Hirao Kohno

The human 32-kDa stress protein induced by exposure to arsenite and cadmium ions is heme oxygenase

Exposure of HeLa and HL60 cells to sodium arsenite or cadmium chloride led to marked increases in cellular heme oxygenase activity. SDS‐polyacrylamide gel clectrophoresis of [35S]methionine‐labeled cellular proteins indicated that these treatments also resulted in the induction of a 32‐kDa protein. Immunoblot analysis further showed that the 32‐kDa protein reacted with anti‐bovine heme oxygenase antibodies. Treatment of the cells with cobaltic chloride or heat induced neither the 32‐kDa protein nor heme oxygenase activity. It is concluded that the 32‐kDa stress protein induced by arsenite and cadmium ions in these human cells is heme oxygenase.

Molecular Characterization of a Newly Identified Heme-binding Protein Induced during Differentiation of urine Erythroleukemia Cells

A heme-binding protein with a molecular mass of 22 kDa, termed p22 HBP, was purified from mouse liver cytosol, using blue Sepharose CL-6B. We identified a cDNA encoding p22 HBP, and sequence analysis revealed that p22 HBP comprises 190 amino acid residues (M r 21,063) and has no homology to any other known heme-binding protein. The p22 HBP mRNA (∼1.0 kilobases) is ubiquitously expressed in various tissues and is extremely abundant in the liver. cDNA allows for expression of active p22 HBP, with a high affinity for 55Fe-hemin, with aK d of 26 ±1.8 nm. TheB max of hemin binding to p22 HBP was 0.55 ± 0.021 mol/mol of protein, a value consistent with one heme molecule binding per molecule of protein. The order of potency of different ligands to compete against 55Fe-hemin binding to p22 HBP was hemin = protoporphyrin IX > coproporphyrin III > bilirubin > palmitic acid > all-trans-retinoic acid. Treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide or hemin resulted in an increase in p22 HBP mRNA. The immunoblot analysis showed that p22 HBP increased with time in dimethyl sulfoxide- and hemin-induced MEL cells. Conversely, transfer of antisense oligonucleotides to p22 HBP cDNA resulted in a decrease of p22 HBP in dimethyl sulfoxide-treated MEL cells, and the heme content in these cells decreased to 66–71% of sense oligonucleotides-transferred cells. Thus, this newly identified heme-binding protein, p22 HBP, may be involved in heme utilization for hemoprotein synthesis and even be coupled to hemoglobin synthesis during erythroid differentiation.

Iron deprivation decreases ribonucleotide reductase activity and DNA synthesis

The effects of the iron-chelator, desferrioxamine, and monoclonal antibodies against transferrin receptors on DNA synthesis and ribonucleotide reductase activity were examined in human leukemia K562 cells. Treatment of the cells with desferrioxamine resulted in decreases of ribonucleotide reductase activity, DNA synthesis, and cell growth. Exposure of the cells to anti-transferrin receptor antibody, 42/6, which blocks iron supplement into cells caused decreases of ribonucleotide reductase activity and DNA synthesis, in a parallel fashion. Decreases of ribonucleotide reductase activity and DNA synthesis by 42/6 were restored by the addition of ferric nitriloacetate. These results indicate that ribonucleotide reductase activity is dependent on the iron-supply and also regulates cell proliferation.

Site-directed mutagenesis of human ferrochelatase: Identification of histidine-263 as a binding site for metal ions

In nature, ferrochelatase catalyzes the insertion of ferrous ion into the porphyrin macrocycle of protoporphyrin IX to exclude two protons to form protoheme IX: other porphyrin substrates, including mesoporphyrin IX may be used in vitro. Based on the deduced amino-acid sequences, one histidine residue (H263 of human enzyme) is conserved among all ferrochelatases cloned from human to bacterial cells, and three histidine residues (H157, H341 and H388 of human enzyme) are conserved among eukaryotic ferrochelatases; no cysteine residue is conserved. To attempt to clarify the binding site of ferrous ion, we converted four highly conserved histidine residues in human ferrochelatase to alanine, using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli, and iron- and zinc-chelating activities were examined. Mutants H157A and H388A lost most of their activities and concomitantly the enzyme became susceptible to proteolytic degradation. Kinetic studies with the residual activities showed no significant change of Km values for metal ions or for mesoporphyrin IX. Mutation at H341 did not alter the enzyme activities. Iron- and zinc-chelating activities of mutant H263A were reduced to 30% and 21% of the activities of the wild type, respectively. Moreover, this mutation resulted in 18- and 3.4-fold increases in Km values toward ferrous and zinc ions, respectively, while the Km value for mesoporphyrin remained unchanged. These results indicate that the binding site for metal ions in ferrochelatase is distinct from that for the porphyrin, and suggest that histidine-263 contributes significantly to the binding of metal ions. Maintenance of the structure of the protein molecule may involve functions related to histidine-157 and -388.

CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells

The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. Previous studies demonstrated that CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34+CD38− SRCs. However, the purification level of rare CD34− SRCs in 18 lineage marker-negative (Lin−) CD34− cells (1/1000) is still very low compared with that of CD34+CD38− SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a high degree of purification of rare human CD34− SRCs. Using 18Lin−CD34− cells, we analyzed the expression of candidate positive markers by flow cytometric analysis. We finally identified CD133 as a reliable positive marker of human CB-derived CD34− SRCs and succeeded in highly purifying primitive human CD34− HSCs. The limiting dilution analysis demonstrated that the incidence of CD34− SRCs in 18Lin−CD34−CD133+ cells was 1/142, which is the highest level of purification of these unique CD34− HSCs to date. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin−CD34− cells, as well as 18Lin−CD34+ cells, in their positive fractions, indicating its functional significance as a common cell surface maker to isolate effectively both CD34+ and CD34− SRCs.

Molecular cloning, sequencing and expression of cDNA encoding human coproporphyrinogen oxidase.

A complete cDNA clone encoding human coproporphyrinogen (coprogen) oxidase, the sixth enzyme in the heme biosynthetic pathway, has been isolated from a human placenta cDNA library. The cDNA had an open reading frame of 1062 base pairs encoding a protein of 354 amino acid residues (M(r) 40,291). Amino acid sequencing showed that the mature enzyme consists of 323 amino acid residues (M(r) 36,842) with a putative leader peptide of 31 amino acid residues. The human enzyme showed an 86% identity to the mouse enzyme. In addition, the recombinant enzyme which did not contain leader peptide was actively expressed in Escherichia coli. The isolation and expression of cDNA for human coprogen oxidase should facilitate studies of the structure of the gene as well as characterization of molecular lesions causing hereditary coproporphyria.

Mouse coproporphyrinogen oxidase is a copper-containing enzyme: expression in Escherichia coli and site-directed mutagenesis

We previously isolated cDNA for mouse coproporphyrinogen oxidase (CPO) and provided evidence for the induction of mRNA during differentiation of murine erythroleukemia cells (Kohno et al. (1993) J. Biol. Chem. 268, 21359-21363). To better understand the structure and the mechanisms of reaction of the enzyme, we expressed mouse CPO in Escherichia. coli and purified it to a homogeneity. Analysis of the metal content revealed that the recombinant mouse CPO contains one copper atom per polypeptide chain. When the bacterial cells were treated with D-penicillamine, a copper chelator, formation of the active CPO was partially reduced. Addition of Cu2+ in minimal medium resulted in 6-fold higher level of CPO activity. These results suggest that expression of active mouse CPO in E. coli depended on the presence of Cu2+ in the culture medium. To elucidate the apparent involvement of Cu2+ in enzyme function, a series of mutant enzymes, whose highly conserved histidine and cysteine residues were individually converted to alanine residue, were prepared by site-directed mutagenesis. Mutant enzymes were expressed in E. coli and their activities examined. Mutation at histidine 158 resulted in a complete loss of enzyme activity, yet the enzyme protein was expressed at a comparable level. Concomitantly, only a trace amount of Cu2+ was detected in the purified H158A enzyme. We propose that mouse CPO is copper-containing enzyme and Cu2+ interacts with a conserved histidine residue.

Cytokine-Dependent Modification of IL-12p70 and IL-23 Balance in Dendritic Cells by Ligand Activation of Vα24 Invariant NKT Cells

CD1d-restricted invariant NKT (iNKT) cells play crucial roles in various types of immune responses, including autoimmune diseases, infectious diseases and tumor surveillance. The mechanisms underlying their adjuvant functions are well understood. Nevertheless, although IL-4 and IL-10 production characterize iNKT cells able to prevent or ameliorate some autoimmune diseases and inflammatory conditions, the precise mechanisms by which iNKT cells exert immune regulatory function remain elusive. This study demonstrates that the activation of human iNKT cells by their specific ligand α-galactosylceramide enhances IL-12p70 while inhibiting the IL-23 production by monocyte-derived dendritic cells, and in turn down-regulating the IL-17 production by memory CD4+ Th cells. The ability of the iNKT cells to regulate the differential production of IL-12p70/IL-23 is mainly mediated by a remarkable hallmark of their function to produce both Th1 and Th2 cytokines. In particular, the down-regulation of IL-23 is markedly associated with a production of IL-4 and IL-10 from iNKT cells. Moreover, Th2 cytokines, such as IL-4 and IL-13 play a crucial role in defining the biased production of IL-12p70/IL-23 by enhancement of IL-12p70 in synergy with IFN-γ, whereas inhibition of the IFN-γ-promoted IL-23 production. Collectively, the results suggest that iNKT cells modify the IL-12p70/IL-23 balance to enhance the IL-12p70-induced cell-mediated immunity and suppress the IL-23-dependent inflammatory pathologies. These results may account for the long-appreciated contrasting beneficial and adverse consequence of ligand activation of iNKT cells.

Dietary restriction: effects of short-term fasting on protein uptake and cell death/proliferation in the rat liver

Dietary restriction (DR) is known to prolong life in laboratory animals. Intermittent (alternate-day) fasting or short-term repeated fasting has also been reported to increase the life span of animals. In the present study, we investigated the changes or induction of abnormalities of protein metabolism in rats during fasting, and measured asialoglycoprotein uptake and cell death/proliferation in the liver of rats receiving fasting and refeeding. In the results, liver weight decreased significantly after 48 h of fasting and increased during the refeeding period, returning to the pre-fasting level by 12 h of refeeding. Cell death, determined by single stranded DNA (ssDNA) staining method, increased during the fasting period, and returned to the pre-fasting level during the refeeding period. Cell proliferation, determined using antibodies (Ab) against proliferating cell nuclear antigen, decreased during the fasting period, and increased during the refeeding period. Changes in cell death and cell proliferation were inversely related. However, there was no significant difference in asialoglycoprotein uptake by the whole liver between the ad libitum (AL)-fed rats and 48 h fasted rats. Thus, neither the changes in liver weight nor cell death/proliferation affected asialoglycoprotein uptake on a living body. These results suggest that episodes of 48 h fasting do not induce protein metabolism abnormalities in the liver.

Overexpression in Escherichia coli, and one-step purification of the human recombinant ferrochelatase.

Ferrochelatase (EC 4.99.1.1), a mitochondrial inner membrane-bound protein, is the terminal enzyme of heme biosynthesis. The cDNA encoding the human mature ferrochelatase was placed under transcriptional control of T7 RNA polymerase in an Escherichia coli expression system. The bacteria produced large amounts of 42 kDa protein which reacted with anti-ferrochelatase antibodies. Expressed ferrochelatase exhibited iron- and zinc-chelating activities, and was found as a soluble protein. The recombinant enzyme has been purified to apparent homogeneity with a high yield, by one-step purification involving Blue-Sepharose chromatography. The purified enzyme which showed a molecular weight of about 40,000 by gel-filtration, functioned in a monomeric form. Km value for both mesoporphyrin IX and protoporphyrin IX with zinc was 12.5 microM. Km values for iron and zinc with mesoporphyrin IX were 6.7 microM and 11.8 microM, respectively. Zinc-chelating activity was markedly stimulated by palmitic acid, but iron-chelating activity remained unchanged. The above results were similar to those reported previously for mammalian ferrochelatase. The overexpression and the simple purification of a functional ferrochelatase exhibiting the same properties as natural enzyme will allow us to elucidate the mechanism of the enzyme reaction and structural changes of the mutated enzyme.