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.
Liu
Wen-Hua
WH
The Open Laboratory for Marine Functional Genomics of State High-Tech Development, Department of Biochemistry, College of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, People's Republic of China.
Wang
Lei
L
Wang
Yi-Liang
YL
Peng
Li-Sheng
LS
Wu
Wen-Yan
WY
Peng
Wen-Lie
WL
Jiang
Xiao-Yu
XY
Tu
Hong-Bin
HB
Chen
Hui-Ping
HP
Ou-Yang
Ping
P
Xu
An-Long
AL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D007274:Injections, Intraperitoneal; D007928:Lethal Dose 50; D051379:Mice; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012615:Sea Anemones; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D003064:Cnidarian Venoms; D017931:DNA Primers; D018076:DNA, Complementary; D009498:Neurotoxins