption spectroscopy and mass spectroscopy. The results showed that the size of C II was about 130 kDa. It absorbed at 223 nm. IR spectrum obtained showed that the triple helical domains of amino-acid sequences were characterized by the repetition of triplets Gly-X-Y. The MS spectrum graphically stated that C II extracted from cow and A. acutus have the similar peptides. The C II of A. acutus was obtained by extraction and purification. Appraisal analysis by SDS-PAGE, UV, IR and MS, C II of A. acutus was consistent with the standard C II of cow. It was proved that the extracted protein was C II.

Mitochondrial mechanisms of apoptosis of human leukemia K562 cells induced by AVVC-1

This study was purpose to investigate apoptosis pathway of leukemia K562 cells induced by anticoagulant fraction from Agkistrodon acutus venom (AVVC-1). The mitochondrial transmembrane potential (ΔΨm) of leukemia K562 cells was detected by flow cytometry with JC-1 single staining. The expression of cytochrome C in the mitochondrial of leukemia K562 cells was analyzed by Western blot after AVVC-1 treatment. The distribution of cytochrome C in leukemia K562 cells was measured by immuno-fluorescence test. The results showed that the potential of mitochondrial membrane decreased after treatment with different concentrations of AVVC-1 (12.5, 25, 50, 100 µg/ml) for 6 h (P < 0.01). The expression level of cytochrome C protein in mitochondria obviously declined after treatment with 30 µg/ml AVVC-1 for 48 h, and the fluorescent intensity of cytochrome C in cytosol was enhanced at the same time. It is concluded that AVVC-1-induced K562 cell apoptosis is related with mitochondrial damage, and cytochrome C may be a useful agent for investigating human leukemia therapy by using AVVC-1.

Abstract 5077: Anti-angiogenesis and anti-cancer activities of ZK002, a C-type lectin-like protein isolated from snake venom.

Abstract Snake venoms contain various biologically active proteins including C-type lectins and -like proteins (CTLs), phospholipases, proteinases and metalloproteinases, that could target diverse cellular processes. Among these active components, CTLs have demonstrated antithrombotic effects through their platelet agglutination and coagulation activities. In our study, we have detected anti-angiogenesis activity from the venom of Deinagkistrodon acutus snake. The partial purified fraction of snake venom with anti-angiogenesis activity was isolated through a stepwise separation in anion-exchange and cation-exchange chromatography. At the same time, the monoclonal antibody was generated and used for further protein purification by affinity chromatography. This highly purified protein was named as ZK002. By MALDI-TOF mass spectrometry determination, the mass of ZK002 is about 30KD. By LC-MS/MS analysis, protein sequence of highly purified ZK002 was determined and compared with the Deinagkistrodon acutus snake protein database. ZK002 is a heterodimer of CTLs consisting of alpha and beta peptide chains. Using In vitro chicken embryo chorioallantoic membrane assay and human umbilical vein endothelial cell (HUVEC) tube formation assay, it showed that ZK002 exhibited an anti-angiogenesis effect. Moreover, ZK002 also demonstrated an anti-cancer effect as shown in the ApcMin/+ mouse model for colorectal cancer. Administration of ZK002 significantly suppressed the total number and the size of tumors and showed no observable adverse effect. To further elucidate the anti-angiogenesis functions of ZK002, we deduced the coding DNA sequences from the peptide sequences of the alpha- and beta-chains, then subcloned individual gene sequence into yeast and mammalian expression vectors. Results showed that the recombinant vectors express secreted peptides with the expected molecular weight. Transient transfection of the alpha- and beta-recombinant vectors individually or in combination inhibit tube formation in HUVEC cells, suggesting that both alpha- and beta-chains possess anti-angiogenesis activity. The success of cloning and expression of recombinant ZK002 would greatly facilitate the future development of ZK002 as anti-angiogenesis and anti-cancer drugs. (This work is supported by the Innovation and Technology Fund (UIM208) of Hong Kong Government) Citation Format: Shan Yu, Xiangrong Dai, William C.S. Tai, Roy C.Y. Choi, Chaofan Liang, Benjamin X.y. Li, Karl W.K. Tsim, Wendy W.L. Hsiao. Anti-angiogenesis and anti-cancer activities of ZK002, a C-type lectin-like protein isolated from snake venom. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5077. doi:10.1158/1538-7445.AM2013-5077

Purification, characterization and gene cloning of Da-36, a novel serine protease from Deinagkistrodon acutus venom

A serine protease termed Da-36 was isolated from crude venom of Deinagkistrodon acutus. The enzyme was a single chain protein with an apparent molecular weight of 36,000 on SDS–PAGE with an isoelectric point of 6.59. Da-36 could clot human plasma by cleaving the Aα, Bβ and γ chains of fibrinogen and also exhibited arginine esterase activity. The proteolytic activity of Da-36 toward TAME was strongly inhibited by PMSF and moderately affected by benzamidine and aprotinin, indicating that it was a serine protease. Meanwhile, Da-36 showed stability with wide temperature (20–50 °C) and pH value ranges (pH 6–10). Divalent metal ions of Ca2+, Mg2+, and Mn2+ had no effects but Zn2+ and Cu2+ inhibited the arginine esterase activity of Da-36. Total DNA was extracted directly from the lyophilized crude venom and the gene (5.5 kbp) coding for Da-36 had been successfully cloned. Sequence analysis revealed that the Da-36 gene contained five exons and four introns. The mature Da-36 was encoded by four separate exons. The deduced mature amino acid sequence of Da-36 was in good agreement with the determined N-terminal sequence of the purified protein and shared high homology with other serine proteases isolated from different snake venoms. Blast search using amino acid sequence of Da-36 against public database revealed that Da-36 showed a maximal identity of 90% with both Dav-X (Swiss-Prot: Q9I8W9.1) and thrombin-like protein 1 (GenBank: AAW56608.1) from the same snake species, indicating that Da-36 is a novel serine protease.