Functional characterization of artemin, a ferritin homolog synthesized in Artemia embryos during encystment and diapause.

Oviparously developing embryos of the crustacean Artemia franciscana encyst and enter diapause, exhibiting a level of stress tolerance seldom seen in metazoans. The extraordinary stress resistance of encysted Artemia embryos is thought to depend in part on the regulated synthesis of artemin, a ferritin superfamily member. The objective of this study was to better understand artemin function, and to this end the protein was synthesized in Escherichia coli and purified to apparent homogeneity. Purified artemin consisted of oligomers approximately 700 kDa in molecular mass that dissociated into monomers and a small number of dimers upon SDS/PAGE. Artemin inhibited heat-induced aggregation of citrate synthase in vitro, an activity characteristic of molecular chaperones and shown here to be shared by apoferritin and ferritin. This is the first report that apoferritin/ferritin may protect cells from stress other than by iron sequestration. Stably transfected mammalian cells synthesizing artemin were more resistant to heat and H(2)O(2) than were cells transfected with vector only, actions also shared by molecular chaperones such as the small heat shock proteins. The data indicate that artemin is a structurally modified ferritin arising either from a common ancestor gene or by duplication of the ferritin gene. Divergence, including acquisition of a C-terminal peptide extension and ferroxidase center modification, eliminated iron sequestration, but chaperone activity was retained. Therefore, because artemin accumulates abundantly during development, it has the potential to protect embryos from stress during encystment and diapause without adversely affecting iron metabolism.

Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis.

We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, since Asx constitutes by far the main amino acid residue. Preliminary sequencing surprisingly suggests that the first 75 N-terminal residues are all Asp. Caspartin self-aggregates spontaneously into multimers. In vitro tests show that it inhibits the precipitation of calcium carbonate. Furthermore, it strongly interferes with the growth of calcite crystals. A polyclonal antiserum raised against caspartin was used to localize this protein in the shell by immunogold. The immunolocalization demonstrates that caspartin is distributed within the prisms and makes a continuous film at the interface between the prisms and the surrounding insoluble sheets. Our finding emphasizes the prominent role of aspartic acid-rich proteins for the building of calcitic prisms among molluscs.

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.

Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin.

Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.

A 49 kDa microtubule cross-linking protein from Artemia franciscana is a coenzyme A-transferase.

Embryos and larvae of the brine shrimp, Artemia franciscana, were shown previously to possess a protein, now termed p49, which cross-links microtubules in vitro. Molecular characteristics of p49 were described, but the protein's identity and its role in the cell were not determined. Degenerate oligonucleotide primers designed on the basis of peptide sequence obtained by Edman degradation during this study were used to generate p49 cDNAs by RT-PCR and these were cloned and sequenced. Comparison with archived sequences revealed that the deduced amino acid sequence of p49 resembled the Drosophila gene product CG7920, as well as related proteins encoded in the genomes of Anopheles and Caenorhabditis. Similar proteins exist in several bacteria but no evident homologues were found in vertebrates and plants, and only very distant homologues resided in yeast. When evolutionary relationships were compared, p49 and the homologues from Drosophila, Anopheles and Caenorhabditis formed a distinct subcluster within phylogenetic trees. Additionally, the predicted secondary structures of p49, 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and glutaconate CoA-transferase from Acidaminococcus fermentans were similar and the enzymes may possess related catalytic mechanisms. The purified Artemia protein exhibited 4-hydroxybutyrate CoA-transferase activity, thereby establishing p49 as the first crustacean CoA-transferase to be characterized. Probing of Western blots with an antibody against p49 revealed a cross-reactive protein in Drosophila that associated with microtubules, but to a lesser extent than did p49 from Artemia.

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.

Functional characterization of the evolutionarily divergent fern plastocyanin.

Plastocyanin (Pc) is a soluble copper protein that transfers electrons from cytochrome b(6)f to photosystem I (PSI), two protein complexes that are localized in the thylakoid membranes in chloroplasts. The surface electrostatic potential distribution of Pc plays a key role in complex formation with the membrane-bound partners. It is practically identical for Pcs from plants and green algae, but is quite different for Pc from ferns. Here we report on a laser flash kinetic analysis of PSI reduction by Pc from various eukaryotic and prokaryotic organisms. The reaction of fern Pc with fern PSI fits a two-step kinetic model, consisting of complex formation and electron transfer, whereas other plant systems exhibit a mechanism that requires an additional intracomplex rearrangement step. The fern Pc interacts inefficiently with spinach PSI, showing no detectable complex formation. This can be explained by assuming that the unusual surface charge distribution of fern Pc impairs the interaction. Fern PSI behaves in a similar way as spinach PSI in reaction with other Pcs. The reactivity of fern Pc towards several soluble c-type cytochromes, including cytochrome f, has been analysed by flavin-photosensitized laser flash photolysis, demonstrating that the specific surface motifs for the interaction with cytochrome f are conserved in fern Pc.