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.

Abstract

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.

Introduction

Earthworms are vital soil invertebrates because their abundance greatly accelerates nutrient mineralization and release of the carbon fixed inside organic debris (Kooch and Jahilvand, 2008). Earthworms also improve soil physical properties, such as aggregation and porosity, owing to their multidirectional burrowing behavior (Sinha et al., 2010). For these qualities, earthworms are employed as viable biological agents to transform various solid wastes into enriched manures in vermicomposting technology (Sahariah et al., 2014; Hussain et al., 2018). Since earthworms readily respond to contaminants, such as pesticides and heavy metals, they are widely used as test organisms for ecotoxicity and valorization of contaminated wastes (Soobhany et al., 2015; Homa et al., 2016; Roy et al. (2020)).

There is a genuine demand for sustainable technology in large scale remediation of toxic heavy metals from contaminated biomass and soils. Cadmium, particularly, is a well-known hazardous metal with no biological utility (Stürzenbaum et al. (2004)). The environmental concerns of cadmium pollution are aggravated due to high bioavailability of the metal and its long term impact on living organisms (Dallinger and Hockner, 2013; Morgan et al., 2004). It has been reported that some earthworm species, such as Lumbricus rubellus, satisfactorily thrive in highly cadmium contaminated sites (Stürzenbaum et al. (1998); Kowald et al., 2016). Thus, it is opined that some earthworm species are apparently tolerant to cadmium contamination.

Earthworms can accumulate several metal species (Cd, Pb, Hg, and Zn) and store them in benign forms in the chloragogenous tissues (Song et al., 2014; Goswami et al., 2016; Roux et al., 2016). The organically bound metals in the earthworm body remain inert for a long time even after their decay (Stürzenbaum et al. (2004)). Metal accumulation and the typical binding mechanism greatly fluctuate depending on several factors like types of metals, exposure levels, substrate characteristics (pH, temperature, nutrients etc.), type of earthworm species, their physiology, age, and induction of metal chelating proteins (Spurgeon and Hopkin (1999); Nannoni et al., 2011). Whilst the majority of the conclusions concerning molecular mechanisms of metal accumulation and detoxification in earthworms have been drawn from studies conducted with Lumbricus rubellus and Allolobphora chlorotica (Hockner et al., 2015; Homa et al., 2016); the epigeic Eisenia fetida, a popular species for vermicomposting (Bhattacharya and Kim, 2016), has not been used much for this kind of studies. Metal sequestration and detoxification pathway in Eisenia fetida, therefore, needs serious attention.

Earthworms ingest heavy metals along with food materials primarily through their mouth and sometimes through their skins (Dominguez and Edwards (2011)). As on date, it is implicit that chelation vis-à-vis detoxification of metals in earthworms is largely performed by a low molecular weight (6−13 kDa) protein, metallothionein (MT). This cysteine rich protein chelates metals like cadmium through their thiol groups and transport the bound metals to chloragogenous tissues where pollutants are detoxified (Stürzenbaum et al. (2012); Homa et al., 2016). However, MT expression may not change or even down-regulate in some earthworm species (e.g. Dendrobaena octaedra and Eisenia fetida) despite high metal exposure and accumulation (Goswami et al., 2016; Mustonen et al., 2014). Such varying results suggest that accumulation and detoxification mechanism may greatly differ among earthworm species during vermicomposting. Question therefore is – how does metal accumulation take place when metallothionein expression is retarded? To address this, we hypothesized that there could be other metal binding proteins besides MT that act as cargo for delivering bound metals to chloragogenous tissue for storage and detoxification. To the best of our knowledge no report is available about non-MT metal induced proteins in earthworms that are larger than 13−30 kDa. Under these perspectives, we performed an in-vivo experiment with Eisenia fetida. This species is widely used in vermitechnology for remediation of heavy metal contaminated solid wastes and are known for their efficient metal removal potential (Bhattacharya and Kim, 2016). Earthworms were exposed to fluorescence tagged cadmium (hereafter Fl-Cd) and CdCl₂ spiked cow dung based feedstock. Temporal pattern of Cd translocation was monitored in feedstock and earthworm body using fluorescence spectrophotometry and inductively coupled plasma optical emission spectrometry (ICP-OES). A few high molecular weight (> 100 kDa) proteins, capable of binding Cd, were selectively isolated through gel filtration chromatography, electrophoresis, and immunoblotting techniques. Eventually, we purified the most consistently expressed metal induced protein (hereafter MIP) among others. Moreover, some interesting trends were noteworthy in relation to Cd binding when immunofluorescence staining, confocal microscopy and Job’s method of continuous variation were conducted.