Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade

Abstract

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation

“…These predictions strongly support previous evidence that CAHS and MAHS proteins are largely disordered, while by this approach SAHS proteins appear less disordered (Supplemental Information). IDPs lack persistent secondary structure (Theillet et al, 2014; Yamaguchi et al, 2012), which we confirmed for CAHS proteins using nuclear magnetic resonance spectroscopy (NMR). To do this we mapped the chemical environment of the covalent bond between each backbone amide nitrogen and its attached proton based on the heteronuclear single-quantum coherence (HQSC) spectra of the protein.…”

“…These gene families, CAHS, SAHS, and MAHS, were previously identified in a proteomic analysis of tardigrades (Tanaka et al, 2015; Yamaguchi et al, 2012). On the basis of heat solubility experiments and circular dichroism spectropolarimetry, all three families are thought to encode IDPs (Tanaka et al, 2015; Yamaguchi et al, 2012).…”

“…These observations, coupled with the fact that TDPs are induced by drying in H. dujardini , suggests that they play a role in tardigrade stress tolerance (Yamaguchi et al, 2012; Tanaka et al, 2015). However, until now no studies have been conducted to directly examine the effect of environmental conditions on the expression of genes encoding TDPs or their involvement in tardigrade desiccation tolerance.…”

“…On the basis of heat solubility experiments and circular dichroism spectropolarimetry, all three families are thought to encode IDPs (Tanaka et al, 2015; Yamaguchi et al, 2012). Previous bioinformatic studies suggest that not all members of disordered protein families are natively unfolded (Hundertmark and Hincha, 2008).…”

“…These data, combined with heat solubility, circular dichroism, and bioinformatics approaches, show that many, if not all, tardigrade CAHS, and likely MAHS and SAHS proteins, are disordered. We refer to these families (CAHS, SAHS, MAHS) of tardigrade-specific IDPs as TDPs to distinguish them from other IDPs, because, at the sequence level, no homologs of TDPs are found outside the phylum tardigrade (Tanaka et al, 2015; Yamaguchi et al, 2012). …”