Group 3 late Embryogenesis abundant proteins from embryos of Artemia francisana : molecular characteristics, expression and function

Late Embryogenesis Abundant (LEA) proteins are highly hydrophilic, intrinsically disordered proteins whose expression has been correlated with desiccation tolerance in anhydrobiotic organisms. Embryos of the brine shrimp, A. franciscana, contain high titers of group 3 LEA proteins during desiccation-tolerant stages such as diapause and pre-emergence development. Here I report the sequencing of three novel variants of AfrLEA3m mRNA (Afrlea3m_47, Afrlea3m_43 and Afrlea3m_29), whose deduced protein sequences are are predicted to localize to the mitochondrion. These mRNAs are very similar to Afrlea3m, but each has a stretch of sequence that is absent in at least one of the others. In addition Afrlea3m_43 has five single nucleotide changes scattered across its sequence, and Afrlea3m_47 and Afrlea3m_43 have three single nucleotide differences in the section of sequence shared only by these two variants. Protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 is highest in diapause embryos and decreases throughout development to their lowest levels in desiccation-sensitive nauplius larvae. This pattern of protein expression is in agreement with previously reported mRNA expression for AfrLEA2 and AfrLEA3m and supports a role for LEA proteins in desiccation tolerance of embryos. 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. Investigations of protein secondary structure show AfrLEA2 and AfrLEA3m to be intrinsically disordered in solution and that they gain structure during desiccation and in the presence of the solvents TFE and SDS. I also show that during drying recombinant AfrLEA2 and AfrLEA3m confer protection to three desiccation-sensitive enzymes (lactate dehydrogenase, phosphofructokinase and citrate synthase). The degree of protective ability was found to depend on the target enzyme chosen. The strongest degree of stabilization was observed when a given LEA protein was used in the presence of the stabilizing sugar trehalose, which is naturally accumulated by A. franciscana embryos. Finally, AfrLEA2 is shown by immunohistochemistry to reside in the cytoplasm and nucleus of embryonic cells of A. franciscana, and the AfrLEA3m proteins are localized to the mitochondrion. The presence of LEA proteins in multiple subcellular compartments suggests a requirement to protect biological structures in many areas of a cell in order for an organism to survive desiccation stress

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

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 desiccation-intolerant 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. © 2013 Cell Stress Society International

Group 3 late embryogenesis abundant proteins from embryos of Artemia franciscana : structural properties and protective abilities during desiccation.

Group 3 late embryogenesis abundant (LEA) proteins are highly hydrophilic, and their expression is associated with desiccation tolerance in both plants and animals. Here we show that two LEA proteins from embryos of Artemia franciscana, AfrLEA2 and AfrLEA3m, are intrinsically disordered in solution but upon desiccation gain secondary structure, as measured by circular dichroism. Trifluoroethanol and sodium dodecyl sulfate are both shown to induce a-helical structure in AfrLEA2 and AfrLEA3m. Bioinformatic predictions of secondary-structure content for both proteins correspond most closely to conformations measured in the dry state. Because some LEA proteins afford protection to desiccation-sensitive proteins during drying and subsequent rehydration, we tested for this capacity in AfrLEA2 and AfrLEA3m. The protective capacities vary, depending on the target enzyme. For the cytoplasmic enzyme lactate dehydrogenase, neither AfrLEA2 nor AfrLEA3m, with or without trehalose present, was able to afford protection better than that provided by bovine serum albumin (BSA) under the same conditions. However, for another cytoplasmic enzyme, phosphofructokinase, both AfrLEA2 and AfrLEA3m in the presence of trehalose were able to afford protection far greater than that provided by BSA with trehalose. Finally, for the mitochondrial enzyme citrate synthase, 400-mg/mL AfrLEA3m without trehalose provided significantly more protection than the same concentration of either AfrLEA2 or BSA

LEA Proteins during Water Stress: Not Just for Plants Anymore

Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many only assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress

Intracellular localization of group 3 LEA proteins in embryos of Artemia franciscana

Late embryogenesis abundant (LEA) proteins are accumulated by anhydrobiotic organisms in response to desiccation and improve survivorship during water stress. In this study we provide the first direct evidence for the subcellular localizations of AfrLEA2 and AfrLEA3m (and its subforms) in anhydrobiotic embryos of Artemia franciscana. Immunohistochemistry shows AfrLEA2 to reside in the cytoplasm and nucleus, and the four AfrLEA3m proteins to be localized to the mitochondrion. Cellular locations are supported by Western blots of mitochondrial, nuclear and cytoplasmic fractions. The presence of LEA proteins in multiple subcellular compartments of A. franciscana embryos suggests the need to protect biological structures in many areas of a cell in order for an organism to survive desiccation stress, and may explain in part why a multitude of different LEA proteins are expressed by a single organism

Occurrence of mitochondria-targeted Late Embryogenesis Abundant (LEA) gene in animals increases organelle resistance to water stress.

Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species

Diapause and anhydrobiosis in embryos of Artemia franciscana : metabolic depression, LEA proteins and water stress.

Metabolic depression is typically correlated with extended survival of environmental challenge and energy-limitation in early life stages of various invertebrates and vertebrates. Diapause is an ontogenetically-programmed reduction of development and often metabolism seen in many invertebrates. When embryos of Artemia franciscana enter the state of diapause, the overall metabolic depression is estimated to be greater than 99%. These embryos also contain trehalose and express multiple isoforms of Late Embryogenesis Abundant (LEA) proteins, constituents often present in a number of such anhydrobiotic animals. The mRNA levels for LEA proteins are highest in diapause and post-diapause embryos that possess desiccation tolerance, but are very low in desiccation-intolerant nauplius larvae. Stable transfection of human HepG2 cells with AfrLEA2 and AfrLEA3m was performed to evaluate the possibility of improved survivorship during drying. A trehalose transporter was used for intracellular loading of this disaccharide. LEA proteins improved desiccation tolerance in mammalian cells during acute drying and immediate rehydration

Structure of the Calcium-Rich Signature Domain of Human Thrombospondin-2

Thrombospondins (TSPs) are secreted glycoproteins that play key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6 Å resolution crystal structure of the glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module. These elements interact extensively to form three striking structural regions termed the stalk, wire, and globe. The TSP-2 signature domain is stabilized by these interactions and by a network of 30 bound Ca2+ ions and 18 disulfide bonds. The structure suggests how genetic alterations of TSPs result in disease