An abundant LEA protein in the anhydrobiotic midge, PvLEA4, acts as a molecular shield by limiting growth of aggregating protein particles

Abstract

LEA proteins are found in anhydrobiotes and are thought to be associated with the acquisition of desiccation tolerance. The sleeping chironomid Polypedilum vanderplanki, which can survive in an almost completely desiccated state throughout the larval stage, accumulates LEA proteins in response to desiccation and high salinity conditions. However, the biochemical functions of these proteins remain unclear. Here, we report the characterization of a novel chironomid LEA protein, PvLEA4, which is the most highly accumulated LEA protein in desiccated larvae. Cytoplasmic-soluble PvLEA4 showed many typical characteristics of group 3 LEA proteins (G3LEAs), such as desiccation-inducible accumulation, high hydrophilicity, folding into α-helices on drying, and the ability to reduce aggregation of dehydration-sensitive proteins. This last property of LEA proteins has been termed molecular shield function. To further investigate the molecular shield activity of PvLEA4, we introduced two distinct methods, turbidity measurement and dynamic light scattering (DLS). Turbidity measurements demonstrated that both PvLEA4, and BSA as a positive control, reduced aggregation in α-casein subjected to desiccation and rehydration. However, DLS experiments showed that a small amount of BSA relative to α-casein increased aggregate particle size, whereas PvLEA4 decreased particle size in a dose-dependent manner. Trehalose, which is the main heamolymph sugar in most insects but also a protectant as a chemical chaperone in the sleeping chironomid, has less effect on the limitation of aggregate formation. This analysis suggests that molecular shield proteins function by limiting the growth of protein aggregates during drying and that PvLEA4 counteracts protein aggregation in the desiccation-tolerant larvae of the sleeping chironomid.

Keywords: CD; CHO; Chinese hamster ovary; DLS; Desiccation tolerance; Dynamic light scattering; FTIR; Fourier transform infrared; G3LEA; GFP; IDP; IPTG; LDH; LEA; LEA proteins; Molecular shield; The sleeping chironomid; Trehalose; circular dichroism; dynamic light scattering; green fluorescent protein; group 3 LEA protein; intrinsically disordered protein; isopropyl-β-D-thiogalactopyranoside; l-lactate dehydrogenase; late embryogenesis abundant.