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The earthworm Enchytraeus is able to survive in cadmium (Cd)-polluted environments. Upon Cd exposure, the worms express a gene encoding the putative non-metallothionein 25-kDa cysteine-rich protein (CRP), which contains eight repeats with highly conserved cysteines in Cys-X-Cys and Cys-Cys arrangements exhibiting 36-53% identities to the 6-7-kDa metallothioneins of different organisms. Here, we demonstrate that the CRP protein confers a highly Cd-resistant phenotype to a Cd-hypersensitive yeast strain. Cd resistance increases with increasing numbers of expressed CRP repeats, but even one 3-kDa CRP repeat still mediates Cd resistance. Site-directed mutagenesis reveals that each single cysteine within a given repeat is important for Cd resistance, though to a different extent. However, replacement of other conserved amino acids such as Pro(136) and Asp(196) at the CRP repeat junctions does not affect Cd resistance. Our data indicate (i) that the non-metallothionein CRP protein is able to detoxify Cd and (ii) that this is dependent on the availability of sulfhydryl groups of the conserved cysteines.

Inducibility of the mouse gene imap38 in the spleen has been recently described to correlate with resistance to Plasmodium chabaudi malaria. Here, we characterize the human ortholog gene himap1. The HIMAP1 34 kDa protein is localizable at the endoplasmic reticulum in transfected cells. It contains a GTP-binding domain, but it does not bind GTP, in contrast to mouse IMAP38. The himap1 gene belongs to a gene family clustered on chromosome 7q32-36 within a region highly syntenic to the mouse imap38 locus on chromosome 6B. The himap genes 1, 2, 3, and 4 display a conserved intron/exon structure. The mRNA of the himap1 gene is predominantly expressed in the spleen, in lymph nodes to a lesser extent, and only at very low levels in diverse cancer cell lines. In accordance, imap-like genes in mice and plants are associated with proliferative and apoptotic events suggesting a role in the control of cell death/survival.

We characterize the mouse gene imap38 and its inducibility by Plasmodium chabaudi malaria among different lymphoid tissues and mouse strains of different H-2 complex and non-H-2 background. Imap38 is a single copy gene assigned to chromosome 6B. It consists of only one exon of 1900 base pairs encoding a highly basic 25.8-kDa protein. Confocal laser scanning microscopy localizes differently tagged IMAP38 proteins in nuclei of transfected cells. Reporter gene assays reveal that the 1730-base pair 5'-flanking region, containing an RSINE1 repeat immediately adjacent to initiation site +1, exhibits promoter activity in nonmurine cells, while it is largely repressed in diverse mouse cell lines, which corresponds to the situation in mouse tissues. P. chabaudi malaria induces imap38 expression almost exclusively in the spleen but not in other lymphoid organs. Parasite lysates are able to induce imap38 in the spleen, but not in spleen cells ex vivo. Activation of spleen cells by LPS and other stimuli is not sufficient to induce imap38. Inducibility of imap38 requires signals from both parasites and the intact spleen, and it is controlled by genes of that non-H-2 background, which also controls development of protective immunity against P. chabaudi malaria.

Cadmium accumulation and its effect on gene expression have been investigated at sublethal cadmium concentrations in the soil oligochaete Enchytraeus buchholzi. This worm is capable of accumulating cadmium to large amounts, which coincides with the induction of a mRNA isolated as a cDNA clone by differential screening of a cDNA library constructed from cadmium-treated enchytraeids. The cDNA clone designated CRP1 is 1474 base pairs in length and contains a 753-base pair open reading frame, encoding a novel Cys-rich non-metallothionein protein. In vitro translation of the in vitro transcribed CRP1 results in a protein with a molecular mass of 25 kDa and an pI of approximately 7.5. These values are consistent with those predicted from the deduced amino acid sequence. The CRP protein contains 27% Cys, most of them arranged in Cys-X-Cys and Cys-Cys segments. The sequence is also characterized by a 31-amino-acid motif, which is tandemly repeated along the sequence. Northern blot analysis reveals that the CRP gene is not constitutively expressed in untreated worms, but rather it is rapidly induced by cadmium. The CRP gene may be a promising candidate gene for monitoring bioavailable cadmium at subtoxic levels in terrestric environments.

Enchytraeus buchholzi (Oligochaeta) were exposed to various concentrations of CdCl2 in agar and aqueous solution. The Cd uptake was determined by atomic absorption spectroscopy as well as Cd effects on survival, reproduction, and mRNA synthesis by in vitro translation of total RNA in a rabbit reticulocyte lysate system. Although Cd was rapidly accumulated by the worms, any acute toxic Cd effects at concentrations below 4 mg Cd/liter were not detectable. However, such subtoxic Cd concentrations caused the induction of an mRNA species coding for a nonmetallothionein 33-kDa protein as revealed by 2D electrophoresis of in vitro translated in vitro translated proteins using [35S]methionine. The Cd-induced synthesis of this transcriptionally regulated protein might be a preindicator for a Cd intoxication in enchytraeids.