A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates.

We have isolated two Ca(2+)-dependent, galactose-binding polypeptides from the budding tunicate, Polyandrocarpa misakiensis. Based on their partial amino acid sequences, full-length cDNAs were cloned. One of them was identical with a tunicate C-type lectin (TC14-2) reported previously. The other was a novel C-type lectin, referred to as TC14-3. In living animals, they appeared to be coupled. This complex of lectins, when applied in vitro to tunicate multipotent cells of epithelial origin, blocked cell proliferation and induced cell aggregation. The aggregates expressed a homolog of the integrin alpha-chain and other differentiation markers specific for epithelial cells. Recombinant TC14-3 could reproduce all the activities of native lectins by itself, which was accelerated by recombinant TC14-2. The inhibitory activity of TC14-3 on cell growth was completely abolished by the addition of 50 microM D-galactose. Anti-TC14-3 monoclonal antibody showed that the antigen was expressed constitutively by the multipotent epithelial and mesenchymal cells. These results provide evidence that in P. misakiensis a C-type lectin plays a novel, cytostatic role in regulating cell growth, cell adhesion and cell differentiation during asexual reproduction.

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression.

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.

Functional retinoid receptors in budding ascidians.

A homolog of retinoid X receptors (RXR), named PmRXR, was cloned from the budding ascidian, Polyandrocarpa misakiensis. Gel-shift assays revealed that PmRXR and a previously identified P. misakiensis retinoic acid receptor (PmRAR) formed a complex to bind vertebrate-type retinoic acid response element (RARE). Transfection assays were carried out using a reporter gene containing a RARE upstream of lacZ. Two chimeric effector genes were constructed by placing PmRXR and PmRAR cDNA fragments (containing the DNA-binding, ligand-binding and ligand-dependent transactivation domains) downstream of the human RXR alpha and RAR alpha cDNA (covering the N-terminal coding region), respectively. Each chimeric cDNA was ligated to a notochord-specific enhancer. In case the embryos were transfected with all three transgenes and treated with retinoic acid (RA), the reporter gene was activated in the notochord cells. The result suggests that the PmRXR/PmRAR complex functions as an RA-dependent transcriptional activator. The PmRXR mRNA was detected in a mesenchymal cell type, called glomerulocyte, in the developing Polyandrocarpa bud. As this cell type has been shown to express PmRAR mRNA, it seems possible that the PmRXR/PmRAR complex mediates RA signaling in this cell type to induce the expression of genes involved in the morphogenesis of the developing bud.

A retinoic acid-inducible modular protease in budding ascidians.

Retinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein-protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase-TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species.

Free radical generation from heterocyclic amines by cytochrome b5 reductase in the presence of NADH.

We previously reported findings that NADPH/cytochrome P450 reductase can generate superoxide anion radical (O2*-) from heterocyclic amines (HCA) and from many anticancer agents in vitro. Here we present more evidence in which O2*- is generated when recombinant human cytochrome b5 reductase (rh-Cytb5Rd) was incubated with HCAs such as IQ and MeIQ in the presence of NADH in vitro. This indicates that free radical generation by rh-Cytb5Rd in the presence of HCA may add new insight into the damage of DNA in addition to the previously known mechanism: interaction of activated HCA-intermediates to form DNA adduct.

Serine protease inhibitors expressed in the process of budding of tunicates as revealed by EST analysis.

To identify genes expressed during budding of the tunicate Polyandrocarpa misakiensis, we isolated and sequenced 624 clones from a directionally constructed cDNA library to prepare a catalog of expressed sequence tags (ESTs). A total of 233 ESTs matched genes of known sequence in the SwissProt database. About 24% out of them showed high similarity to ribosomal proteins, twice the value (12%) of pre-budding animals. ESTs involved in the respiratory chain also appeared with significant redundancy, suggesting that tunicate budding is accompanied by the enhancement of energy conversion as well as protein synthesis. Serine protease inhibitor (serpin) afforded another striking example of a gene that was highly expressed in the process of budding. The deduced amino acid sequences of five serpin cDNAs all had two consensus signatures of the Kazal's type of secretory protease inhibitor, one of which had an active site for trypsin and the other for elastase. In line with this, recombinant GST-fusion protein showed both trypsin and elastase inhibitor activities. In accordance with the EST analysis, the hemolymph taken from the budding stage showed the highest activity of trypsin inhibitor. We discuss a possible role that Polyandrocarpa serpins may play in bud development by counteracting trypsin-like serine protease, which could facilitate dedifferentiation of formative tissues.

Immunohistochemical study of cytochrome b5 in human adrenal gland and in adrenocortical adenomas from patients with Cushing's syndrome.

Cytochrome b5, a component of the electron transfer system increases the relative activity of 17,20-lyase to 17alpha-hydroxylase of P450c17 in vitro. In the present study, immunohistochemical analysis of cytochrome b5 was performed in the human adrenal gland and in adrenocortical adenomas from patients with Cushing's syndrome. In the human adrenal gland, cytochrome b5 was stained in all three adrenocortical layers but the staining was most remarkable in the zona reticularis. All of the adenomas were composed mainly of compact cells, which exhibited immunoreactive staining for cytochrome b5 as well as for P450c17 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The distribution of b5 in the adenomas was correlated with that of P450c17 rather than with that of 3beta-HSD. The immunoreactive staining for cytochrome b5 appeared to be more prominent in the two adenomas that produced relatively high concentrations of adrenal androgens than in adenomas that produced low concentrations of adrenal androgens. These results immunohistochemically support the functional association of b5 with androgen production through interaction with P450c17 and the previous finding that higher concentrations of cytochrome b5 are associated with greater production of adrenal androgens in adrenocortical adenomas from patients with Cushing's syndrome.

Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 studied by site-directed mutagenesis.

Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 was studied by site-directed mutagenesis. The target residues for mutagenesis were selected on the basis of the previously reported chemical cross-linking study of these two proteins, which implicated possible charge-pair interactions between Lys-41, Lys-125, Lys-162, and Lys-163 of the enzyme, and Glu-47, Glu-48, Glu-52, Glu-60, Asp-64 (group A), and heme propionate of cytochrome b5. Mutant reductases that lost one of the above-listed Lys residues showed higher K(m) values for cytochrome b5 and lower kcat values than those of the wild type, suggesting that all of the examined Lys residues participate in binding with cytochrome b5 as reported previously. In contrast, a removal of one of (or even all of) the group A residues from cytochrome b5 by mutagenesis caused no significant effect on the catalytic properties of cytochrome b5. Additional elimination of another set of negative residues (Glu-41, Glu-42, Asp-57, and Glu-63 (Group B)), which are also located close to heme, elevated the K(m) value by more than five folds. These results suggest that there should be other acidic residue(s) than group A in cytochrome b5 which participate in binding with NADH-cytochrome b5 reductase.

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH-cytochrome b5 reductase gene.

Hereditary methaemoglobinaemia, caused by deficiency of NADH-cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C-to-T substitution in codon 8 3 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C-to-T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life-span of erythrocytes.

Mitochondrial NADH- or NADPH-linked aquacobalamin reductase activity is low in human skin fibroblasts with defects in synthesis of cobalamin coenzymes.

Mammalian livers have been reported to contain NADH- and NADPH-linked aquacobalamin reductases, which are distributed in both mitochondria and microsomes. The four aquacobalamin reductase isozymes have been purified and characterized from rat liver. It is unclear which aquacobalamin reductase among the four reductase isozymes participates in the synthesis of cobalamin coenzymes. To clarify the physiological roles of the aquacobalamin reductase isozymes, human mutant fibroblasts (cblC and cblA cells) with defects in cobalamin reductases involved in the coenzyme synthesis were used. In the cblC cells, the activity of the mitochondrial NADH-linked aquacobalamin reductase was reduced significantly, compared with normal human fibroblasts but the mitochondrial NADPH-linked enzyme was not. The reduced specific activity of the NADH-linked enzyme was not due to reduction in levels of the enzyme, but in its affinity for NADH. Although there was not a significant difference in the mitochondrial NADH-linked enzyme activity between normal and cblA cells, the activity of the mitochondrial NADPH-linked enzyme was not detectable in the mutant cells. These results indicate that the defects in the mitochondrial NADH- and NADPH-linked aquacobalamin reductases underlie cblC and cblA disorders, respectively.

Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia.

Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. From this mutant allele, the product without the catalytic portion of the enzyme is generated. The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll. The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart. The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.

Cloning and sequence analysis of a full-length cDNA of rat adrenodoxin.

A cDNA clone which covers the entire coding region for the precursor of adrenodoxin was isolated from a rat adrenal cDNA library. This precursor consists of amino-terminal 64 residues of extrapeptide for transport into mitochondria and the following 124 residues of mature peptide region. The amino acid sequence of rat mature adrenodoxin showed 85-98% homology with mouse, human, chicken, porcine, bovine and sheep counterparts, whereas that of the extrapeptide showed significantly lower values.

Two different activities of phospholipase A2 in porcine aortic cytosol.

Two different activities of phospholipase A2 (PLA2s, EC 3.1.1.4) which was activated at micro molar concentration of free Ca2+ were identified in cytosolic fraction of porcine aortic tissue. The two enzymes behaved very similarly during purification on ion-exchange, hydrophobic, gel filtration and affinity columns, and finally the active peaks of these enzymes were separated from each other on a hydroxyapatite HPLC column. Both of the enzymes hydrolyzed efficiently phosphatidylcholine (PtdCho) containing arachidonoyl residue as the substrate, but showed the apparent differences in head group specificity of phospholipids and also in the sensitivity against high Ca2+ concentration. This is the first report for the identification and characterization of high molecular weight cytosolic PLA2s in porcine aortic tissue.

Transient kinetics of intracomplex electron transfer in the human cytochrome b5 reductase-cytochrome b5 system: NAD+ modulates protein-protein binding and electron transfer.

Transient kinetics of reduction and interprotein electron transfer in the human cytochrome b5 reductase-cytochrome b5 (b5R-b5) system was studied by laser flash photolysis in the presence of 5-deazariboflavin and EDTA at pH 7.0. Flash-induced reduction of the FAD cofactor of b5R by deazariboflavin semiquinone (in the absence of b5) occurred in a rapid second-order reaction (k2 = 3.1 x 10(8) M-1 s-1) and resulted in a neutral (blue) FAD semiquinone. The heme of cytochrome b5 (in the absence of b5R) was also rapidly reduced in this system with k2 = 3.1 x 10(8) M-1 s-1. When the two proteins were mixed at low ionic strength, a strong complex was formed. Although the heme of complexed b5 could be directly reduced by deazariboflavin semiquinone, the second-order rate constant was nearly an order of magnitude smaller than that of free b5 (k2 = 3.4 x 10(7) M-1 s-1). In contrast, access to the FAD of b5R by the external reductant was decreased by considerably more than an order of magnitude (k2 < 1 x 10(7) M-1 s-1). When an excess of b5R was titrated with small increments of b5 and then subjected to laser flash photolysis in the presence of deazariboflavin/EDTA, interprotein electron transfer from the b5R FAD semiquinone to the heme of b5 could be observed. At low ionic strength (I = 16 mM), the reaction showed saturation behavior with respect to the b5 concentration, with a limiting first-order rate constant for interprotein electron transfer k1 = 375 s-1, and a dissociation constant for protein-protein transient complex formation of approximately 1 microM. The observed rate constants for interprotein electron transfer decreased 23-fold when the ionic strength was increased to 1 M, indicating a plus-minus electrostatic interaction between the two proteins. Saturation kinetics were also observed at I = 56, 96, and 120 mM, with limiting first-order rate constants of 195, 155, and 63 s-1, respectively. In the presence of NAD+, the transient protein-protein complex was stabilized by approximately a factor of two, and limiting first-order rate constants of 360 s-1 were obtained at both I = 56 mM and I = 96 mM and 235 s-1 at I = 120 mM. Thus, NAD+ appears to stabilize as well as to optimize the protein-protein complex with respect to electron transfer. Another effect of NAD+ is to appreciably slow autoxidation and disproportionation of the FAD semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome b5-like hemoprotein/cytochrome b5 reductase complex in rat liver mitochondria has NADH-linked aquacobalamin reductase activity.

Rat liver mitochondrial NADH-linked aquacobalamin reductase was characterized to clarify its enzymological properties. Most of the enzyme was solubilized with 10 g/L Triton X-100 from rat liver mitochondrial membranes. The elution behavior of the solubilized enzyme was identical to that of NADH-cytochrome c reductase (b-type cytochromes/cytochrome b5 reductase complex) during DEAE-Sepharose Fast Flow column chromatography. By mixing both purified cytochrome b5-like hemoprotein (outer membrane-cytochrome b) and cytochrome b5 reductase, cob(II)alamin was formed from aquacobalamin and NADH. These results provide evidence that the outer membrane-cytochrome b/cytochrome b5 reductase complex has the activity of the NADH-linked aquacobalamin reductase in rat liver mitochondria. Some properties of the NADH-linked aquacobalamin reductase were studied using the function of rat liver mitochondrial membranes. The specific activity (109.5 +/- 14.3 nmol.min-1.mg protein-1) of the enzyme was shown under physiological conditions (pH 7.1 at 40 degrees C). The optimal pH and temperature for activity were 7.1 and 40 degrees C, respectively. The apparent Km values were 41.9 mumol/L for aquacobalamin in the presence of 0.2 mmol/L NADH and 14.4 mumol/L for NADH in the presence of 0.1 mmol/L aquacobalamin. The enzyme was specific for aquacobalamin, because cyanocobalamin could not be reduced by the enzyme.

Cloning and nucleotide sequence of a cDNA of the human erythrocyte NADPH-flavin reductase.

A cDNA of human erythrocyte NADPH-flavin reductase was cloned from a lambda gt 11 human reticulocyte cDNA library by polymerase chain reaction using degenerate primers and followed by a plaque hybridization. The nucleotide sequence of the cDNA contains an open reading frame of 621 base pairs which encodes 206 amino acid residues including an initial methionine. The amino acid sequence deduced from the base sequence coincided well with peptide sequence determined for the purified human erythrocyte NADPH-flavin reductase. A homologous sequence to the FMN-binding site of flavodoxins was found at the amino-terminal region.

An in-frame deletion of codon 298 of the NADH-cytochrome b5 reductase gene results in hereditary methemoglobinemia type II (generalized type). A functional implication for the role of the COOH-terminal region of the enzyme.

The nucleotide sequence was determined for the gene of NADH-cytochrome b5 reductase of a patient of type II hereditary methemoglobinemia found in Yokohama, Japan. An in-frame deletion of 3 base pairs corresponding to codon 298 (TTC) was identified in the patient. The patient was homozygous for the mutation as shown by hybridization experiments using allele-specific oligonucleotides. The mutation causes deletion of Phe-298, which is the third to the COOH-terminal residue, indicating that in this mutant enzyme the sequence of this region has changed from -Cys-Phe-Val-Phe-COOH to -Cys-Val-Phe-COOH. The mutant enzyme, whose Phe-298 was deleted (F298 delta), was prepared by means of a bacterial expression system and site-directed mutagenesis. The kcat/Km value (NADH) of the enzyme was 5.7 s-1 M-1, which corresponds to 0.4% of that of the wild type. Moreover, the enzyme was much less thermostable than the wild type. To examine further the role of the COOH-terminal portion of the enzyme, various mutant enzymes were also prepared and characterized. The enzymatic properties of F298L, F300L, and F298L/F300L were essentially the same as that of the wild type. The kinetic properties of F298A, and F300A were not greatly affected, but the stability of the enzymes was somewhat impaired. Since Val-299 is naturally Ala in steer enzyme, no specific residues in the carboxyl-terminal region (298-300) are essential to the enzyme function. The instability of the F298/F300A double mutant indicates that the hydrophobicity of the carboxyl-terminal region of the enzyme might be important to maintain the conformation of the enzyme. high impairment of the activity of the F298 delta, F298stop, and F300stop mutants might be caused by the loss of the residue(s) in the carboxyl-terminal portion. These results indicate that the hydrophobicity, but not the specific amino acid residues, of the carboxyl-terminal portion of the enzyme is important for the stability of the enzyme.

Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.