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.

Stress tolerance during diapause and quiescence of the brine shrimp, Artemia

“…LEA proteins, often enriched in repeated motifs, are hydrophilic, mostly unstructured, flexible proteins within the group of intrinsically disordered proteins; however, when drying, many LEA proteins reversibly assume structure stabilized by hydrogen bonding and electrostatic interactions (Goyal et al 2003;Chakrabortee et al 2011;Hundertmark et al 2012;Hatanaka et al 2013). LEA proteins were first discovered in maturing plant seeds (Dure et al 1981) and have since been found in desiccation-tolerant cyanobacter (Close and Lammers 1993), bacteria and Archaea (Campos et al 2013), nematodes (Browne et al 2002;Gal et al 2004;Goyal et al 2005a;Erkut et al 2013), rotifers (Tunnacliffe et al 2005), an insect (Kikawada et al 2006;Hatanaka et al 2013), and a crustacean (Hand et al 2007;Sharon et al 2009).…”

“…LEA proteins were first discovered in maturing plant seeds (Dure et al 1981) and have since been found in desiccation-tolerant cyanobacter (Close and Lammers 1993), bacteria and Archaea (Campos et al 2013), nematodes (Browne et al 2002;Gal et al 2004;Goyal et al 2005a;Erkut et al 2013), rotifers (Tunnacliffe et al 2005), an insect (Kikawada et al 2006;Hatanaka et al 2013), and a crustacean (Hand et al 2007;Sharon et al 2009). LEA proteins are induced by and mediate the effects of dehydration/ rehydration (Gal et al 2004;Tunnacliffe et al 2005;Kikawada et al 2006), osmotic stress (Close and Lammers 1993; Gal et al 2004;Kikawada et al 2006), temperature fluctuation (Gal et al 2004), and freezing-thawing (Hand et al 2011;Campos et al 2013;Toxopeus et al 2014).…”

“…LEA proteins are induced by and mediate the effects of dehydration/ rehydration (Gal et al 2004;Tunnacliffe et al 2005;Kikawada et al 2006), osmotic stress (Close and Lammers 1993; Gal et al 2004;Kikawada et al 2006), temperature fluctuation (Gal et al 2004), and freezing-thawing (Hand et al 2011;Campos et al 2013;Toxopeus et al 2014). Protein aggregation is inhibited by LEA proteins, either by chaperoning (Grelet et al 2005) or more probably by molecular shielding, the formation of electrostatic and/or physical barriers that prevent protein interactions (Goyal et al 2005b;Chakrabortee et al 2011;Hatanaka et al 2013). LEA proteins may stabilize membranes by interaction with phospholipids (Jaspard et al 2012), and they strengthen vitrified sugar glasses of trehalose or sucrose, raising the transition temperature and sustaining glasses upon exposure to increased heat (Hand et al 2011).…”

A Simple Method for the Decapsulation of Dormant Resting Eggs for Nuclear Staining in the Monogonont Rotifer, Brachionus plicatilis Muller

“…Desiccation tolerance involves a serious of coordinated events involving late embryogenesis abundant (LEA) and heat shock proteins (HSPs) that are activated during water loss to prevent the drying out of cells [16][17][18]. In plants, pollen and seeds, survive desiccation by recruiting soluble non-reducing carbohydrate and LEAs to form intracellular glass that slow down intracellular trafficking and stabilize macromolecules [19].…”

Digested disorder, Quarterly intrinsic disorder digest (October-November-December, 2013)

“…Hatanaka et al 47 studied PvLEA4 and observed high hydrophilicity and a tendency to form a-helices when desiccated. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy revealed that these proteins are intrinsically disordered when hydrated, and take on a more structured form upon dehydration, potentially contributing to their ability to reduce aggregated particles during drying, a function known as molecular shielding.…”