Inn-Ho Tsai | National Taiwan University (original) (raw)

Papers by Inn-Ho Tsai

European journal of biochemistry, Dec 1, 1999

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 k... more To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca 2+ . The clottable protein contains 44% a helices and 26% b sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4±9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man 8 GlcNAc and Man 9 GlcNAc were released upon endo-b-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.

Biochemical Journal, Nov 1, 1995

Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic ne... more Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its complete primary structure was solved by an automated N-terminal sequencing and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1,g/ml and showed lethality in mice (LD50= 1.2,ug/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filtration experiment, and dissociates into monomers during SDS/PAGE in the absence of Ca2l. However, * To whom correspondence should be addressed. The sequence data reported in this paper have been deposited in the EMBL/GenBank Databases under the accession number X77645.

PubMed, Oct 1, 1999

Phospholipases A2 were purified from the venoms of Asian monotypic crotalinae snakes including Ca... more Phospholipases A2 were purified from the venoms of Asian monotypic crotalinae snakes including Callosellasma, Hypnale, Deinagkistrodon, and Tropidolaemus by a combination of gel filtration and reversed-phase chromatographic methods. One to four isoforms of the enzyme were found in each of the venoms. The venom enzymes were subjected to N-terminal sequencing up to the 30th amino acids, and their molecular weights were analyzed by electrospray-ionization mass spectrometry. Homologous antiplatelet phospholipase with a conserved Glu 6 residue was found in each of the venoms. Basic phospholipases with Trp 6 (W6) but without detectable enzyme activities were also isolated from the venom of C. rhodostoma, H. hypnale, and T. wagleri. These W6 enzymes showed strong heparin-binding affinity and capable of inducing edema in rat paws. The fact that the venoms of Callosellasma and Hypnale contain similar types of phospholipases is in accord with recent reports that these two taxa formed a clade. Deinagkistrodon venom does not contain phospholipase variants other than the Glu-6 subtype as Trimeresurus, Agkistrodon, and Protobothrops venoms do. Interestingly, the Glu-6 enzyme from T. wagleri venom has a molecular weight of 15,319 Daltons, higher than those of most other venom phospholipases. Our results show that new types of the enzyme are more likely to be found in the venom of monotypic species; the amino acid sequence data or the subtypes of venom-phospholipases are potentially useful as markers or a character system for studying higher-order systematics of venomous snakes.

Toxicon, Nov 1, 1998

of site directed mutagenesis on the activity of recombinant trimucrotoxin, a neurotoxic phospholi... more of site directed mutagenesis on the activity of recombinant trimucrotoxin, a neurotoxic phospholipase from Trimeresurus mucrosquamatus venom. Toxicon 36, 1591±1597, 1998.Ð Trimucrotoxin, the basic phospholipase A 2 from Trimeresurus mucrosquamatus venom, is neurotoxic and myotoxic, and structurally similar to crotoxin B subunit. To investigate the amino acid residues responsible for its neurotoxicity, we have mutated its interface-recognition residues including a conserved Asn6 in all the Crotalinae neurotoxic phospholipases. The wild-type and the mutants were expressed in E. coli as fusion-proteins and activated in vitro by factor Xa cleavage after folding. The completion of folding and activation were checked with electrospray ionization mass spectrometry and circular dichroism measurement. Enzymatic activities and neurotoxicities toward the chick tissue of four trimucrotoxin mutants (N6A, N6E, N6R and 6E7T8L) were compared with those of the wild type which was as active as that was isolated from the venom. Mutants N6A and N6E retained more than half of the original enzymatic activity but their neurotoxicities reduced to 33% and 10% that of the wild type, respectively. Mutants N6R and 6E7T8L retained 20±25% of the enzyme activity toward the anionic micellar substrate but were inactive toward the zwitterionic micellar substrate, and their neurotoxicities were less than 3% of that of the wild type. These results demonstrate the importance of residues 6±8 in trimucrotoxin for its neuronal speci®city and the speci®city toward potential substrates.

Journal of Biological Chemistry, Feb 1, 1982

Band 3, the anion transport protein of the human erythrocyte, provides the site of association of... more Band 3, the anion transport protein of the human erythrocyte, provides the site of association of certain glycolytic enzymes with the membrane. We have now demonstrated that glyceraldehyde-3-P dehydrogenase is inhibited, reversibly and completely, when membrane bound. The inhibition was competitive with respect to NAD+ and arsenate, but was noncompetitive with glyceraldehyde-3-P. Peptide fragments containing the NH2-terminal 23 residues of band 3 also inhibited the enzyme and displaced it from ghosts. Thus, the red cell membrane binding site for glyceraldehyde-3-P dehydrogenase is the same as that for aldolase, the polyanionic NH2-terminal region of the band 3 polypeptide.

$Research paper thumbnail of Crystallization and preliminary X-ray diffraction studies of the neurotoxic, heterodimeric phospholipase A<sub>2</sub>from the Taiwan viper (<i>Vipera russelli formosensis</i>)$

Acta Crystallographica Section D-biological Crystallography, May 1, 1999

The 28 kDa heterodimeric complex from Taiwan viper (F4/F7 complex) is composed of a neurotoxic ph... more The 28 kDa heterodimeric complex from Taiwan viper (F4/F7 complex) is composed of a neurotoxic phospholipase A2 (F4) and a non-toxic PLA2-like component (F7). Despite a high sequence identity (65%), the biological and pharmacological activities of F4 and F7 are contrasting. The complex is a structural analogue of Vipoxin found in the venom of the Bulgarian viper Vipera ammodites meridionalis. It is unclear how and why such varied bioactivities are expressed in these similar components. The F4/F7 complex has been crystallized using hanging-drop vapour diffusion and macroseeding techniques. The space group is monoclinic P21 with unit-cell dimensions a = 74.92, b = 85.13, c = 78.16 A and beta = 95.12 degrees. X-ray intensity data to 2.0 A resolution have been collected at 120 K and the structure has been solved using the molecular-replacement method. There are four F4/F7 complex molecules in the asymmetric unit, which do not exhibit any local point-group symmetry.

Toxin Reviews, 2007

The reaction mechanism of the 14 kDa secreted phospholipases A 2 (PLAs) and examples of venom PLA... more The reaction mechanism of the 14 kDa secreted phospholipases A 2 (PLAs) and examples of venom PLAs with diminished catalytic activities are reviewed. Evolutionary strategies to reduce the venom PLA catalytic power and new function gains are discussed. Down-regulations of the enzymatic activities appear to be due to: 1) retention of interfacial binding, but with selective alternation of active site residues in basic PLAs; 2) mutations at both the interfacial binding sites and catalytic sites of strong anticoagulating PLAs which bind to the coagulation factor; and 3) either substitution or truncation of the interface binding sites in acidic subunits of heterodimeric PLA-neurotoxins to generate chaperon like molecules.

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...

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 ...

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.

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.

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

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.

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.

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.

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.

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.