Marilisa Riggio

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish).

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT-PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.

High affinity copper transport protein in the lizard Podarcis sicula: molecular cloning, functional characterization and expression in somatic tissues, follicular oocytes and eggs

Copper (Cu) is an essential element required in many biological processes including cellular growth and development. The molecular mechanisms involved in copper homeostasis include proteins that play a role in Cu uptake. Genes encoding high affinity copper transporters (Ctr) have been identified in yeast, plant and mammalian cells. Analysis of copper and zinc content in growing ovarian follicles and ovulated eggs of the reptilian Podarcis sicula demonstrated that the levels of both metals rise during oocyte growth, reaching the maximum in ovulated eggs. By exploiting the remarkable evolutionary conservation of the primary structure of Ctr proteins, cDNA encoding a Ctr was isolated from the liver of the lizard P. sicula by reverse transcriptase PCR and RACE strategy by using primers designed based on consensus motifs present in mammalian Ctr. The predicted protein sequence contains three transmembrane domains and a putative hydrophilic extracellular amino-terminal domain. Besides complementing the respiratory deficiency of yeast cells defective in high affinity Cu transport, expression of lizard Ctr1(1) in Hek293 cells stimulates Cu uptake.Gene expression assessed by Northern blot hybridization of RNA from different tissues of P. sicula shows the highest levels of transcript in both intestine and liver. The profile of Ctr1 mRNA in growing ovarian follicles and eggs demonstrates that the transcript accumulates during the oocyte growth and reaches the highest levels in ovulated eggs. These results suggest that lizard Ctr1 protein may function in Cu acquisition in growing oocytes and eggs.

Molecular cloning and sequence determination of a novel aspartic proteinase from Antarctic fish.

In the present report, we describe a novel aspartic proteinase from the liver of two Antarctic fish species. The nucleotide sequences of the cDNA obtained from the two fishes show 90% identity with each other but only 58% identity with aspartic proteinases from other sources. Sequence analysis shows features for the Antarctic enzymes which are not present in related enzymes of other organisms.

Responses to cadmium intoxication in the liver of the wall lizard Podarcis sicula.

This study examined the cytological and molecular effects of cadmium, a toxic heavy metal, in the liver of the Italian wall lizard Podarcis sicula. Cadmium was administered in single dose, by diet, to induce a concentration comparable with that measured in animals living in contaminated sites. For comparison, cadmium was also administered in multiple doses by food (chronic) or in a single dose intraperitoneally (i.p.); the effects were followed at regular time intervals up to 30 days post treatments. Atomic absorption spectrometry analysis demonstrated cadmium ion uptake and accumulation in the parenchyma with an estimated half-life of approximately 8 days. Cytological analyses revealed that the metal induced oedema, activated metallothionein expression in Kupffer cells and extracellular matrix production in fat storing cells. It also caused swelling and alteration in lipid and sugar metabolism in hepatocytes. In conclusion, in the wall lizard cadmium is toxic to the liver even at very low concentrations, the response is not strictly dose and time dependent and almost no recovery occurs in short (30 days) time periods.

Molecular cloning and sequencing of metallothionein in squamates : New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.

Oestrogen-induced expression of a novel liver-specific aspartic proteinase in Danio rerio (zebrafish)

Aspartic proteinases are a group of endoproteolytic proteinases active at acidic pH and characterized by the presence of two aspartyl residues in the active site. They include related paralogous proteins such as cathepsin D, cathepsin E and pepsin. Although extensively investigated in mammals, aspartic proteinases have been less studied in other vertebrates. In a previous work, we cloned and sequenced a DNA complementary to RNA encoding an enzyme present in zebrafish liver. The sequence resulted to be homologous to a novel form of aspartic proteinase firstly described by us in Antarctic fish. In zebrafish, the gene encoding this enzyme is expressed only in the female liver, in contrast with cathepsin D that is expressed in all the tissues examined independently of the sex. For this reason we have termed the new enzyme liver-specific aspartic proteinase (LAP). Northern blot analyses indicate that LAP gene expression is under hormonal control. Indeed, in oestrogen-treated male fish, cathepsin D expression was not enhanced in the various tissues examined, but the LAP gene product appeared exclusively in the liver. Our results provide evidence for an oestrogen-induced expression of LAP gene in liver. We postulate that the sexual dimorphic expression of the LAP gene may be related to the reproductive process.

Metallothionein in Antarctic Fish

Zinc occupies a prominent position among transition and d10 elements because of the role played in many biological processes [1,2], including catalysis [3], transcription and translation [4]. The functional role of zinc depends on the large variety of metallorganic complexes produced by this element as a result of its interaction with different protein families [3,5].

Metal accumulation and transport in the ovary of the lizard Podarcis sicula

Abstract The molecular characterisation of a CTR protein is reported in the ovary of the lizard Podarcis sicula; this protein proves to be homologous with the mammalian high affinity copper transporter CTRL Gene expression assessed by Northern blot hybridisation of RNA from growing ovarian follicles and eggs demonstrated that the transcript accumulated during the oocyte growth and reached the highest levels in ovulated eggs. Analysis of the copper content paralleled the profile of CTR1 mRNA, with a rapid increase of the metal concentration during oocyte maturation. These data suggest that the P. sicula CTR1 protein may function in the uptake and storage of the copper to be used during embryonic development.

Aspartic proteinases from Antarctic fish. A biochemical and molecular approach

Abstract Cathepsin D was purified to homogeneity from the liver of Antarctic icefish by anion‐exchange chromatography followed by affinity chromatography on concanavalin‐A Sepharose. The N‐terminal sequence of this proteinase was used to design a primer to be employed in reverse‐transcriptase polymerase chain reaction together with liver RNA from two Antarctic Notothenioidei, the red‐blooded Trematus bernacchii and the haemoglobinless icefish Chionodraco hamatus. The reaction mixture analysed by agarose gel electrophoresis, showed two bands of 1400 bp and 1200 bp. The open reading frame of the cloned cDNA, containing the 1400 bp fragment, encoded an aspartic proteinase showing sequence similarity with cathepsin D of other vertebrates. The nucleotide sequence of the 1200 bp fragment showed 58% identity with aspartic proteinase from other sources, but also features not found in homologous enzymes from other organisms. The temperature dependence of kinetic parameters was determined for the purified icefish cathepsin D. At temperatures between 8 and 50° C, the icefish proteinase had a higher specificity constant (kcat/Km) than human cathepsin D. The stability of both enzymes was measured at 50° C and half‐lives of 55 and 3 min were derived for icefish and human cathepsin D, respectively.