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

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.

缓步动物（Tardigrades）可通过可逆切换至无代谢状态，耐受近乎完全的脱水环境。该能力被称为脱水休眠（anhydrobiosis）。处于脱水休眠状态时，缓步动物能够耐受包括太空在内的多种极端环境，但其背后的分子基础仍未得到充分阐释。胚胎发育晚期丰富（Late embryogenesis abundant, LEA）蛋白是一类热可溶性蛋白，可在其他脱水休眠生物的脱水条件下抑制蛋白质聚集，但其与缓步动物脱水休眠的相关性尚未明确。本研究聚焦于LEA蛋白特有的热可溶性特性，以处于脱水休眠状态的缓步动物为材料开展热可溶性蛋白质组学研究，最终鉴定出5种高丰度热可溶性蛋白。所有这些蛋白与LEA蛋白均无序列相似性，且可形成两个具有独特亚细胞定位的新型蛋白质家族。根据其亚细胞定位特征，我们将其分别命名为细胞质丰富热可溶性（Cytoplasmic Abundant Heat Soluble, CAHS）蛋白家族与分泌型丰富热可溶性（Secretory Abundant Heat Soluble, SAHS）蛋白家族。两类蛋白家族仅在缓步动物中保守存在，未在其他动物门中被发现。尽管在水合状态下，CAHS蛋白呈内在无序结构，SAHS蛋白则富含β折叠结构，但两类家族的蛋白均如LEA蛋白一般，在缺水条件下会发生构象转变，形成α螺旋结构。CAHS蛋白中存在两个保守的19肽基序重复序列，可在α螺旋中形成两亲性条纹，这提示它们在缺水条件下可作为分子防护因子发挥作用；不过CAHS与LEA蛋白的α螺旋电荷分布模式存在差异。缓步动物或许演化出了具备热可溶性特性的新型蛋白质家族，本研究揭示了这类脱水休眠动物中一类全新的主要热可溶性蛋白库。