HLA-A2 mutants immunoselected in vitro. Definition of residues contributing to an HLA-A2-specific serological determinant.

The HLA-A2-specific mouse monoclonal antibody BB7.2 plus complement has been used to immunoselect variant clones of the lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, and -B27). Members of one class of variant clones appear to express cell surface HLA-A2 molecules that display reduced reactivity with the selecting antibody, but normal or near normal reactivities with some other HLA-A2-specific monoclonal antibodies and human alloantisera. The HLA-A2 heavy chains derived from two of these variant clones were characterized by comparative double-label tryptic peptide mapping in conjunction with microsequence analysis. These heavy chains were found to carry distinct mutations in the same peptide in the molecule. We conclude that residues within this short segment of the polypeptide contribute to an HLA-A2-specific serological determinant.

Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder.

Involvement of PLAGL2 in activation of iron deficient- and hypoxia-induced gene expression in mouse cell lines.

We searched iron-deficient inducible cDNA, using subtraction cloning and mRNA from desferrioxamine-treated mouse macrophage Raw264.7 cells. We identified a pleomorphic adenoma gene like 2 (PLAGL2), one of PLAG superfamily proteins exhibiting antiproliferative properties on tumor cells. Mouse PLAGL2 consists of 496 amino acids with seven C2H2 zinc-fingers. PLAGL2 mRNA was induced in RAW264.7 cells, mouse erythroleukemia cells and Balb/c 3T3 cells when they were treated with desferrioxamine. Hypoxia also increased PLAGL2 mRNA. Expression of PLAGL2 in COS-7 cells led to nuclear localization. PLAGL2 had potential binding ability to GC-rich oligonucleotide and activated transcription of a gene with the binding sequence in transient reporter assay, a finding consistent with a case seen in a PLAGL2 homolog, ZAC-1. Transient co-transfection of PLAGL2 or ZAC1 cDNA and a reporter containing a lactate dehydrogenase A (LDHA) promoter carrying the hypoxia inducible factor-1 responsive element led to an increase in the basal transcription in Balb/c 3T3 and HepG2 cells. Activation in transcription from the LDHA promoter increased by desferrioxamine treatment or hypoxia was further enhanced when PLAGL2 was expressed. We propose that PLAGL2 is involved in the cell cycle arrest and apoptosis of tumor cells by regulating iron depletion- or hypoxia-inducible gene expression.

Non-enzymatic heme formation in the presence of fatty acids and thiol reductants.

Non-enzymatic heme formation from equimolar amounts of porphyrin and iron was investigated. When mesoporphyrin IX and iron citrate were incubated with oleic acid and dithiothreitol at 37 degrees C in vacuo, mesoheme was formed in a high yield. When protoporphyrin IX and deuteroporphyrin IX were used, protoheme and deuteroheme were formed, respectively. Cysteine or 2-mercaptoethanol instead of dithiothreitol also resulted in the formation of heme. Linoleic acid was as effective as oleic acid, but at 37 degrees C, saturated fatty acids and phospholipids gave low yields. When incubation was at 70 degrees C saturated fatty acids as well as unsaturated fatty acids produced a large amount of heme. The optimum pH was 8.8. By increasing the concentration of Triton X-100 to 0.1%, heme formation decreased, and at concentrations above this level, completely disappeared. The conditions of non-enzymatic heme reaction presented here seem to be useful in elucidation of the mechanism of metalloporphyrin formation.

Characterization of sterol-ester hydrolase in Saccharomyces cerevisiae.

A homgenate of Saccharomyces cerevisiae grown under semi-anaerobic as well as aerobic conditions was found to catalyze the hydrolysis of fatty acid esters of sterols in the presence of Triton X-100. The enzyme levels in cells grown under various conditions were similar and the enzyme had a broad substrate specificity for sterol esters. The enzyme was localized in the mitochondrial fraction for the aerobically grown cells and in the mitochondrial and cytosolic fractions for the semi-anaerobically grown cells.

Expression of haptoglobin receptors in human hepatoma cells.

The uptake of radio-labeled hemoglobin-haptoglobin complex (Hb-Hp) by human hepatoma PLC/PRF/5 and HepG2 cells was investigated in an attempt to characterize the uptake process and intracellular transport. Human hepatoma cells took up Hb-Hp in a receptor-mediated manner. Scatchard analysis of binding revealed that PLC/PRF/5 and HepG2 cells exhibited about 21,000 and 63,000 haptoglobin receptors/cell, with a dissociation constant (Kd) of 8.0 and 17 nM, respectively. Human hepatocytes in primary culture also expressed about 84,000 receptors/cells, with a Kd of 7.4 nM. The hemoglobin-haptoglobin complex was internalized and subsequently the internalized Hb-Hp was slowly degraded in the cells. Preincubation of the cells with Hb-Hp resulted in a decrease in binding of the radioactive Hb-Hp to the cell surface, and was accompanied with an accumulation of intracellular receptors. The uptake of Hb-Hp by the cells was not inhibited by 100 microM chloroquine or by 10 mM methylamine, but was inhibited by 50 microM monodansylcadaverine. Hemoglobin-heme taken up by the cells induced microsomal heme oxygenase. Thus, human hepatoma PLC/PRF/5 and HepG2 cells can take up Hb-Hp by haptoglobin receptor-mediated endocytosis and Hb-Hp probably causes translocation of the haptoglobin receptors from the cell surface to the cell interior where they can be degraded. The internalized heme-moiety of hemoglobin can regulate the expression of heme oxygenase.

Quantitative aspects of free and esterified sterols in Saccharomyces cerevisiae under various conditions.

For extraction of free and esterified sterols from yeast cells, a method was devised in which both forms of sterols were extracted with light petroleum after the treatment of the cells with acetone, and then with dimethylsulfoxide. The content of sterol esters in the cells under aerobic conditions markedly increased with time, amounting to 95% of the total sterols under some conditions. However, the formed sterol esters were decreased, accompanied with an increase of free sterols, when the cells were put under anaerobic conditions. Variations of radioactivities of both sterols which had been labeled in the side chain by incubation of the cells with [Me[-14C]methionine were examined on the cells grown under various conditions. No variation was observed on the cells under aerobic conditions. On the other hand, the labeled esters were hydrolyzed to yield free sterols in the cells under anaerobic conditions. In the cells under aerobic conditions, the free sterols were found to consist mainly of ergosterol, whereas the esterified sterols contained considerable amounts of zymosterol, lanosterol, and other intermediate sterols besides ergosterol.

Characterization of sterol-ester synthetase in Saccharomyces cerevisiae.

Cell-free extracts of Saccharomyces cerevisiae grown under aerobic as well as semi-anaerobic conditions were found to catalyze the synthesis of fatty acid ester of sterol from cholesterol, fatty acid, ATP and CoA, or from cholesterol and fatty acyl-CoA. This result indicates that the enzyme involved in the formation of the ester is acyl-CoA:sterol O-acyltransferase (EC 2.3.1.26). The enzyme had a broad substrate specificity for sterols and acyl-CoAs. The enzyme levels in the cells grown under aerobic and semi-anaerobic conditions were almost equal. The enzyme was located in the microsomal fraction of the aerobically grown cells.

Characterization of ferrochelatase in kidney and erythroleukemia cells.

Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.5 and 12.7 microM, respectively. The Km values for iron and zinc with mesoporphyrin IX were 3.51 and 3.17 microM, respectively. The purified enzyme showed a single band with an apparent molecular mass of 42,000 daltons (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbit antibody against the purified enzyme markedly inhibited activities of the enzyme from both the kidney and liver. Immunoblot analysis showed that the antibody reacted with the renal as well as the hepatic enzymes showing the same molecular weight. Peptide mapping with trypsin or alpha-chymotrypsin showed that digested peptides of renal enzyme were similar to those of hepatic enzyme. Ferrochelatase activity in mouse erythroleukemia (MEL) cells increased in parallel with an increase of heme synthesis by treatment with dimethylsulfoxide. Using immunoblotting techniques, the amount of the enzyme in the MEL cells has been shown to increase by the induction, showing a molecular mass of 41 kDa which was the same as that of the mouse hepatic enzyme. Comparative structural analysis of the enzyme of MEL cells and that of mouse liver by peptide mapping showed that the partial digestive peptides of both enzymes exhibited a similar pattern. These results strongly suggest that ferrochelatase in kidney, liver and erythroid cells can be of one type.

Enhancement of glutathione levels in mouse peritoneal macrophages by sodium arsenite, cadmium chloride and glucose/glucose oxidase.

Glutathione content of mouse peritoneal macrophages markedly increased when they were exposed to insulting agents like sodium arsenite, cadmium chloride, and glucose/glucose oxidase which generates hydrogen peroxide. This increase was attributed to the induction of the cystine transport activity by these agents. The transport activity for other amino acids was not induced, but rather diminished by these agents. Heat shock treatment did not induce the cystine transport activity, nor did it augment glutathione. Since glutathione protects cells against the cytotoxic effects of these agents, the induction of the cystine transport activity constitutes a protective mechanism related to the stress caused by the agents. The protein component(s) for cystine transport may fall into the category of the stress protein.

A new immunoblotting assay for thymidylate synthetase and its application to the regulation of enzyme activity in regenerating rat liver.

A highly sensitive and specific immunoblot assay has been developed to quantitate the content of rat liver thymidylate synthetase (EC 2.1.1.45). Applying the method, it is demonstrated that the increase of the activity of thymidylate synthetase in liver regeneration after partial hepatectomy is due to the de novo synthesis of the enzyme protein. Administration of cycloheximide, phenoxybenzamine, phorbol 12-myristate 13-acetate, nifedipine, dexamethasone or indomethacin to partially hepatectomized rats prevented the synthesis of thymidylate synthetase in regenerating liver. Thyroparathyroidectomy also inhibited the increase of the enzyme in liver regeneration. These observations are discussed in relation to the signal transduction concerning the alpha 1-receptor, which was shown to regulate liver regeneration in our previous papers.

Association of ferrochelatase with Complex I in bovine heart mitochondria.

The location of ferrochelatase in bovine heart mitochondria has been studied. When the mitochondria were fractionated into Complexes I, II and III, ferrochelatase activity was only found in Complex I. Complex I also showed heme synthesis from ferric ion in the presence of NADH as an electron donor. Immunoblot experiments confirmed the presence of ferrochelatase in Complex I, but not in Complexes II or III. Some phospholipids, including phosphatidylserine and cardiolipin, stimulated NADH-dependent heme synthesis from ferric ion. When purified ferrochelatase was incubated with the low molecular weight form of NADH dehydrogenase prepared from Complex I, heme synthesis from ferric ion occurred by the addition of NADH. FMN markedly elevated the synthesis. These results indicate that ferrous ion is produced by NADH oxidation in Complex I and is then utilized for heme synthesis by ferrochelatase.

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.

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.

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.

Screening for ferrochelatase mutations: molecular heterogeneity of erythropoietic protoporphyria.

The DNA of 21 patients from 19 unrelated families with erythropoietic protoporphyria (EPP) were screened for the 6 ferrochelatase point mutations so far described. The mutation previously described by us (A >> T transversion at position -3 of the donor site of intron 10, causing exon 10 skipping) was detected in two additional unrelated EPP patients: in these patients, cDNA lacking exon 10 was also detected. The mutation described by Nakahashi et al. as responsible for exon 2 skipping (C >> T transition at position -23 of the acceptor site of intron 1), although also observed in some normal individuals, was invariably observed in all EPP patients tested and may thus play some role in the pathogenesis of EPP. Thus, it does not appear that this mutation is the primary mechanism underlying exon 2 skipping. None of the other four previously described mutations were detected. These data demonstrate the heterogeneity of the ferrochelatase locus and of the genetic defect in EPP.

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.

A novel mutation in erythropoietic protoporphyria: an aberrant ferrochelatase mRNA caused by exon skipping during RNA splicing.

An aberrant ferrochelatase mRNA lacking exon 10 was found in a patient with erythropoietic protoporphyria (EPP). In her genomic DNA an A-->T transversion at position -3 of the donor site of intron 10 appeared to be responsible for the exon skipping. Both the patient and her sister were heterozygous for this mutation.

Homozygous variegate porphyria: 20 y follow-up and characterization of molecular defect.

The long-term follow-up of a homozygous variegate porphyria patient revealed severe photosensitivity accompanied by mild sensory neuropathy and IgA nephropathy. A 35T to C transition in exon 2 (I12T) and a 767C to G transversion in exon 7 (P256R) of the protoporphyrinogen oxidase gene were identified from both alleles of the patient's cDNA and genomic DNA samples. Both prokaryotic and eukaryotic expression studies showed that the first mutation in the evolutionary conserved region resulted in a decrease in the protoporphyrinogen oxidase activity in contrast to the polymorphic substitution in exon 7, which affected the function of the enzyme assayed in Escherichia coli but not COS-1 cells.

Structure and transcriptional regulation of the mouse ferrochelatase gene.

Ferrochelatase (EC.4.99.1.1), the final step in the biosynthesis of heme, is widely expressed in various tissues and is induced in erythroid cells. We determined the structure of the mouse ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. The gene spans about 25 kb and consists of 11 exons. The exon/intron boundary sequences conform to consensus acceptor (GTn)/donor (nAG) sequences, and exons in the gene encode functional protein domains. The promoter region contains multiple Sp1 sites, a CACCC box and GATA-1 binding sites. Function analysis of the promoter by transient transfection assay demonstrated that one Sp1 binding site located at -37/-32 is essential for basic expression of the ferrochelatase gene in both mouse erythroleukemia (MEL) and non-erythroid EL4 cells. In addition, the region (-66/-51) containing a CACCC box and the neighboring GC box partly contributes to the inducible activity of the reporter in MEL cells upon induction with dimethylsulfoxide. It appears that at least two promoter regions of the mouse ferrochelatase gene function in basic and inducible expression.