Anlong Xu

EST analysis of gene expression in the tentacle of Cyanea capillata

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltage‐dependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.

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.

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3,5-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (), having 61.7% beta-sheet and no alpha-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD(50) of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5+/-160.5%. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.

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.

Expression and purification of Src I from sea anemone Sagartia rosea as a recombinant non-fusion protein

The cDNA coding for an acidic actinoporin, Sagartia rosea cytolysin I (Src I), has been isolated, cloned into pGEM-T Easy Vector, and sequenced. The region encoding matured Src I was also cloned into prokaryotic expression vector pBV220 and expressed in Escherichia coli as a non-fusion protein in the form of inclusion body. Through washing and denaturation-renaturation step, the recombinant Src I was purified by Q Sepharose Fast Flow ion exchange chromatography and Phenyl Sepharose hydrophobic interaction chromatography. The two-step purification of Src I was effective, simple, and suitable for isolating large amount of high purity (above 95%) recombinant Src I.

cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.

Effects of recombinant neurotoxins on single Na(+) channels in isolated rat hippocampal neurons.

Four recombinant neurotoxins Hk2a, Hk7a, Hk8a, Hk16a, originally from a sea anemone species Anthopleura sp., were obtained by fusion expression of their genes in Escherichia coli. These neurotoxins were composed of 47 amino acid residues, among which the differences were found at positions 14, 22, 25, and 37, respectively. The effects of the four neurotoxins on single‐channel current of sodium in rat hippocampal neurons were studied by cell‐attached patch clamp. Each neurotoxin 2 μM could modulate the sodium channel by prolonging the opening dwell time and increasing the open probability, but did not change the amplitude of sodium channel currents. Based on the studies of the structure–function relationship, we found that Hk7a displayed the biggest increase of the open probability because His14 (from Arg14) makes its structure seem more compact in comparison with the other three toxins and Ap‐A. Phe25 (Hk8a, Hk16a), which varied from Ala25 (Hk2a, Hk7a), showed that phenyl group might interfere with other key amino acid residue to decrease the activity of toxins. Arg37 (from His37) in Hk8a contributed to decrease of open probability. In our work, it was shown that these important amino acid sites might provide a reliable proof for the future pharmaceutical design.