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

Abstract

Late embryogenesis abundant (LEA) proteins are highly hydrophilic, low complexity proteins whose expression has been correlated with desiccation tolerance in anhydrobiotic organisms. Here, we report the identification of three new mitochondrial LEA proteins in anhydrobiotic embryos of Artemia franciscana, AfrLEA3m_47, AfrLEA3m_43, and AfrLEA3m_29. These new isoforms are recognized by antibody raised against recombinant AfrLEA3m, the original mitochondrial-targeted LEA protein previously reported from these embryos; mass spectrometry confirms all four proteins share sequence similarity. The corresponding messenger RNA (mRNA) species for the four proteins are readily amplified from total complementary DNA (cDNA) prepared from embryos. cDNA sequences of the four mRNAs are quite similar, but each has a stretch of sequence that is absent in at least one of the others, plus multiple single base pair differences. We conclude that all four mitochondrial LEA proteins are products of independent genes. Each possesses a mitochondrial targeting sequence, and indeed Western blots performed on extracts of isolated mitochondria clearly detect all four isoforms. Based on mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis migration, the cytoplasmic-localized AfrLEA2 exists primarily as a homodimer in A. franciscana. Quantification of protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 as a function of development shows that cellular concentrations are highest in diapause embryos and decrease during development to low levels in desiccationintolerant nauplius larvae. When adjustment is made for mitochondria matrix volume, the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide guidance for the design of in vitro functional studies with these proteins.

Molecular approaches for improving desiccation tolerance: insights from the brine shrimp Artemia franciscana

“…Three new LEA proteins, predicted to be mitochondrial-targeted, were identified in anhydrobiotic embryos of A. franciscana (AfrLEA3m_47, AfrLEA3m_43, and AfrLEA3m_29), and the corresponding mRNA species were isolated and the cDNA amplified (Boswell et al 2014a). All three of these new mitochondrial LEA proteins (plus a fourth mitochondrial isoform previously reported, AfrLEA3m) are apparently products of independent genes based on mass spectrometry and sequence comparisons.…”

“…All three of these new mitochondrial LEA proteins (plus a fourth mitochondrial isoform previously reported, AfrLEA3m) are apparently products of independent genes based on mass spectrometry and sequence comparisons. AfrLEA2, predicted to be cytoplasmically-localized based on bioinformatics (Hand et al 2007), was shown to exist as a homodimer in A. franciscana (Boswell et al 2014a). Quantification of protein expression for these proteins showed that cellular concentrations are highest in diapause embryos and decrease during development to low levels in desiccation-intolerant nauplius larvae (Fig.…”

“…We found that the titer of the cytoplasmic-localized LEA protein (AfrLEA2) ranged from 0.8 to 1.8 mg/g cellular water across development, and the combined value for mitochondrial-targeted LEA proteins (AfrLEA3m, AfrLEA3m_29, AfrLEA3m_43) was 1.2–2.2 mg/ml matrix volume for post-diapause and diapause embryos, respectively. These units should be reasonably physiological considering the subcelluar compartments in which the proteins reside (for methods of calculation, please see Boswell et al 2014a). We find the work of Tolleter et al (2007) on the mitochondrial targeted LEAM in pea seeds to be particularly germane in this regard.…”

Insights into Late Embryogenesis Abundant (LEA) Proteins in Plants: From Structure to the Functions

“…They have been found in prokaryotes Deinococcus radiodurans [56], Haemophilus influenza [57] and in Caenorhabditis elegans (Ce-LEA-1), whose expression is correlated with the survival of this nematode under conditions of desiccation, osmotic, and heat stress [58]. Other anhydrobiotic organisms such as the nematodes Steinernema feltiae [36] [59] and Aphelencus avenae [37], as well as the bdelloid rotifer Philodina roseola [60], the chironomid Polypedilum vanderplanki [61] and the crustacean A. franciscana [62] [63] also accumulate group 3 LEA proteins in their desiccated states.…”

Small Heat Shock Proteins and Diapause in the Crustacean, Artemia franciscana

“…How proteins are protected from irreversible denaturation during stress associated with diapause in A. franciscana has received considerable attention. A. franciscana cysts contain high levels of trehalose (Clegg and Jackson 1998 ;Clegg 2001 ) and in concert with late embryogenesis abundant (LEA) proteins (Sharon et al 2009 ;Menze et al 2009 ;Hand et al 2011 ;Warner et al 2010Warner et al , 2012Wu et al 2011 ;Boswell et al 2014 ;Toxopeus et al 2014 ) this non-reducing sugar may protect embryos either by the formation of amorphous glasses, known as vitrifi cation, or by replacing H 2 O lost during dehydration (Clegg 2001 ;Hand et al 2011 ;Hengherr et al 2011 ). Cysts of A. franciscana are endowed with large amounts of two cystspecifi c molecular chaperones, namely artemin, a heat tolerant ferritin homologue (Tanguay et al 2004 ;Chen et al 2007 ;Hu et al 2011 ;Shahangian et al 2011 ;King et al 2014 ) and p26, a sHsp (Jackson and Clegg 1996 ;Liang and MacRae 1999 ;Sun et al 2004Sun et al , 2006King and MacRae 2012 ).…”