Aspolin, a novel extremely aspartic acid-rich protein in fish muscle, promotes iron-mediated demethylation of trimethylamine-N-oxide

Trimethylamine-N-oxide (TMAO) is abundant in marine fish. Formaldehyde synthesis by TMAO demethylation during storage markedly deteriorates fish meat. In the present work, we cloned the extremely aspartic acid-rich proteins from skeletal muscle of a commercially important species, walleye pollack, in the course of molecular identification of trimethylamine-N-oxide demethylase (TMAOase). One of the cDNAs, designated as aspolin1, encodes an extremely aspartic acid-rich protein of 228 amino acids which is converted to the TMAOase after processing between Ala42 and Asp43. Mature aspolin1/TMAOase protein contains 179 Asp in 186 total amino acids. The other cDNA, designated as aspolin2, has a common nucleotide sequence with aspolin1 in the 5' part and encodes a protein which has an additional Asp polymer and a C-terminal cysteine-rich region. The amino acid sequence of the C-terminal cysteine-rich region of aspolin2 is highly homologous to the mammalian histidine-rich Ca2+-binding protein. Aspolin1/TMAOase and aspolin2 mRNA was most abundant in the skeletal muscle. A lower level of the mRNA was also detected in kidney, heart, spleen, and brain. Synthetic Asp polymer showed marked TMAOase activity in the presence of Fe2+, whereas a monomer and oligomers did not. Purified TMAOase protein bound to Fe2+ with low affinity, which may be responsible for the catalytic activity. Poly aspartic acid-Fe2+ complex generated after death would be involved in formaldehyde synthesis by the demethylation of TMAO during the storage of fish meat.     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Neurotoxicity and physicochemical properties of Abeta mutant peptides from cerebral amyloid angiopathy: implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease

Cerebral amyloid angiopathy (CAA) due to beta-amyloid (Abeta) is one of the specific pathological features of familial Alzheimer's disease. Abeta mainly consisting of 40- and 42-mer peptides (Abeta40 and Abeta42) exhibits neurotoxicity and aggregative abilities. All of the variants of Abeta40 and Abeta42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the Abeta40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type Abeta40. Similar tendency was observed for Abeta42 mutants at positions 22 and 23 whose neurotoxicity was 50-200 times stronger than that of the corresponding Abeta40 mutants, suggesting that these Abeta42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-Abeta42 and D23N-Abeta42 was similar to that of wild-type Abeta42, E22Q-Abeta42 and E22K-Abeta42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of Abeta mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that beta-sheet content of the Abeta mutants correlates with their aggregation. However, beta-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue beta-turn significantly enhanced the aggregative ability.     

Differences in the betaC-S lyase activities of viridans group streptococci

betaC-S Lyase catalyzes the alpha,beta-elimination of L-cysteine to hydrogen sulfide, which is one of the main causes of oral malodor and is highly toxic to mammalian cells. We evaluated the capacity of six species of oral streptococci to produce hydrogen sulfide. The crude enzyme extract from Streptococcus anginosus had the greatest capacity. However, comparative analysis of amino acid sequences did not detect any meaningful differences in the S. anginosus betaC-S lyase. The capacity of S. anginosus purified betaC-S lyase to degrade L-cysteine was also extremely high, while its capacity to degrade L-cystathionine was unremarkable. These findings suggest that the extremely high capacity of S. anginosus to produce hydrogen sulfide is due to the unique characteristic of betaC-S lyase from that organism.     

Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

Establishment of a swine monocyte cell line

A swine monocyte cell line was established from peripheral blood sample collected from a healthy adult male pig. The cloned cells grow actively in forming monolayers in both glass and plastic cell culture flasks with the growth medium reported previously (Kadoi, 2000) at 36.5 degrees C incubation. The plating efficiency is more than 95%. Densely grown cells in flasks show an epithelioid morphology. The fundamental properties of the cells were examined for cytological definition as monocytes. A positive property detected was guinea pig complement receptor, porcine IgG receptor, non-specific esterase, and acid phosphatase. A significant phagocytic activity proved by the inoculation of Saccharomyces cerevisiae is also one of the characteristics observed in the LPS-activated cells.     

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu²⁺ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO₄. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO₄. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU g^–1 total protein) when the strain was grown in the absence of CuSO₄. However, the activity was stimulated (25.8 mU g^–1 total protein) when cells were grown with 1 mM CuSO₄ and further enhanced to 110 mU g^–1 total protein with 10 mM CuSO₄. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO₄ in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO₄ in strain N6.