Yuriko Ohta

IDENTIFICATION OF S100B PROTEIN AS COPPER-BINDING PROTEIN AND ITS SUPPRESSION OF COPPER-INDUCED CELL DAMAGE

We have isolated from bovine brain a protein with a high capacity to inhibit the copper ion-catalyzed oxidation ofl-ascorbate and identified it as S100b protein, an EF-hand calcium-binding protein, by sequencing its proteolytic peptides. Copper binding studies showed that this protein has four copper-binding sites per dimeric protein molecule with a dissociation constant of 0.46 μm and that in the presence ofl-ascorbate, copper ions bind to a total of six binding sites with a great increase in affinity. Furthermore, we examined whether S100b protein can prevent copper-induced cell damage. Bovine S100b protein was found to suppress dose-dependently the hemolysis of mouse erythrocytes induced by CuCl2. We transformed Escherichia coli cells with pGEX-5X-3 vector containing a cDNA for rat S100b protein, so that this protein could be expressed as a fusion protein with glutathioneS-transferase. The transformed cells were demonstrated to be markedly resistant to a treatment with CuCl2 plus H2O2 as compared with the control cells expressing glutathione S-transferase alone. These results indicate that S100b protein does suppress oxidative cell damage by sequestering copper ions.

RANDOM NUCLEOTIDE SUBSTITUTIONS IN PRIMATE NONFUNCTIONAL GENE FOR L-GULONO-GAMMA -LACTONE OXIDASE, THE MISSING ENZYME IN L-ASCORBIC ACID BIOSYNTHESIS

Humans and other primates have no functional gene for L-gulono-gamma-lactone oxidase that catalyzes the last step of L-ascorbic acid biosynthesis. The 164-nucleotide sequence of exon X of the gene was compared among human, chimpanzee, orangutan, and macaque, and it was found that nucleotide substitutions had occurred at random throughout the sequence with a single nucleotide deletion, indicating that the primate L-gulono-gamma-lactone oxidase genes are a typical example of pseudogene.

Copper-catalyzed autoxidations of GSH and L-ascorbic acid: mutual inhibition of the respective oxidations by their coexistence.

Glutathione (GSH) is known to inhibit copper-catalyzed autoxidation of L-ascorbic acid (AA); in this study, AA was found to conversely inhibit copper-catalyzed autoxidation of GSH. To elucidate the mechanism of the mutual inhibition of the autoxidations of these two reducing substances in their coexistence, we have kinetically investigated these phenomena. The study of the former phenomenon revealed that GSH forms a 1:1 chelate with Cu(+) and thereby prevents the autoxidation of AA. By the analysis of the latter phenomenon, it was postulated that the inhibition of GSH oxidation by AA is due to rapid reduction of thiyl radical of GSH by AA rather than competition of AA with GSH in the reduction of Cu(2+). The effect of GSH on the formation of hydroxyl radical by the copper-catalyzed autoxidation of AA was also studied and it was found that the hydroxyl radical formation was delayed dose-dependently by GSH with time lags comparable to those of the oxidation of AA. Because there are several lines of evidence that redox-active copper ions are released from tissues under pathological conditions, it is possible that such copper ions coexist with AA and GSH in vivo, and in such a situation, GSH may exert an inhibitory effect on the hydroxyl radical formation caused by the autoxidation of AA.

C-Mannosylated peptides derived from the thrombospondin type 1 repeat interact with Hsc70 to modulate its signaling in RAW264.7 cells

The thrombospondin type 1 repeat (TSR) is a functional module of proteins called TSR superfamily proteins (e.g., thrombospondin, F-spondin, mindin, etc.) and includes a conserved Trp-x-x-Trp (W-x-x-W) motif, in which the first Trp residue is preferably modified by C-mannosylation. We previously reported that synthesized C-mannosylated TSR-derived peptides (e.g., C-Man-WSPW) specifically enhanced lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells. In this study, we searched for the proteins that bind to C-mannosylated TSR-derived peptides in RAW264.7 cells and identified heat shock cognate protein 70 (Hsc70). The binding affinity of Hsc70 for C-mannosylated peptides in solution was higher than that for the peptides without C-mannose. The binding was influenced by a nucleotide-induced conformational change of Hsc70, and C-mannosylated peptides preferred the substrate-binding domain of Hsc70. Furthermore, in RAW264.7 cells, addition of Hsc70 stimulated cellular signaling to produce tumor necrosis factor-alpha, via transforming growth factor-beta-activated kinase 1, and the Hsc70-induced signaling was enhanced more in the presence of the peptides with C-mannose than that without C-mannose, suggesting functional interaction between Hsc70 and the C-mannosylated peptides in the cells. Together, these results demonstrate a novel function of the C-mannosylation of TSR-derived peptides in terms of interaction with Hsc70 to regulate cellular signaling.

Identification by bacterial expression of the yeast genomic sequence encoding L‐galactono‐γ‐lactone oxidase, the homologue of L‐ascorbic acid‐synthesizing enzyme of higher animals

Bakers yeast possesses L‐galactono‐γ‐lactone oxidase that is similar in enzymic properties to L‐gulono‐γ‐lactone oxidase, the terminal enzyme of L‐ascorbic acid biosynthesis in higher animals. Computer‐assisted comparison of amino acid sequences revealed the occurrence of a homologue of rat L‐gulono‐γ‐lactone oxidase in the database of the proteins encoded by the Saccharomyces genome. The gene encoding the homologue was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S‐transferase in Escherichia coli cells with expression vector pGEX‐5X‐3. The expressed protein was found to show L‐galactono‐γ‐lactone oxidase activity and also to have covalently bound flavin, as is the case with purified L‐galactono‐γ‐lactone oxidase. Thus the gene for this enzyme was unequivocally identified.

Occurrence of two missense mutations in Cu‐ATPase of the macular mouse, a menkes disease model

We have investigated the genetic defect of the Cu‐ATPase gene (Atp7a) in the macular mouse, a genetic model of classical Menkes disease. Northern blot analysis showed that its placenta and kidney possess a normal amount of the Cu‐ATPase mRNA of the normal size; sequencing analysis revealed two missense mutations, His674Arg and Ser1381Pro, in a PCR‐amplified cDNA for mutant Cu‐ATPase. The latter mutation was suspected to affect the function of the ATPase, because it lies in the transmembrane segment that is thought to form a channel for the transportation of copper ions.

Increased cellular resistance to oxidative stress by expression of cyanobacterium catalase‐peroxidase in animal cells

To exploit prokaryotic antioxidant enzymes for protection of animal cells from oxidative damage, we expressed catalase‐peroxidase of cyanobacterium Synechococcus PCC 7942 in 104C1 cells. The gene for this enzyme was inserted into the mammalian expression vector pRc/CMV. The stable transfectants obtained had higher specific activities of catalase and as a result became more resistant to H2O2 or paraquat than the parental cells. Subcellular fractionation and immunoblot analysis revealed that the expressed catalase‐peroxidase was confined to the cytosol; this localization may be the basis for the effective protection of the transfectants from the oxidative cell damage.