Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

Sae Tanaka,Yoshihiro Miwa,Takeo Kubo,Takekazu Kunieda,Toshiaki Katayama,Junko Tanaka,Daiki D Horikawa,Atsushi Toyoda,Kazuharu Arakawa,John R Battista

doi:10.1371/journal.pone.0118272

Abstract

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Highlights

Tardigrades are microscopic invertebrates comprising the phylum Tardigrada
As candidate proteins involved in protecting mitochondria from desiccation stress, we detected an Late Embryogenesis Abundant (LEA) protein potentially targeted to the mitochondria from our transcriptome database of R. varieornatus
Similar to most LEA proteins, RvLEAM is highly hydrophilic (GRAVY score, -0.94) and a hydropathy plot showed no clear hydrophobic region over the entire sequence, suggesting that RvLEAM localized in the mitochondrial matrix rather than integrating into the mitochondrial membrane (Fig. 1a)

Summary

Introduction

Tardigrades are microscopic invertebrates comprising the phylum Tardigrada. all tardigrade species are primarily aquatic and require water to grow and reproduce offspring, some terrestrial species can tolerate almost complete desiccation by entering an ametabolic dehydrated state, referred to as anhydrobiosis [1]. RvLEAM improved the metabolic activity of human cells 2-fold (100% increase) at 300 mM sucrose (upshift of ~300 mOsm). A cytosolic LEA protein of the anhydrobiotic nematode transfected to human T-REx293 cells improved metabolic activity by 20% to 70% compared to uninduced cells after 2-d exposure to hyperosmotic medium (400 mOsm upshift with various solutes) [16]. The improvement, was only observed within a particular osmotic range of 150 to 200 mM sucrose (upshift of 150~200 mOsm) with up to a 20% increase. In tardigrades, both heat soluble proteins, MAHS and RvLEAM, likely localize in the mitochondria. MAHS protein might require other molecules, like RvLEAM, for efficient protection, as MAHS protein alone provided significant, but comparatively less, improvement of cellular activity in the hyperosmotic condition

Methods

Results

Conclusion