Chee Hong Tan

Lophozozymus pictor toxin: A fluorescent structural isomer of palytoxin

Purified Lophozozymus pictor toxin (LPTX) shares many properties similar to palytoxin (PTX). LPTX and palytoxin isolated from Palythoa caribaeorum (C-PTX) have similar mol. wts of approx. 2680 on ionspray mass spectrometry (MS). In addition, antibodies against PTX could recognize and bind LPTX. Mixed mode high-performance liquid chromatography (HPLC) of LPTX, C-PTX and H-PTX (isolated from Palythoa tuberculosa) showed a major PTX component common to all three with the characteristic PTX-like UV spectrum at a retention time (Rt) of 17 min. However, LPTX exhibits fluorescence but PTX of equivalent toxicity does not. LPTX showed a unique peak at Rt of approx. 22 min on mixed mode HPLC. In addition, LPTX and C-PTX showed different ion fragmentation patterns on MS/MS. These results suggest that LPTX and the palytoxins are structural isomers, containing at least one difference which gives rise to fluorescence in LPTX.

Four new postsynaptic neurotoxins from Naja naja sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis

cDNAs encoding 4 short chain alpha-neurotoxins from Malayan spitting cobra (Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to alpha-neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups, 10E11T and 10Q11A, which could be further subdivided into 10E11T28S: 10E11T28G and 10Q11A28S; 10Q11A28G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.

SIX ISOFORMS OF CARDIOTOXIN IN MALAYAN SPITTING COBRA (NAJA NAJA SPUTATRIX) VENOM : CLONING AND CHARACTERIZATION OF CDNAS

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Molecular cloning of a cardiotoxin structural gene from Malayan spitting cobra (Naja naja sputatrix)

The structural gene and cDNA encoding cardiotoxin in Naja naja sputatrix have been cloned and characterized with a view to study the gene protein relationships and also to produce pure protein in large amounts. Using the polymerase chain reaction on the total RNA isolated from the venom glands, the structural gene (180 bp) has been synthesized and expressed in Escherichia coli to produce a fusion protein with beta-galactosidase. Immunoblotting using polyclonal antibodies raised against the total venom in rabbits demonstrated the presence of cross-reacting proteins in plaques produced by recombinant lambda gt11 phages.

Isolation of a novel fluorescent toxin from the coral reef crab, Lophozozymus pictor

A novel toxin has been isolated from an aqueous extract of the coral reef crab Lophozozymus pictor by a combination of ion exchange and two-dimensional thin layer chromatography. The isolated toxin exhibited bioactivities similar to palytoxin (toxicity response of mice, cytotoxicity towards HeLa cells and release of potassium ions from rat erythrocytes). Unlike palytoxin it gave an intense blue fluorescence under ultraviolet light. A fluorescence scan showed that the toxin had a maximum excitation wavelength at 285 nm and a maximum emission wavelength at 355 nm. The toxin was distinguishable from palytoxin when analysed by high performance capillary electrophoresis and reverse phase high performance liquid chromatography.

Bioactivity and mechanism of action of Lophozozymus pictor toxin

The bioactivity of Lophozozymus pictor toxin (LPTX) and the possible mechanism of action of the purified toxin are described. LPTX is found to possess palytoxin-like bioactivities. Besides exhibiting cytotoxic and haemolytic properties, LPTX causes the release of K+ from erythrocytes and inhibits 2-[14C]deoxy-D-glucose uptake into HeLa cells. Although LPTX acts on HeLa cell and erythrocyte membranes, it does not interact with mitochondrial or liposomal membranes containing different phospholipid compositions. Ouabain, but not sphingomyelin, is able to prevent the toxic effects of LPTX. This antagonistic effect of ouabain on LPTX suggests that the toxin might mediate its toxic effects via the membrane Na+/K(+)-ATPase but not through interaction with membrane lipids.

Localization of toxins in the poisonous mosaic crab, Lophozozymus pictor (Fabricius, 1798) (Brachyura, Xanthidae)

A hot aqueous extraction method was used to extract toxins from different parts of the poisonous coral reef crab, Lophozozymus pictor. Contrary to an earlier report that crab toxins do not show localization, the toxicity of L. pictor was found to be consistently high in the gut and hepatopancreas, whereas the muscle was less toxic and the carapace was mildly or non-toxic. Crabs kept in captivity for 10 days showed reduction in toxicity in all parts studied with almost complete loss of toxicity after 24 days.

An agar-diffusion method for quantitative determination of ribonuclease

Abstract 1. 1. A sensitive agar-diffusion slide method has been developed for the assay of ribonuclease activity in microvolumes of samples. 2. 2. The method involves the diffusion of ribonuclease from small wells cut into an RNA-agar-coated microscopic glass slide. 3. 3. Diffusion of ribonuclease results in the production of distinct zones of clearing around the wells after acid precipitation of the unhydrolysed RNA. 4. 4. Conditions for optimal ribonuclease activity on the agar-diffusion slides were determined and the assay was applied to crude tissue extracts.