Metal induced non-metallothionein protein in earthworm: A new pathway for cadmium detoxification in chloragogenous tissue

Highlights: • Fluorescence probed Cd helped to isolate a novel metal binding protein in E. fetida. • 41–42 % Cd removal was achieved with 4–5 folds rise in accumulation in worm biomass. • Glutamic acid dominant ∼150 kDa protein exhibited an uncommon Cd binding mechanism. • Confocal microscopy revealed that the protein detoxified Cd in chloragogenous cells. Earthworms neutralize toxic metals by a small (∼13 kDa) cysteine rich metal binding protein, metallothionein (MT). Although the rate of metal accumulation and MT expression does not correlate well, the reason behind such inconsistency has not yet been deciphered. The present investigation clearly demonstrates that expression of some non-MT metal induced proteins is responsible for such incongruity. Applying selective protein isolation techniques in fluorescence tagged cadmium exposed (135 mg/kg) earthworms we were able to purify a 150 kDa metal induced protein (MIP) among others. After 60 days of exposure cadmium accumulation in earthworm intestines was significant. Immunofluorescence staining followed by confocal microscopy exhibited that MIP accumulates ingested cadmium in the intestinal region and eventually deposits the metal in the chloragogenous tissue. We determined the N-terminal sequence of 15 amino acid residues and after bioinformatics analysis, it was concluded that MIP is most probably a glutamic acid rich, novel cadmium binding protein. To further validate the binding mechanism, we conducted paper chromatography and continuous variation experiments which evidenced that cadmium readily binds to glutamic acid. The present finding is the first in-vivo evidence of a non-metallothionein cadmium binding protein induced in the intestines of earthworm exposed to a cadmium rich environment.