Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

Abstract

Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporter was used for intracellular loading of this disaccharide. Cells were rapidly and uniformly desiccated to low water content (<0.12 g H 2 O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, compared with 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.water stress | biopreservation | intrinsically disordered proteins | osmolyte | Artemia franciscana

Quantification of cellular protein expression and molecular features of group 3 LEA proteins from embryos of Artemia franciscana

“…This predicted location was confirmed in HepG2 cells transfected with the green fluorescent protein-tagged protein (Li et al 2012). Recombinant AfrLEA2 has a total molecular mass of 43.1 kDa (38.9 kDa plus 4.2 kDa for a 6×-His tag and associated sequence) but migrates on SDS gels at a calculated mass of 49.3±0.9 (mean±SD; n =3) (Fig.…”

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress

“…Another wheat (Triticum aestivum) LEA protein was shown to increase cold tolerance when overexpressed in Arabidopsis (NDong et al, 2002). Recently, a dramatic effect was reported for a brine shrimp (Artemia franciscana) mitochondrial LEA protein expressed in cultured human hepatoma cells (Li et al, 2012b). Indeed, membrane integrity of transgenic cells was preserved after a rapid and uniform cell desiccation, and with the combination of intracellular loading of trehalose before drying, cells maintained almost normal proliferation rates after rehydration.…”

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

“…Either trehalose or AfrLEA2 improves membrane integrity and survival of transfected HepG2 cells upon spin drying, with protection reaching 98 % when they are used together. Unexpectedly, AfrLEA3m acting alone increases transfected cell survival to almost this level, indicating that AfrLEA3m is important to mitochondrial integrity upon water loss (Li et al 2012), a view strengthened by the presence of more group 3 LEA proteins, AfrLEA3m_47, AfrLEA3m_43, and AfrLEA3m_29, in A. franciscana, all with mitochondrial localization signals . These LEA proteins cross-react with antibody to AfrLEA3m, but their corresponding cDNAs are sufficiently different that each is liable to be the product of a distinct gene.…”

“…AfrLEA3m mRNA is more abundant in diapause-destined embryos than in nauplii, and mitochondria containing trehalose and AfrLEA3m display enhanced freeze tolerance. Transfection with AfrLEA2 and AfrLEA3m cDNA under tetracycline (Tet) control, along with a constitutively expressed trehalose transporter 1 cDNA, yields HepG2 cells with AfrLEA2 in the cytoplasm and AfrLEA3m in the mitochondria, with the latter enriched in trehalose (Li et al 2012). Either trehalose or AfrLEA2 improves membrane integrity and survival of transfected HepG2 cells upon spin drying, with protection reaching 98 % when they are used together.…”