Inn-Ho Tsai

National Taiwan University, Biochemistry, Faculty Member

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Papers by Inn-Ho Tsai

Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species

Toxins

Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and i... more Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A2, 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrha...

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Full sequencing and comparison of five venom metalloproteases of Trimeresurus gracilis: The PI-enzyme is most similar to okinalysin but the PIII-enzyme is most similar to Crotalus venom enzymes

Toxicon

Determination of the structure of two novel echistatin variants and comparison of the ability of echistatin variants to inhibit aggregation of platelets from different species

Biochemical Journal, 1995

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi an... more Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These proteins were found to be about 85% similar in amino acid sequence to echistatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alpha. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD) but inhibit the aggregation of platelets from human and other mammals with different potencies. Echistatin beta and alpha are far more effective on platelets from humans and guinea pigs than those from rabbits and rats whereas echistatin gamma is less discriminating of the platelets of the species tested. This species-dependent platelet sensitivity to echistatin beta and gamma could be attributed to the variations in residues 15, 21, 22 and 27, which are close to or within the RGD loop, rather than to the C-terminal variations after residue 46. Taking advantage of the ...

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Terminal disialylated multiantennary complex-type N-glycans carried on acutobin define the glycosylation characteristics of the Deinagkistrodon acutus venom

Glycobiology, 2010

Glycosylation analysis of nonmammalian sources often springs surprises and conjures up intriguing... more Glycosylation analysis of nonmammalian sources often springs surprises and conjures up intriguing views of evolutionary adaptation. Many of the constituents of snake venoms are known to be glycosylated and yet very few were fully characterized and accorded specific functions. In the process of glycomic screening through the venoms from Asian pit vipers, a partially O-acetylated NeuAcα2-8NeuAcα2-3Galβ1-4GlcNAcβ1-terminal epitope was found to be the predominant glycosylation characteristic of the snake venom produced by the monotypic Deinagkistrodon acutus, with acutobin, a highly specific fibrinogenase, being identified as a primary protein carrier. Full structural definition and glycosylation site mapping were completed through advanced mass spectrometry analyses at both the glycan and glycopeptide levels in conjunction with chemical and enzymatic cleavages. Although similar occurrence of such terminal disialyl cap on the N-glycans of several mammalian glycoproteins has been implicated, most of these correspond to only minor constituents of the full glycomic heterogeneity and remain poorly characterized. In contrast, each antennae of the hybrid-and complex-type N-glycans derived from acutobin was found to be rather homogeneously disialylated. With up to eight sialic acids evenly distributed on nonextended tetraantennary core structure, these unusual N-glycans are among those of highest sialic acid density ever identified without actually carrying polysialic acid chains. It remains to be tested whether they may serve as multivalent disialyl ligands for several of the human Siglecs and thus meddle with the natural immuno-recognition systems of snakebite victims, apart from affecting the general efficacy of acutobin as anticoagulant in biomedical applications.

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Russtoxin: A new family of two-component phospholipase A 2 toxins from Russell’s vipers

Peptides, 1994

The Hemolymph Coagulation System of Shrimps

Current Aspects of Blood Coagulation, Fibrinolysis, and Platelets, 1993

The effects of different anticoagulants on the shrimp coagulation systems were studied. Metal ion... more The effects of different anticoagulants on the shrimp coagulation systems were studied. Metal ion chelators, Zn2+, Hg2+, serine protease inhibitors and some transglutaminase-inhibitors were found to inhibit the coagulation. We have purified the clottable proteins (CP) from the hemolymph of marine shrimps Penaeus monodon and Penaeus japonicus by DEAE-ion exchanger and gel-filtration. The purified CP was cross-linked to form an insoluble clot in vitro by the shrimp hemocyte transglutaminase (TG) in the presence of 10−4 M Ca2+. Being a dimer of 92 kDa subunits, the shrimp TG is homologous to Factor XIIIa of the mammalian coagulation system, but very unstable especially in presence of Ca2+. The soluble CP-dimers or oligomers are composed of glycoprotein subunits of 180 kDa, which contain 24% glucose and 9% Nacetylglucosamine. Its N-terminal amino acid sequence up to the 25th residue is LQPGLEYQYRYSARVASGIPSINRQ, bearing 72 and 28% similarity to the lobster CP and the C. elegans vitellogenin, respectively. Using the rabbit antiserum raised against the shrimp CP for western-blot analysis we found that the CP was present only in the hemolymph and hemopoietic nodule tissue but not in the midgut or the muscle of the shrimp. The rocket immunoelectrophoretic analyses showed specific changes of the hemolymph CP-concentration under different physiological conditions. The anti-CP antiserum also precipitated the CP’s in hemolymphs of other penaeid and metapenaeid shrimps but not those of lobster, fresh water prawn (Macrobrachium rosenbergii) or marine crab.

Group-II phospholipase A2 enzymes

Venomous Reptiles and their Toxins: Evolution, Pathophysiology and Biodiscovery (pp.335-340). Reino Unido: Oxford University Press, UK, May 1, 2015

Phospholipase A2 are esterolytic enzymes that catalyze the hydrolysis of 1,2-diacyl-3-sn-glycerop... more Phospholipase A2 are esterolytic enzymes that catalyze the hydrolysis of 1,2-diacyl-3-sn-glycerophospholipids at the C2 position, resulting in the formation of lysophospholipids and free fatty acid. They constitute one of the largest families of lipid hydrolyzing enzymes. PLA2s are known to be one of the most potent components found in snake venoms, where they exhibit a diverse array of pharmacological activities.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Structural and functional relationships of anticoagulant viperid venom phospholipases A2

Glycoprotein Ib-binding Protein from the Venom of Deinagkistrodon acutus – cDNA Sequence, Functional Characterization, and Three-Dimensional Modeling

Thrombosis and Haemostasis, 2000

SummaryAgkicetin-C, a potent glycoprotein Ib antagonist from the venom of the Chinese pit viper, ... more SummaryAgkicetin-C, a potent glycoprotein Ib antagonist from the venom of the Chinese pit viper, Deinagkistrodon acutus, has been purified and characterized (5). It is a disulfide-linked heterodimer containing subunits of 132 and of 123 amino acid residues. Herein, the complete amino acid sequences were resolved by cloning and nucleotide sequencing of the cDNAs. The sequences of its subunits are homologous to those of other snake venom proteins of the C-type (Ca2+-dependent) lectin superfamily. A three-dimensional model of agkicetin-C was constructed based on the crystal structure of habu coagulation factor IX/X-binding protein. By careful alignment of all the related sequences available and comparing the 3D-model of agkicetin-C with structures of other homologous proteins of different functions, some variable residues of agkicetin-C were identified, which possibly are responsible for the specificity of this distinct subtype of the C-type lectin-like venom proteins.

Revised amino acid sequences of the three major phospholipases A2 from Bungarus fasciatus (banded krait) venom

Toxicon, 1990

The structures of three cardiotoxin-like proteins obtained from the venom of Bungarus fasciatus (... more The structures of three cardiotoxin-like proteins obtained from the venom of Bungarus fasciatus (banded krait) were elucidated previously (Lu and Lo, 1980, 1981). Since their molecular sizes are similar to that of phospholipase A2 and since they show weak phospholipase A2 activities (Chang et al., 1983), a further study of their primary structures was carried out. Fractions Va, Vb-2 and VI, corresponding to the former V-2, V-3 and VI were determined to be typical phospholipases A2. Among 118 amino acid residues, they all have in common a Pro29 between Gly28 and Gly30, the latter two residues being implicated in Ca2+ binding together with Tyr26 and Asp47.

Structure of the heterodimeric neurotoxic complex viperotoxin F (RV-4/RV-7) from the venom of Vipera russelli formosensis at 1.9 Å resolution

Acta Crystallographica Section D Biological Crystallography, 2003

The presynaptic viperotoxin F is the major lethal component of the venom of Vipera russelli formo... more The presynaptic viperotoxin F is the major lethal component of the venom of Vipera russelli formosensis (Taiwan viper). It is a heterodimer of two highly homologous (65% identity) but oppositely charged subunits: a basic and neurotoxic PLA 2 (RV-4) and an acidic non-toxic component with a very low enzymatic activity (RV-7). The crystal structure of the complex has been determined by molecular replacement and re®ned to 1.9 A Ê resolution and an R factor of 22.3% with four RV-4/RV-7 complexes in the asymmetric unit, which do not exhibit any local point-group symmetry. The complex formation decreases the accessible surface area of the two subunits by $1425 A Ê 2. Both PLA 2 s are predicted to have very low, if any, anticoagulant activity. The structure of viperotoxin F is compared with that of the heterodimeric neurotoxin vipoxin from the venom of another viper, V. ammodytes meridionalis. The structural basis for the differences between the pharmacological activities of the two toxins is discussed. The neutralization of the negative charge of the major ligand for Ca 2+ , Asp49, by intersubunit salt bridges is probably a common mechanism of self-stabilization of heterodimeric Viperinae snake-venom neurotoxins in the absence of bound calcium.

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A Novel Heparin-dependent Inhibitor of Activated Protein C That Potentiates Consumptive Coagulopathy in Russell's Viper Envenomation

Journal of Biological Chemistry, 2012

Background: The reason behind the development of consumptive coagulopathies in Russell's viper bi... more Background: The reason behind the development of consumptive coagulopathies in Russell's viper bite patients is still elusive. Results: An inhibitor of activated protein C (a major physiological anticoagulant) synergizes with the venom's procoagulating enzymes. Conclusion: Down-regulation of activated protein C in Russell's viper envenomation is associated with consumptive coagulation. Significance: The discovery provides new insights into the pathogenesis of Russell's viper venom-induced coagulopathies.

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Sequence determination and bioinformatic comparison of ten venom serine proteases of Trimeresurus gracilis, a Taiwanese endemic pitviper with controversial taxonomy

Toxicon, 2022

Trimeresurus gracilis (Tgc) is endemic to Taiwan and shown to be closely related with Ovophis oki... more Trimeresurus gracilis (Tgc) is endemic to Taiwan and shown to be closely related with Ovophis okinavensis by previous phylogenetic analyses, but their taxonomic status remain controversial. Here, we cloned and sequenced ten of its venom serine-proteases (designated as Tgc-vSPs). All the Tgc-vSPs conserve the catalytic triads, six appear to be kallikrein-like (KNs) and four are plasminogen-activator homologs (PAHs and PAs). They are studied under four structural categories: (1) highly similar Tgc-KN1, Tgc-KN2 and Tgc-KN3, with four predicted N-glycosylation sites; (2) Tgc-KN4, with a single N -glycosylation site; (3) Tgc-KN5 and Tgc-KN6, with two distinct N-glycosylation sites; (4) Tgc-PAH1/PAH2, TgcPA3, and Tgc-PA4, with two conserved N-glycosylation sites. Additionally, Tgc-KN1, Tgc-KN4 and Tgc-PAH1 were purified by reversed-phase HPLC and identified by peptide-mass-fingerprinting. Results of BLAST and sequence alignments reveal that Tgc-KN1∼3 and Tgc-KN6 are most like the vSPs of rattlesnakes, while the sequences of Tgc-KN4, KN5 and Tgc-PAH1/PAH2 match closely to the partial sequences of three O. okinavensis vSPs. Thus, our results reveal non-overlapping similarities of Tgc-vSPs to the O. okinavensis vSPs and vSPs of the New World pitvipers. In addition, molecular phylogenetic analyses of the plasminogen-activator like vSPs reveal separate evolution of two clusters of the enzymes with distinct functions.

$Research paper thumbnail of {"__content__"=>"Tracing the evolution of venom phospholipases A in Gloydius strauchii and related pitvipers: A tale of two acidic isozymes.", "sub"=>{"__content__"=>"2"}}$

{"__content__"=>"Tracing the evolution of venom phospholipases A in Gloydius strauchii and related pitvipers: A tale of two acidic isozymes.", "sub"=>{"__content__"=>"2"}}

Toxicon : official journal of the International Society on Toxinology, 2018

Two acidic Asp-PLAs with Glu substitution and a neutral Lys-PLA (designated Gst-K49) were cloned ... more Two acidic Asp-PLAs with Glu substitution and a neutral Lys-PLA (designated Gst-K49) were cloned from G. strauchii venom glands, their full amino acid sequences were deduced. The predominant acidic PLA (designated Gst-E6a) contains 124 residues and the MW substitutions, while the minor acidic PLA (designated Gst-E6b) contains 122 residues and the VA substitutions. Their sequences are most similar to those of the respective orthologous PLAs of G. intermedius venom. Gst-E6a and Gst-E6b appear to be paralogs and possibly have different predatory targets or functions. The LC-MS/MS results indicate the presence of only three PLA gene products in the crude venom, the relative expression levels were in the order of Gst-E6a ≫ Gst-E6b > Gst-K49, as confirmed by qPCR results. In contrast to other Gloydius, G. strauchii venom does not contain neurotoxic or basic anticoagulant Asp-PLAs, but Gst-K49 is the first Lys-PLA identified in Gloydius venoms. However, its venom content is relatively l...

Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications

Journal of proteomics, Jan 5, 2017

Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomi... more Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1-4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like ...

Trimucytin: A collagen-like aggregating inducer isolated from Trimeresurus mucrosquamatus snake venom

Thrombosis and …, 1993

Trimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus sna... more Trimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytin- and collagen-induced platelet aggregation and ATP release.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of venom phospholipases A2 from six Daboia russelli geographic samples revealed the existence of two types of the Asia viper

Toxicon, 1997

Structures of Azemiops feae venom phospholipases and Cys-rich-secretory protein and implications for taxonomy and toxinology

Toxicon : official journal of the International Society on Toxinology, Jan 18, 2016

The Azemiops snakes are pit-less and phylogenetically located at the Crotalinae and Viperinae div... more The Azemiops snakes are pit-less and phylogenetically located at the Crotalinae and Viperinae divergence. cDNAs encoding five Azemiops venom phospholipase (sPLA2) molecules were cloned and sequenced; their signal-peptides were similar to those of crotalid sPLA2s. Based on their calculated pI-values and residue-49 substitutions, they were designated as Af-E6, Af-N49a, Af-N49a1, Af-N49a2, and Af-N49b, respectively. The first three isoforms, comprising 3-4% of the venom proteins, were purified by reversed-phase HPLC. Af-E6 is catalytically active and has >80% sequence-similarity to other Glu(6)-PLA2 (a pitviper venom-marker). Results of phylogenetic analyses reveal that acidic Af-N49a and Af-N49a1 are rather unique and loosely linked with crotalid PLA2s, while Af-N49b is related to the viperid PLA2s with Ser(1) substitution. Notably, the Asn(49)-substitutions in these molecules imply catalytic-independent mechanisms. The 3D-models of Af-E6 and Af-N49a have surface electropotential m...