Molecular characterization of a small heat shock/alpha-crystallin protein in encysted Artemia embryos

Molecular chaperones protect cells during stress by limiting the denaturation/aggregation of proteins and facilitating their renaturation. In this context, brine shrimp embryos can endure a wide variety of stressful conditions, including temperature extremes, prolonged anoxia, and desiccation, thus encountering shortages of both energy (ATP) and water. How the embryos survive these stresses is the subject of continuing study, a situation true for other organisms facing similar physiological challenges. To approach this question we cloned and sequenced a cDNA for p26, a molecular chaperone specific to oviparous Artemia embryos. p26 is the first representative of the small heat shock/alpha-crystallin family from crustaceans to be sequenced, and it possesses the conserved alpha-crystallin domain characteristic of these proteins. The secondary structure of this domain was predicted to consist predominantly of beta-pleated sheet, and it appeared to lack regions of alpha-helix. Unique properties of the nonconserved amino terminus, which showed weak similarity to nucleolins and fibrillarins, are enrichments in both glycine and arginine. The carboxyl-terminal tail is the longest yet reported for a small heat shock/alpha-crystallin protein, and it is hydrophilic, a common attribute of this region. Site-specific differences between amino acids from p26 and other small heat shock/alpha-crystallin proteins bring into question the functions proposed for some of these residues. Probing of Southern blots disclosed a multi-gene family for p26, whereas two size classes of p26 mRNA at 0.7 and 1.9 kilobase pairs were seen on Northern blots, the larger probably representing nonprocessed transcripts. Examination of immunofluorescently stained samples with the confocal microscope revealed that a limited portion of intracellular p26 is found in the nuclei of encysted embryos and that it resides within discrete compartments of this organelle. The results in this paper demonstrate clearly that p26 is a novel member of the small heat shock/alpha-crystallin family of proteins. These data, in concert with its restriction to embryos undergoing oviparous development, suggest that p26 functions as a molecular chaperone during exposure to stress, perhaps able to limit protein degradation and thus ensure a ready supply of functional proteins when growth is reinitiated.