Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-ß-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause.

Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis-life without water-during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H2O2) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.

Complexity of the heat-soluble LEA proteome in Artemia species.

The brine shrimp Artemia is a well known stress tolerant invertebrate found on most continents. Under certain conditions females produce cysts (encysted gastrulae) that enter diapause, a state of obligate dormancy. During developmental formation of diapause embryos several different types of stress proteins accumulate in large amounts, including the late embryogenesis abundant (LEA) proteins. In this study we used a combination of heterologous group 3 LEA antibodies to demonstrate that the heat-soluble proteome of the cysts contains up to 12 distinct putative group 3 LEA proteins that complement the group 1 LEA proteins found previously. Most antibody-positive, heat-soluble proteins were larger than 50 kDa although antibody positive proteins of 20-38 kDa were also detected. Both nuclei and mitochondria had distinct complements of the putative group 3 LEA proteins. A few small group 3 LEA proteins were induced by cycles of hydration-dehydration along with one protein of about 62 kDa. The expression of group 3 LEA proteins, unlike members of group 1, was not restricted to encysted diapause embryos. Three to five putative group 3 LEA proteins were expressed in gravid females and in larvae. Cysts of different species from various geographic locations had distinct complements of group 3 LEA proteins suggesting rapid evolution of the LEA proteins or differences in the type of group 3 Lea genes expressed. Our results demonstrate the potential importance of group 3 LEA proteins in embryos and other life cycle stages of this animal extremophile.

Evidence for multiple group 1 late embryogenesis abundant proteins in encysted embryos of Artemia and their organelles.

The presence of late embryogenesis abundant (LEA) proteins in plants and animals has been linked to their ability to tolerate a variety of environmental stresses. Among animals, encysted embryos of the brine shrimp Artemia franciscana are among the most stress resistant eukaryotes, and for that reason it is considered to be an extremophile. The study presented here demonstrates that these embryos contain multiple group 1 LEA proteins with masses of 21, 19, 15.5 and 13 kDa. The LEA proteins first appear in diapause-destined embryos, beginning at ∼4 days post-fertilization, but not in nauplii-destined embryos. After resumption of embryonic development, the LEA proteins decline slowly in the desiccation resistant encysted stages, then disappear rapidly as the embryo emerges from its shell. LEA proteins are absent in fully emerged embryos, larvae and adults. They are abundant in mitochondria of encysted embryos, but barely detectable in nuclei and absent from yolk platelets. LEA proteins were also detected in dormant embryos of six other species of Artemia from hypersaline environments around the world. This study enhances our knowledge of the group 1 LEA proteins in stress tolerant crustacean embryos.

Characterization of a group 1 late embryogenesis abundant protein in encysted embryos of the brine shrimp Artemia franciscana.

Late embryogenesis abundant (LEA) proteins are hydrophilic molecules that are believed to function in desiccation and low-temperature tolerance in some plants and plant propagules, certain prokaryotes, and several animal species. The brine shrimp Artemia franciscana can produce encysted embryos (cysts) that enter diapause and are resistant to severe desiccation. This ability is based on biochemical adaptations, one of which appears to be the accumulation of the LEA protein that is the focus of this study. The studies described herein characterize a 21 kDa protein in encysted Artemia embryos as a group 1 LEA protein. The amino acid sequence of this protein and its gene have been determined and entered into the NCBI database (no. EF656614). The LEA protein consists of 182 amino acids and it is extremely hydrophilic, with glycine (23%), glutamine (17%), and glutamic acid (12.6%) being the most abundant amino acids. This protein also consists of 8 tandem repeats of a 20 amino acid sequence, which is characteristic of group 1 LEA proteins from non-animal species. The LEA protein and its gene are expressed only in encysted embryos and not in larvae or adults. Evidence is presented to show that the LEA protein functions in the prevention of drying-induced protein aggregation, which supports its functional role in desiccation tolerance. This report describes, for the first time, the purification and characterization of a group 1 LEA protein from an animal species.

Further characterization of the cathepsin L-associated protein and its gene in two species of the brine shrimp, Artemia.

The major cysteine protease in embryos and larvae of the brine shrimp Artemia franciscana is a heterodimer composed of a cathepsin L-like polypeptide of 28.5 kDa and a 31.5 kDa polypeptide called the cathepsin L-associated protein or CLAP. In a previous study, CLAP was shown to be a cell adhesion protein containing two Fas I domains and two GTP/ATP binding sites known as Walker A and B motifs. Here, we have characterized CLAP and its genes to better understand the role of this protein in Artemia development. The polymerase chain reaction was used to investigate the structure of the CLAP gene in two species of Artemia, the New World bisexual diploid A. franciscana and the Old World parthenogenetic tetraploid Artemia parthenogenetica. The protein coding region of the CLAP gene from each species was 99.5% identical for a protein of 332 amino acids, while the 3' non-coding region, representing nearly 45% of the gene, was only 86% identical between the two related species. However, while the CLAP gene is intronless in A. franciscana, in A. parthenogenetica the gene contained a mini-intron of 30 base pairs in the 3' non-coding region. The sequences representing the CLAP gene in A. franciscana and A. parthenogenetica have been entered into the NCBI database as AY757920 and DQ100385, respectively. Northern blot analysis showed that while the cathepsin L gene is expressed constitutively in Artemia franciscana embryos and young larvae, the CLAP gene is not expressed in late embryos and young larvae. In contrast, Western blots indicated that CLAP is present in developing embryos and young larvae, at least to the first larval molt, supporting results obtained previously showing CLAP's resistance to degradation by its dimeric partner, cathepsin L. At the protein level we showed that the GTP/ATP binding sites in CLAP are functional with rate constants of 0.024 and 0.022 for GTP and ATP hydrolase activity, respectively. GTP but not ATP also had a slight stimulatory effect on cathepsin L activity of the heterodimeric protease containing CLAP. Our results support the hypothesis that CLAP plays an important role in targeting and expression regulation of cathepsin L activity during early development of Artemia.

Characterization of a cathepsin L-associated protein in Artemia and its relationship to the FAS-I family of cell adhesion proteins.

We reported previously that the major cysteine protease in embryos and larvae of the brine shrimp, Artemia franciscana, is a heterodimeric protein consisting of a catalytic subunit (28.5 kDa) with a high degree of homology with cathepsin L, and a noncatalytic subunit (31.5 kDa) of unknown function. In the study reported here the noncatalytic subunit, or cathepsin L-associated protein (CLAP), was separated from cathepsin L by chromatography on Mono S and found to contain multiple isoforms with pIs ranging from 5.9 to 6.1. Heterodimeric and monomeric cathepsin L showed similar activity between pH 5 and 6.5, while the heterodimer was about twice as active as monomeric cathepsin L below pH 5. The heterodimer was more stable than the monomer between pH 6 and 7.4 and at 30-50 degrees C. Artemia CLAP and cathepsin L are present in nearly equimolar amounts at all stages in the life cycle and most abundant in encysted eggs and embyros. Moreover, CLAP, either free or as a complex with cathepsin L, was resistant to hydrolysis by cathepsin L. Two clones coding for CLAP were isolated from an Artemia embryo cDNA library and sequenced. Both clones have nearly identical open reading frames, but show differences at the 5'- and 3'-termini. Each cDNA clone has an extensive 3'-untranslated region containing 70-72% A+T. The deduced amino acid sequence of CLAP cDNA revealed two domains which were very similar to domains in fasciclin I and other cell adhesion proteins. The nucleotide sequences of clones 1 and 2 have been entered into the NCBI database (AY307377 and AY462276). This study supports the view that the noncatalytic subunit of the heterodimeric cysteine protease in Artemia stabilizes cathepsin L at various pH and temperatures normally inconsistent with cathepsin L from other organisms, and that CLAP serves as a docking mechanism for cathepsin L at nonlysosomal sites in Artemia embryos.

Molecular characterization of artemin and ferritin from Artemia franciscana.

Embryos of the brine shrimp, Artemia franciscana, exhibit remarkable resistance to physiological stress, which is temporally correlated with the presence of two proteins, one a small heat shock/alpha-crystallin protein termed p26 and the other called artemin, of unknown function. Artemin was sequenced previously by Edman degradation, and its relationship to ferritin, an iron storage protein, established. The isolation from an Artemia expressed sequence tag library of artemin and ferritin cDNAs extends this work. Artemin cDNA was found to contain an ORF of 693 nucleotides, and its deduced amino-acid sequence, except for the initiator methionine, was identical with that determined previously. Ferritin cDNA is 725 bp in length with an ORF of 516 nucleotides. Artemin amino-acid residues 32-185 are most similar to ferritin, but artemin is enriched in cysteines. The abundance of cysteines and their intramolecular spatial distribution suggest that artemin protects embryos against oxidative damage and/or that its function is redox regulated. The conserved regions in artemin and ferritin monomers are structurally similar to one another and both proteins assemble into oligomers. However, modeling of the quaternary structure indicated that artemin multimers lack the central space used for metal storage that characterizes ferritin oligomers, implying different roles for this protein. Probing of Northern blots revealed two artemin transcripts, one of 3.5 kb and another of 2.2 kb. These transcripts decreased in parallel and had almost disappeared by 16 h of development. The ferritin transcript of 0.8 kb increased slightly during reinitiation of development, then declined, and was almost completely gone by 16 h. Clearly, the loss of artemin and ferritin during embryo development is due to transcriptional regulation and proteolytic degradation of the proteins.