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Kavi Ratanabanangkoon - One of the best experts on this subject based on the ideXlab platform.
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A novel in vitro potency assay of antisera against Thai Naja kaouthia based on nicotinic acetylcholine receptor binding
Scientific Reports, 2017Co-Authors: Kavi Ratanabanangkoon, Kae Yi Tan, Sukanya Eursakun, Choo Hock Tan, Pavinee Simsiriwong, Kritsada Pruksaphon, Bunkuea Chantrathonkul, Wongsakorn Wongwadhunyoo, Sirida Youngchim, Nget Hong TanAbstract:Snake envenomation is an important medical problem. One of the hurdles in antivenom development is the in vivo assay of antivenom potency which is expensive, gives variable results and kills many animals. We report a novel in vitro assay involving the specific binding of the postsynaptic neurotoxins (PSNTs) of elapid snakes with purified Torpedo californica nicotinic acetylcholine receptor (nAChR). The potency of an antivenom is determined by its antibody ability to bind and neutralize the PSNT, thus preventing it from binding to nAChR. The PSNT of Naja kaouthia (NK3) was immobilized on microtiter wells and nAChR was added to bind with it. The in vitro IC50 of N. kaouthia venom that inhibited 50% of nAChR binding to the immobilized NK3 was determined. Varying concentrations of antisera against N. kaouthia were separately pre-incubated with 5xIC50 of N. kaouthia venom. The remaining free NK3 were incubated with nAChR before adding to the NK3 coated plates. The in vitro and in vivo median effective ratio, ER50s of 12 batches of antisera showed correlation (R 2) of 0.9809 (p
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a novel in vitro potency assay of antisera against thai Naja kaouthia based on nicotinic acetylcholine receptor binding
Scientific Reports, 2017Co-Authors: Kavi Ratanabanangkoon, Kae Yi Tan, Sukanya Eursakun, Choo Hock Tan, Pavinee Simsiriwong, Kritsada Pruksaphon, Bunkuea Chantrathonkul, Wongsakorn WongwadhunyooAbstract:Snake envenomation is an important medical problem. One of the hurdles in antivenom development is the in vivo assay of antivenom potency which is expensive, gives variable results and kills many animals. We report a novel in vitro assay involving the specific binding of the postsynaptic neurotoxins (PSNTs) of elapid snakes with purified Torpedo californica nicotinic acetylcholine receptor (nAChR). The potency of an antivenom is determined by its antibody ability to bind and neutralize the PSNT, thus preventing it from binding to nAChR. The PSNT of Naja kaouthia (NK3) was immobilized on microtiter wells and nAChR was added to bind with it. The in vitro IC50 of N. kaouthia venom that inhibited 50% of nAChR binding to the immobilized NK3 was determined. Varying concentrations of antisera against N. kaouthia were separately pre-incubated with 5xIC50 of N. kaouthia venom. The remaining free NK3 were incubated with nAChR before adding to the NK3 coated plates. The in vitro and in vivo median effective ratio, ER50s of 12 batches of antisera showed correlation (R 2) of 0.9809 (p < 0.0001). This in vitro assay should be applicable to antisera against other elapid venoms and should reduce the use of live animals and accelerate development of life-saving antivenoms.
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Venomics of Naja kaouthia (Vietnam) venom and TF under RP-HPLC.
2016Co-Authors: Kavi Ratanabanangkoon, Kae Yi Tan, Sukanya Eursakun, Choo Hock Tan, Pavinee Simsiriwong, Teeraporn Pamornsakda, Witthawat Wiriyarat, Chaiya Klinpayom, Nget Hong TanAbstract:Venomics of Naja kaouthia (Vietnam) venom and TF under RP-HPLC.
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A study of Thai cobra (Naja kaouthia) bites in Thailand.
Military Medicine, 2005Co-Authors: Nualnong Wongtongkam, Henry Wilde, Chitr Sitthi-amorn, Kavi RatanabanangkoonAbstract:ABSTRACT This study evaluated factors affecting the severity of bite site necrosis and systemic symptoms resulting from envenomation among patients bitten by Thai cobras (Naja kaouthia) in Thailand. We studied 45 victims prospectively. An additional 40 medical records were obtained for a retrospective study. Collected data included gender of the victims, anatomic locations of bites, where attacks took place, and predisposing factors and how they might have affected the clinical course. Most patients were asymptomatic or mildly symptomatic. Neurotoxic symptoms and respiratory failure developed in 31.11% and 12.50% in the prospective and retrospective groups, respectively. Only one patient died, from the effects of prolonged respiratory failure. There was some degree of tissue necrosis at the bite site for almost all victims. One victim required amputation of a digit in the retrospective study, and 33.60% of the prospective group and 20% of the retrospective group required minor surgical debridement. Snakeb...
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Preparations of toxic components from Naja kaouthia venom by selective heat denaturation.
Journal of Natural Toxins, 1998Co-Authors: T. Saetang, Porntip Suttijitpaisal, Kavi RatanabanangkoonAbstract:A simple procedure to prepare the toxic components from Naja kaouthia venom for use as immunogens has been studied. The aim was to produce serum rich in antitoxins. By heating the venom (1-6 mg/ml) at 100 degrees C for 10 min at pH 5.0, at least 10 proteins with MW greater than 25,000 daltons were precipitated and removed. The toxic components, i.e., postsynaptic toxins, Direct Lytic Factor (DLF), and phospholipase A2 were relatively stable to this treatment; however, their activities were progressively lost as the heating time was prolonged. The LD50S of the heated (100 degrees C, 10 min) and the unheated venom were 0.37 and 0.325 mg/kg, respectively. As compared to the unheated venom, immunization of rabbits with the heated venom resulted in a 3.38-fold increase in precipitable antibodies against N. kaouthia toxin 3 and a 1.85-fold increase in neutralizing capacity. This toxin preparation should be useful as an immunogen or as a starting material for chemical modification prior to immunization in the production of potent therapeutic antiserum.
Lawan Chanhome - One of the best experts on this subject based on the ideXlab platform.
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Genome-wide SNP analysis of Siamese cobra (Naja kaouthia) reveals the molecular basis of transitions between Z and W sex chromosomes and supports the presence of an ancestral super-sex chromosome in amniotes.
Genomics, 2020Co-Authors: Nararat Laopichienpong, Lawan Chanhome, Worapong Singchat, Siwapech Sillapaprayoon, Narongrit Muangmai, Sudarath Baicharoen, Sunutcha Suntrarachun, Surin Peyachoknagul, Ekaphan Kraichak, Tariq EzazAbstract:Elucidation of the process of sex chromosome differentiation is necessary to understand the dynamics of evolutionary mechanisms in organisms. The W sex chromosome of the Siamese cobra (Naja kaouthia) contains a large number of repeats and shares amniote sex chromosomal linkages. Diversity Arrays Technology provides an effective approach to identify sex-specific loci that are epoch-making, to understand the dynamics of molecular transitions between the Z and W sex chromosomes in a snake lineage. From a total of 543 sex-specific loci, 90 showed partial homology with sex chromosomes of several amniotes and 89 loci were homologous to transposable elements. Two loci were confirmed as W-specific nucleotides after PCR amplification. These loci might result from a sex chromosome differentiation process and involve putative sex-determination regions in the Siamese cobra. Sex-specific loci shared linkage homologies among amniote sex chromosomes, supporting an ancestral super-sex chromosome.
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Complete mitochondrial genome of Siamese cobra (Naja kaouthia) determined using next-generation sequencing
Mitochondrial DNA Part B, 2019Co-Authors: Worapong Singchat, Lawan Chanhome, Prapatsorn Areesirisuk, Siwapech Sillapaprayoon, Narongrit Muangmai, Sudarath Baicharoen, Sunutcha Suntrarachun, Surin Peyachoknagul, Kornsorn SrikulnathAbstract:Siamese cobra (Naja kaouthia) exhibits highly toxic venom, which causes morbidity and mortality. Accurate species identification through molecular approaches is very important to administer correct antivenoms. The Siamese cobra mitogenome contains 17,203 bp with slight AT bias (58.2%) containing 37 genes in identical order to snake mitogenomes; no tandem repeat was found in the control region. Phylogenetic analysis indicated that Siamese and other cobras had highly supported monophyletic clades similar to the genus Naja and close relationships with other elapid snakes. Our results will facilitate clinical diagnosis and enrich genomic resources for future evolutionary studies and conservation management.
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The survey of internal parasites of consumed - Siamese cobra (Naja kaouthia) in Thailand.
Veterinary Parasitology: Regional Studies and Reports, 2017Co-Authors: Taksa Vasaruchapong, Panithi Laoungbua, Tanapong Tawan, Lawan ChanhomeAbstract:In Asia and Africa, snake meat and organs are commonly prepared for consumption as traditional medicine or aphrodisiac regardless of the awareness of zoonotic diseases. Parasitic zoonosis can be contracted from the consumption of raw reptile and amphibian meat. Therefore to study this further, forty wild-caught Siamese cobras (Naja kaouthia) which were supplied to exotic restaurants in Thailand were randomized for fecal and blood examination as well as necropsy to dissect for the presence of parasites. Six groups of internal parasites and one blood parasite were found. The prevalence of rhabditids, Kalicephalus spp. and pentastomids were 82.5% (32/40), 60% (24/40) and 22.5% (9/40), respectively. Pentastomids are one of the potential zoonotic parasites reported in Thailand and other countries. But there is no report of pentastomids in Siamese cobra. Therefore, the further species identification of pentastome should be performed to raise community awareness on snake consumption.
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Surgical Removal of Foreign Bodies in the Gastrointestinal Tract of Monocellate Cobra, Naja kaouthia
Thai Journal of Veterinary Medicine, 2013Co-Authors: Taksa Vasaruchapong, Lawan ChanhomeAbstract:A wild-captured female monocellate cobra (Naja kaouthia) had abnormal posture and move with difficulty as consequences of swelling at the middle third of the body. The swelling area was firm on palpation with average size of 10x4 cm. The shape of the swelling area was similar to a small bottle which was obviously recognized on the dorsal recumbency. Therefore, the foreign body obstruction in gastrointestinal tract was diagnosed without radiographic examination. Digital manipulation was performed, but could not move the foreign bodies. Therefore, gastrotomy was considered. The foreign bodies were a plastic bottle and a piece of cloth which were removed. Feeding was withdrawn for one week with parenteral supportive treatment with fluid. The snake fully recovered 1 month after surgery.
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reproductive patterns of captive male and female monocled cobra Naja kaouthia lesson 1831
Zoological Studies, 2012Co-Authors: Panas Tumkiratiwong, Lawan Chanhome, Worawitoo Meesuk, Anchalee AowpholAbstract:We monitored the morphology and histology of male and female reproductive systems and plasma levels of male testosterone and female estradiol to describe the reproductive pattern of captive monocled cobras (Naja kaouthia). Gonads were collected in May and Nov. 2007, and monthly blood samples were collected from Jan. to Dec. 2007, from captive snakes at the Queen Saovabha Memorial Institute, Bangkok, Thailand. Male testes were hypertrophic in May corresponding to spermatogenetic events and had regressed in Nov. in the absence of spermatogenetic events. We found that spermatozoa were stored in the epididymides in both May and Nov. Male testosterone levels peaked in Oct. preceding the Nov. mating time. In May, the granulose layer of previtellogenic follicles contained 3 distinct cell types of small, intermediate, and large pyriform cells. Intermediate and large pyriform cells had disappeared from the granulosa layer of the vitellogenic follicles in Nov. Three follicular types of atretic, previtellogenic, and vitellogenic follicles were common inside the ovaries in Nov. The corpora lutea had regressed to a corpora atretica in Nov. The plasma level of female estradiol surged in Nov. coincident with the mating time and vitellogenic events. We suggest that the reproductive pattern of captive monocled cobras exhibits either postnuptial spermatogenesis or a dissociated reproductive pattern.
Zakhar O. Shenkarev - One of the best experts on this subject based on the ideXlab platform.
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Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors.
Toxicon, 2016Co-Authors: Ekaterina N. Lyukmanova, Mikhail A. Shulepko, Zakhar O. Shenkarev, Igor E. Kasheverov, Anton O. Chugunov, D.s. Kulbatskii, Mikhail Yu. Myshkin, Yuri N. Utkin, Roman G. Efremov, Victor I. TsetlinAbstract:Abstract ‘Three-finger’ toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with 125 I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.
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structural insight into specificity of interactions between nonconventional three finger weak toxin from Naja kaouthia wtx and muscarinic acetylcholine receptors
Journal of Biological Chemistry, 2015Co-Authors: Ekaterina N. Lyukmanova, Mikhail A. Shulepko, Zakhar O. Shenkarev, Alexander S Paramonov, Anton O. ChugunovAbstract:Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
Ekaterina N. Lyukmanova - One of the best experts on this subject based on the ideXlab platform.
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Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors.
Toxicon, 2016Co-Authors: Ekaterina N. Lyukmanova, Mikhail A. Shulepko, Zakhar O. Shenkarev, Igor E. Kasheverov, Anton O. Chugunov, D.s. Kulbatskii, Mikhail Yu. Myshkin, Yuri N. Utkin, Roman G. Efremov, Victor I. TsetlinAbstract:Abstract ‘Three-finger’ toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with 125 I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.
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structural insight into specificity of interactions between nonconventional three finger weak toxin from Naja kaouthia wtx and muscarinic acetylcholine receptors
Journal of Biological Chemistry, 2015Co-Authors: Ekaterina N. Lyukmanova, Mikhail A. Shulepko, Zakhar O. Shenkarev, Alexander S Paramonov, Anton O. ChugunovAbstract:Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
Yuri N. Utkin - One of the best experts on this subject based on the ideXlab platform.
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Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors.
Toxicon, 2016Co-Authors: Ekaterina N. Lyukmanova, Mikhail A. Shulepko, Zakhar O. Shenkarev, Igor E. Kasheverov, Anton O. Chugunov, D.s. Kulbatskii, Mikhail Yu. Myshkin, Yuri N. Utkin, Roman G. Efremov, Victor I. TsetlinAbstract:Abstract ‘Three-finger’ toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with 125 I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.
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Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors
FEBS Journal, 2009Co-Authors: Dmitry Y. Mordvintsev, Victor I. Tsetlin, Dmitry I. Rodionov, Yakov L. Polyak, Jan Jakubík, Vladimir Dolezal, Evert Karlsson, Yuri N. UtkinAbstract:Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ-specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX-binding sites. Specific [125I]WTX binding was partially inhibited by both alpha-cobratoxin, a blocker of the alpha7 and muscle-type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0+/-0.8 microM and was inhibited by alpha-cobratoxin but not by a short-chain alpha-neurotoxin antagonist of the muscle-type nAChR, suggesting a specific interaction with the alpha7-type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 microM did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N-methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G-protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G-proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.
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Behavioural effects in mice and intoxication symptomatology of weak neurotoxin from cobra Naja kaouthia
Basic & Clinical Pharmacology & Toxicology, 2007Co-Authors: Dmitry Y. Mordvintsev, Victor I. Tsetlin, Alexey Ya. Ogay, Dmitry I. Rzhevsky, Arkady N. Murashev, Dmitry I. Rodionov, Mariya V. Makarova, A.a. Kamensky, Natalya G. Levitskaya, Yuri N. UtkinAbstract:Weak neurotoxins belong to the superfamily of three-finger toxins from snake venoms. In general, weak toxins have a low toxicity and, contrary to other three-finger toxins, their molecular targets are not well characterized: in vitro tests indicate that these may be nicotinic acetylcholine receptors. Here, we report the influence of intraperitoneal and intravenous injections of weak neurotoxin from Naja kaouthia venom on mouse behaviour. Dose-dependent suppression of orientation-exploration and locomotion activities as well as relatively weak neurotropic effects of weak neurotoxin were observed. The myorelaxation effect suggests a weak antagonistic activity against muscle-type nicotinic acetylcholine receptors. Neurotoxic effects of weak neurotoxin were related to its influence on peripheral nervous system. The symptomatology of the intoxication was shown to resemble that of muscarinic agonists. Our data suggest that, in addition to interaction with nicotinic acetylcholine receptors observed earlier in vitro, weak neurotoxin interacts in vivo with some other molecular targets. The results of behavioural experiments are in accord with the pharmacological profile of weak neurotoxin effects on haemodynamics in mice and rat indicating the involvement of both nicotinic and muscarinic acetylcholine receptors.
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Weak neurotoxin from Naja kaouthia cobra venom affects haemodynamic regulation by acting on acetylcholine receptors.
Toxicon, 2004Co-Authors: Alexey Ya. Ogay, Victor I. Tsetlin, Dmitry I. Rzhevsky, Arkady N. Murashev, Yuri N. UtkinAbstract:Recent in vitro studies of weak neurotoxins from snake venoms have demonstrated their ability to interact with both muscle-type and neuronal α7 nicotinic acetylcholine receptors (nAChR). However, the biological activity in vivo of weak neurotoxins remains largely unknown. We have studied the influence of weak neurotoxin (WTX) from the venom of cobra Naja kaouthia on arterial blood pressure (BP) and heart rate (HR) in rats and mice. It was found that intravenous injection of WTX induced a dose-dependent decrease in BP and an increase in HR in both species, the rats being more sensitive to WTX. Application of WTX following blockade of nAChRs or muscarinic acetylcholine receptors (mAChR) by hexamethonium or atropine, respectively, showed that both nAChRs and mAChRs are involved in the haemodynamic effects of WTX. Blockade of either nAChRs or mAChRs affected WTX action differently in rats and mice, thus reflecting interspecies differences in haemodynamic regulation.
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The first representative of glycosylated three-fingered toxins. Cytotoxin from the Naja kaouthia cobra venom.
FEBS Journal, 2004Co-Authors: Alexey V. Osipov, M. V. Astapova, Victor I. Tsetlin, Yuri N. UtkinAbstract:There are different glycosylated proteins in snake venoms, but no glycosylated representatives of a large family of three-fingered toxins have previously been detected. A new glycoprotein was isolated from the venom of the Thai cobra Naja kaouthia. MALDI MS of the glycoprotein contained an array of peaks in the range from ≈ 8900 to ≈ 9400 Da indicating its microheterogeneity. Carbohydrate analysis showed the presence of mannose, galactose, N-acetylglucosamine, fucose and neuraminic acid. The N-terminal sequence of the glycoprotein was identical to that of cytotoxin 3 (CX3) from N. kaouthia, and CD spectra of the glycoprotein and CX3 were almost the same. Cleavage of a glycan moiety by N-glycosidase F gave a protein of molecular mass practically coinciding with that of CX3. MALDI MS of the tryptic digest of reduced glycoprotein S-pyridylethylated at cysteine residues, contained peaks corresponding to all tryptic fragments of CX3, with the exception of fragment 24–30. The peak corresponding to this peptide appeared in the mass-spectrum of similarly treated deglycosylated glycoprotein. These data show that the potential N-glycosylation site at Asn29 in CX3 is utilized for glycan attachment and that the glycoprotein is glycosylated CX3. In vivo toxicity of the glycoprotein to the cricket Gryllus assimilis was twofold lower than that of CX3. The cytotoxic activity of the glycoprotein towards HL60 cells was about two orders of magnitude lower than that of CX3, but could be made equal to the CX3 cytotoxicity by deglycosylation. Thus for the first time we have isolated a glycosylated three-fingered snake venom toxin wherein glycosylation appears to modulate its biological activity.
