[Correlation of antitumor effect of recombinant sea snake basic phospholipase A2 to its enzymatic activity].

BACKGROUND & OBJECTIVE: Snake venom phospolipase A2 (PLA(2)), a large family of homologous (14 ku) soluble proteins, exerts diverse pharmacologic activities as well as enzymatic activities. So far, the structure and function of terrestrial snake PLA(2), especially the relationship of its enzymatic and pharmacologic activities have been studied extensively, but the investigation of sea snake PLA(2) are limited. This study was to investigate the in vitro and in vivo antitumor effects of recombinant sea snake basic PLA(2) (rSSBPLA(2)) and its mutants rN48 and rK4 from sea snake Lapemis hardwickii venom, and to explore the influence of 2 residues related with the enzymatic activity on the antitumor effects. METHODS: Site-directed mutagenesis of the 2 conserved residues related with enzymatic activity (His48 mutated to Asn and Asp49 mutated to Lys) was performed. The inhibitory effects of rSSBPLA(2), rN48 and rK49 on proliferation of human myeloid leukemia cell line HL-60, human neuroblastoma cell line SK-N-SH, human gastric cancer cell line MGC-803, and human liver cancer cell line HepG2 were assessed by MTT assay. Their antitumor effects on sarcoma cell line S180 xenograft and EAC ascites cancer model in mice were detected. RESULTS: The relative enzymatic activities of rN48 and rK49 were 0 and 5% of that of rSSBPLA(2). The 50% inhibitory concentration (IC(50)) of rSSBPLA(2) for HL60, SK-N-SH, and MGC-803 cells were (45.28+/-0.09) microg/ml, (57.07+/-0.12) microg/ml, and (69.34+/-0.35) microg/ml, respectively, but it had no inhibitory effect on proliferation of HepG2 cells. rSSBPLA(2) obviously inhibited growth of S180 xenograft in miceû the inhibitory rates were 50.8%, 43.2%, 38.2%, and 55.5%, respectively, under the dose of 2 mg/kg (qd x 10), 2 mg/kg (q2d x 5), 4 mg/kg (qd x 1) and 4 mg/kg (q5d x 2). The inhibitory rate of EAC model was 33.5% under the dose of 4 mg/kg (q5d x 2). The inhibitory rates were significantly higher in test groups than in control groups (P<0.01). rN48 and rK49 had no inhibitory effect on proliferation of the 4 tumor cell lines and on growth of the xenograft tumors. CONCLUSION: The antitumor effect of rSSBPLA(2) may be closely related with its enzymatic activity.

Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E. coli.

Three full-length phospholipase A(2) (PLA(2)) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study. In order to investigate their biological functions, we established a fusion expression system for PLA(2)-9 in E. coli. The open reading frame encoding mature peptide of PLA(2)-9 was subcloned into the vector pTRX. The Trx-PLA(2)-9 fusion protein was expressed as a soluble protein by IPTG induction at 23 degrees C. The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase. The mature recombinant PLA(2)-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA(2)-9 from 1l of bacteria culture was obtained. The catalytic activity of recombinant PLA(2)-9 (rPLA(2)-9) was measured and found to be similar to native enzyme. As the Austrelaps superbus PLA(2), which shares 90% nucleotide sequence similarity to PLA(2)-9, the rPLA(2)-9 displayed the anti-platelet aggregation effect. Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA(2)-9 may be dissociated.

Molecular cloning, expression and characterization of three short chain alpha-neurotoxins from the venom of sea snake--Hydrophiinae Hydrophis cyanocinctus Daudin.

Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin. Sequence analysis showed that these three genes encoded three different short chain alpha-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids. Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys46 and Ser46, respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain alpha-neurotoxins, Asp31 and Arg33. The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E. coli. After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained. These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences. LD50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively. Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually. The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified alpha-neurotoxins, and no significant structural differences in these recombinant proteins are observed.

Cloning and characterization of an acidic cytolysin cDNA from sea anemone Sagartia rosea.

A full-length cDNA of cytolysin (Src I) was isolated from the tentacle of Sagartia rosea (a representative species in China) by reverse transcription polymerase chain reaction. The cDNA with an open reading frame of 648 bp encodes a precursor protein of 216 amino acids, which contains a prepropeptide of 38 amino acids including a signal peptide of 19 amino acids and a propart of 19 amino acids. Lys-Pro at C-terminus of propart is a cleavage site for proline-endopeptidase-like protease. The mature cytolysin has a molecular mass of 19.6 kDa and a pI value of 4.8. Src I is an acidic cytolysin found in sea anemone and shares 75% amino acid sequence similarity to equinatoxin II (Eqt II). The predicted secondary structure of the mature cytolysin comprises 15% alpha-helix, 45% beta-sheet, and 40% random coil. The characteristic amphiphilic alpha-helix of cytolysin is located at the N-terminus of the processed Src I.

EST analysis of mRNAs expressed in neurula of Chinese amphioxus.

Amphioxus, a cephalochordate, is the closest living relative to the vertebrates. In order to investigate the molecular mechanisms of the early embryogenesis of amphioxus, we constructed a neurula embryo cDNA library of Chinese amphioxus (Branchiostoma belcheri tsingtauense) and generated 5235 expressed sequenced tags in the present study. The initial ESTs consisted of 638 clusters and 1855 singletons, which revealed approximately 2493 unique genes in the data set. Of these sequences, 35.52% ESTs matched to known genes, 12.76% matched to other ESTs, and 51.71% had no match to any known sequences in GenBank. Interestingly we found homologous genes related to neural development and human disease. Bioinformatic analysis showed the direct evidence that the gene homologue found only in vertebrates in previous studies also exists in the amphioxus genome. This study provides a preliminary view of the gene information involved in the development of neurula embryos of Chinese amphioxus and helps our understanding of vertebrate evolution at gene level.

[The construction of cDNA expression library from the tentacles of Sagartia rosea].

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.

Identification and Funtional Characterization of Three Postsynaptic Short-chain Neurotoxins from Hydrophiinae, Lapemis hardwickii Gray.

Three cDNA clones, sn12, sn36 and sn160, encoding isoforms of postsynaptic short-chain neurotoxins, were cloned by screening a cDNA library of the venom from Hydrophiinae, Lapemis hardwickii Gray. The sequences of three cDNA clones encoded proteins consisting of 60 amino acid residues. There was only one amino acid substitution among the three isoforms SN12, SN36 and SN160 at the position 46 of mature proteins, and they were Pro(46), His(46) and Arg(46), respectively. The three molecules were expressed in Escherichia coli and the recombinant proteins were characterized. Different LD(50) were obtained, namely 0.0956 mg/kg, 0.3467 mg/kg and 0.2192 mg/kg, when the SN12, SN36 and SN160 were injected into Kunming mice(i.p.). In analgesic effect assayed by the acetic acid-induced writhing method, SN12 and SN160 showed similar analgesic effect, but SN36 had effects significantly different with the other two. Our studies suggested that the amino acid residues on position 46 could affect the combination between the postsynaptic short-chain neurotoxins and the nicotinic acetylchoine receptor, since different amino acid substitution resulted in different biological activities.

Diversity of PLA(2) Genes from Sea Snake Lapemis hardwickii Gray Venom.

Three full-length cDNA from Lapemis hardwickii Gray, (namely PLA(2)-8, PLA(2)-9 and PLA(2)-384), encoding phospholipase A(2) (PLA(2)), were isolated and sequenced. These cDNA sequences were composed of 456 bp, 438 bp and 438 bp, encoding a signal peptide of 27 amino acids and a mature peptide of 125, 119 and 119 amino acids, respectively. The analysis results by computer indicated PLA(2)-8, PLA(2)-9and PLA(2)-384 has a pI of ca. 4.8, 7.8 and 8.4, respectively. Sequence analysis of amino acid and prediction of secondary structure suggested that these isoforms of PLA(2) belong to class I PLA(2) family. PLA(2)-8 was highly homologous (81%) to Notechis scutatus scutatus (Australian tiger snake) PLA(2), PLA(2)-9 and PLA(2)-384 showed fairly high sequence homology (90%) to Enhydrina schistosa (beaked sea snake) PLA(2), indicating that they might have similar functions. These results reflect the diversity of Lapemis hardwickii Gray PLA(2) genes. The successful cloning of these isoenzyme genes of sea snake PLA(2) may provide new information for the study on structure-function relationship of PLA(2) family and its possible molecular mechanism.