Purification and cDNA Cloning of a Cadmium-Binding Metallothionein from the Freshwater Crab Sinopotamon henanense

Abstract

Metallothioneins (MTs) are cysteine-rich, metal-binding proteins that are useful biomarkers for monitoring pollution by heavy metals. In this report, a novel cadmium (Cd)-binding MT (CdMT) from Sinopotamon henanense was purified using acetone precipitation (50-80%), followed by gel-filtration chromatography and anion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and time-of-flight mass spectrometry analysis showed that S. henanense CdMT existed as monomer and dimmer forms, with a monomer molecular weight of 6890 Da and a dimmer molecular weight of 13,766 Da. In addition, the full-length cDNA sequence of S. henanense CdMT was prepared from the gill RNA using reverse transcription-polymerase chain reaction and 3' and 5' rapid amplification of cDNA ends (RACE) methods. Sequence analyses indicated that the isolated cDNA (633 bp) contains an open reading frame of 177 bp that encodes a protein with 59 amino acids. The deduced amino acid sequence has 18 cysteine residues, implying that S. henanense CdMT binds six equivalents of bivalent metal ions (Cd) as opposed to the seven in its mammalian counterparts. The deduced molecular weight of MT without binding metals is 6218 Da. If six bound Cd atoms are counted, the deduced molecular weight of S. henanense CdMT would be 6892 Da, which is very similar to the molecular weight of the purified protein (6890 Da) determined by time-of-flight mass spectrometry analysis. These confirmed our results of MT purification. These present studies will be helpful to increase the database information of heavy-metal-induced MT in terms of crustaceans.