Cloning, expression, and characterization of cadmium-induced metallothionein-2 from the earthworms Metaphire posthuma and Polypheretima elongata.
In this study we report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Complementary (c)DNA was verified as the MT-2 gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2 of both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The cDNAs corresponding to the two megascolecid MT-2 genes were expressed, and the MT-2 proteins were purified for biochemical characterization. The binding of Cu2+ exhibited monophasic kinetics and those of Zn2+ and Cd2+ biphasic kinetics. The proteins bound more tightly to Cd2+ than to Zn2+ and more tightly still to Cu2+. Zn-MT and apo-MT were the most effective at scavenging free radicals, followed by Cd-MT. In conclusion, MT-2s from M. posthuma and P. elongata showed unique sequence features compared to those of lumbricid earthworms. These earthworms could be used to evaluate heavy-metal pollution in soil due to the inducible MT-2 by cadmium exposure.
Department of Biotechnology, National Kaohsiung Normal University, 62 Shenjhong Rd., Yanchao Township, Kaohsiung County 82444, Taiwan.
Shih-Hsiung
SH
Liang
Yu-Ping
YP
Jeng
Yuh-Wen
YW
Chiu
Jiun-Hong
JH
Chen
Bao-Sen
BS
Shieh
Chien-Yen
CY
Chen
Chien-Cheng
CC
Chen
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015415:Biomarkers / Q000378:metabolism; D019256:Cadmium Chloride / Q000378:metabolism / Q000633:toxicity*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000032:analysis; D018076:DNA, Complementary; D004784:Environmental Monitoring / Q000379:methods; D016166:Free Radical Scavengers; D020869:Gene Expression Profiling; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009835:Oligochaeta / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012989:Soil Pollutants / Q000378:metabolism / Q000633:toxicity*; D015032:Zinc / Q000378:metabolism
D015415:Biomarkers; D018076:DNA, Complementary; D016166:Free Radical Scavengers; D011994:Recombinant Proteins; D012989:Soil Pollutants; D003300:Copper; D008668:Metallothionein; D015032:Zinc; D019256:Cadmium Chloride; D003545:Cysteine