Autoantibodies against inosine-5'-monophosphate dehydrogenase 2--characteristics and prevalence in patients with HCV-infection.

BACKGROUND: Indirect immunofluorescence (IIFT) on in house HEp-2 cell preparations revealed a novel antibody giving a granular cytoplasmic pattern not described before, which on two commercial cell preparations revealed a "rings and rods" pattern. This pattern was also observed in four HCV-RNA carriers and prompted the identification of the reactive antigen and the evaluation of the antibody prevalence in HCV-RNA carriers and control groups. METHODS: The antigen's molecular weight was determined by radioimmunoprecipitation of 35S-methionine labeled cell proteins. Expression library screening and sequencing was performed by standard techniques using an oligo(dT)-primed human HeLa cell cDNA expression library. Antibodies against the novel antigen Inositol-5'-monophosphatdehydrogenase 2 (IMPDH2) were analyzed by IIFT, western blot, line blot, and radioimmunoprecipitation assay (RIPA). IIFT was performed on commercial HEp-2 cells and cells cultivated in house for 24 - 60 hours, with or without the IMPDH2 inhibitors mycophenolic acid (MPA) or ribavirin, and subjected to various fixation conditions. Western and line blots were performed with IMPDH2 synthesized in E. coli, RIPA with 35S-methionine-IMPDH2 from in vitro transcription/translation products. Sera screened were positive for HCV-RNA (108), HBV-DNA (100), anti-mitochondrial (31), anti-actin (42), and anti-nuclear antibodies (51) and negative for HCV-RNA (100) and blood donors (100). RESULTS: IMPDH2 is capable of considerable intracellular rearrangements (upon action of inhibitors like MPA and ribavirin), which explains the contrasting immunofluorescence patterns in cells from different sources. By RIPA, proven to be the sole assay suitable for screening of anti-IMPDH2 in human sera, autoantibodies were found in 35.2% of HCV-RNA carriers and in low concentrations in 31% of anti-actin positive patients suspicious of autoimmune hepatitis. Antibodies reacted preferentially with conformational epitopes. Compared to the low concentration of anti-IMPDH2 found in other disease groups, high antibody concentrations were observed in HCV-RNA carriers. CONCLUSIONS: The common occurrence of anti-IMPDH2 in HCV-RNA carriers may be related to ribavirin therapy, causing intracellular aggregation of IMPDH2 thereby altering its immunogenicity. In this study the "rods and rings" immunofluorescence pattern observed could be ascribed to anti-IMPDH2. Anti-IMPDH2 may cause difficulties in interpretation of immunofluorescence patterns in routine autoantibody testing.

Expression and functional characterization of a pHis-tagged human bradykinin B2 receptor in COS-7 cells.

A polyHis-tagged bradykinin (BK) B2 receptor (pHis-BKR) cDNA was constructed and expressed in COS-7 cells. The pHis-BKR is suitable for both immunoprecipitation and immunoblotting with anti-polyHis antibodies and can be easily purified using Ni-NTA columns. Immunochemical detection revealed a molecular mass of approximately 66 kDa. The pHis-BKR is capable of mediating BK-induced stimulation of inositol phosphate formation as well as of mitogen-activated protein kinase (MAPK) activity. Compared with the wild-type receptor (WT-BKR) the tagged receptor showed a slightly enhanced affinity towards BK but a reduced expression level. Despite these modified pharmacological properties the pHis-tagged BKR may be a useful tool for studying BKR modifications and signaling.

Molecular analysis of regulation of gene expression of the human erythroid anion exchanger (AE) 1.

The anion exchange protein AE1 is the most abundant membrane protein in human erythrocytes mediating the electroneutral chloride/bicarbonate exchange. We identified a promoter region in the 5' flanking region of the human AE1 gene which controls transcription in a cell type independent manner. In addition a second, distal promoter element mediates gene expression only in erythroid cells and in dependence upon differentiation. Within this distal promoter region we defined a 44 bp sequence containing a novel CT-rich motif with very strong promoter activity whereas a second 28 bp segment suppresses gene expression.

Inhibition of mouse erythroid band 3-mediated chloride transport by site-directed mutagenesis of histidine residues and its reversal by second site mutation of Lys 558, the locus of covalent H2DIDS binding.

Substitution by site-directed mutagenesis of any one of the histidine residues H721, H837, and H852 by glutamine, or of H752 by serine, inhibits Cl- flux mediated by band 3 expressed in Xenopus oocytes. Mutation of Lys 558 (K558N), the site of covalent binding of H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) in the outer membrane surface, in combination with any one of the His/Gln mutations leads to partial (H721Q; H837Q) or complete (H852Q) restoration of Cl- flux. In contrast, inhibition of Cl- flux by mutation of proline or lysine residues in the vicinity of His 837 at the inner membrane surface cannot be reversed by the second-site mutation K558N, indicating specificity of interaction between Lys 558 and His 837. The histidine-specific reagent diethyl pyrocarbonate (DEPC) is known to inhibit band 3-mediated anion exchange in red blood cells [Izuhara, K., Okubo, K., & Hamasaki, N. (1989) Biochemistry 28, 4725-4728]. It was also found to inhibit transport after expression in the oocyte of wild-type band 3, of the double mutants of the histidines listed above, and of the single mutant H752S. The effects on the wild type and the double mutants were indistinguishable, while the mutant H752S exhibited a considerably reduced sensitivity to inhibition, suggesting that His 752 is the most prominent site of action of DEPC. According to a hydrophobicity plot of band 3 and further independent evidence, Lys 558, the mutated histidines, and Glu 699, the mutation of which was also found to inhibit Cl- flux [Müller-Berger, S., Karbach, D., Kang, D., Aranibar, N., Wood, P. G., Rüterjans, H., & Passow, H. (1995) Biochemistry 34, 9325-9332], are most likely located in five different transmembrane helices. The interactions between Lys 558 and the various histidines suggest that these helices reside in close proximity. Together with the helix carrying Glu 699, they could form an access channel lined with an array of alternating histidine and glutamate residues. Together with a chloride ion bridging the gap between His 852 and His 837, they could have the potential to form, at low pH, a transmembrane chain of hydrogen bonds. The possible functional significance of such channel is discussed.

Expression of the anion exchanger (AE) gene family in human brain. Identification of a new AE protein: AE0.

We present the first identification of proteins of the anion exchanger (AE) gene family expressed in human brain. Expression was established by the reverse transcriptase-polymerase chain reaction (RT-PCR), performed on RNA isolated from frontal cortex tissue. The erythroid form AE1, the major non-erythroid form AE2, and a novel member of the AE family, which we named AE0, were identified. Immunohistochemical analysis revealed the highest expression of these proteins in large pyramidal neurons in the frontal cortex and hippocampus. The sequence of the membrane domain of AE0 is identical to that of the major erythroid species AE1, except for the third extracellular loop, which contains a 25 amino acid insertion. This insertion is identical to a sequence in the third extracellular loop of AE2. Expression of AE0 is not restricted to brain tissue, since we could also detect AE0-mRNA in T-lymphocytes and reticulocytes. Chromosomal mapping indicates that the AE0 gene is most likely located on human chromosome 22. We did not find any indications for qualitative changes in AE1, AE2, or AE0 in Alzheimer brain tissue.

Anion exchanger 1 in human kidney and oncocytoma differs from erythroid AE1 in its NH2 terminus.

Acid-secreting intercalated cells of the kidney collecting duct and tumor cells of renal oncocytoma express an anion exchanger that is immunologically related but not identical to the chloride-bicarbonate anion exchanger of erythrocytes (AE1). In this study, we have mapped the binding site of a monoclonal antibody against erythroid AE1 that does not react with either intercalated cells or oncocytoma. The epitope is located close to the NH2 terminus of AE1, indicating that AE1 in intercalated cells and oncocytoma differs in its NH2 terminus from erythroid AE1. This conclusion was supported by an antibody directed against residues 1-14 of erythroid AE1 that does not react with intercalated cells in oncocytoma. Polymerase chain reaction performed with mRNA from a human kidney revealed that the sequence containing the codons for Met-1 and Met-33 in erythroid mRNA is missing in the kidney transcript, whereas the sequence coding for Met-66 is present. DNA sequence data derived from cloning the 5' end of the human kidney AE1 mRNA clearly showed that the 5' untranslated region comprises part of intron 3, the complete exon 4 that is followed by exon 5 containing Met-66 as the site of translation initiation. Altogether, the results indicate that AE1 in the human kidney is an amino-terminally truncated form of erythroid AE1 that is restricted to the basolateral membrane domain of the acid-secreting intercalated cells of the collecting duct and is also expressed in oncocytoma.

Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.

The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.

Amplification and direct sequencing of a cDNA encoding human cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

Cytosolic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase (E.C. 4.1.3.5) is a highly regulated enzyme involved in isoprenoid biosynthesis and therefore a potential target for cholesterol-lowering drugs. Up to now, primary structure data have only been available for chicken, rat and hamster HMG-CoA synthase. Using in vitro amplification and direct sequencing, we have determined the nucleotide sequence of the coding region of the human cytosolic 3-hydroxy-3-methylglutaryl CoA synthase cDNA.

Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA.

In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5'-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3'-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins.

Complete nucleotide sequence of band 3 related anion transport protein AE2 from human kidney.

The cDNA coding for the complete human band 3 related anion exchange protein AE2 has been cloned from human kidney mRNA. The protein is encoded by a mRNA of approx. 3885 nucleotides containing an open reading frame of 3720 nucleotides. The AE2 protein consists of 1240 amino acid residues and has a molecular mass of 136,805 Da.

Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous beta 1 subunits of the sodium pump.

Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.

Expression and characterization of recombinant second domain variants of human mucus proteinase inhibitor (MPI).

The second, carboxyterminal domain of human mucus proteinase inhibitor (MPI) represents a strong antagonist of trypsin, chymotrypsin and leucocyte elastase. To modulate the inhibitory specificity and chemical stability of this domain, mutants have been prepared by site-directed mutagenesis of a cDNA fragment encoding for the carboxyterminal half of the inhibitor, followed by expression in E. coli. Inhibition assays with the purified recombinant domains revealed the possibility to create variants for potential pharmaceutical use.

Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain.

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. This monomeric protein (107 amino acids) comprises two homologous domains. It is generally thought that Leu19-Arg20-Tyr21 in the NH2-terminal domain represent the trypsin inhibitory activity, whereas Leu72-Met73-Leu74 in the COOH-domain represent the chymotrypsin and elastase inhibitory activity. Besides Met73, antileukoprotease contains three additional methionine residues all located in the COOH-terminal domain. Treatment of antileukoprotease with different amounts of methionine-selective reagents such as myeloperoxidase in the presence of H2O2 and Cl-, or cis-platinumdiammine dichloride resulted in a dose-dependent inactivation of all inhibitory activities, suggesting that methionine residues are involved in these activities. By using specific synthetic substrates, it was observed that elastase is able to displace trypsin from the inhibitor molecule, indicating that the trypsin and elastase inhibitory sites are located close to each other or at the same site. Incubation of antileukoprotease or its recombinant COOH-terminal domain with an antileukoprotease-specific monoclonal antibody (MoAb15) resulted in a strong selective increase of the trypsin inhibitory activity. The results presented reveal strong evidence that the inhibitory activities of antileukoprotease against trypsin, chymotrypsin and elastase are represented by its COOH-terminal domain, and that methionine residues are involved in interactions with these proteinases.

The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases.

Human mucus proteinase inhibitor (MPI) consists of 107 amino acids arranged in two domains showing high homology to each other. This protein is an inhibitor of different serine proteinases including trypsin, chymotrypsin, leukocyte elastase and cathepsin G. On the basis of sequence comparisons it has been suggested that the first domain inhibits trypsin, whereas the second one was thought to be active against chymotrypsin and elastase. To prove the location of the different inhibitory activities gene fragments for both domains have been cloned separately and expressed in Escherichia coli. Inhibition assays with the isolated recombinant domains showed that the second domain is active against chymotrypsin, neutrophil elastase and trypsin, whereas for the first domain only a weak activity against trypsin could be detected. These results suggest that the inhibitory activities of the native molecule towards these three proteinases are all located in the second domain.

Cloning and expression of human salivary-gland kallikrein in Escherichia coli.

CDNA clones for human kallikrein have been identified in a cDNA library constructed from mRNA of human salivary gland. The entire coding sequence for preprokallikrein and for the 5'- and 3'-untranslated regions were isolated by using a mixture of oligonucleotides corresponding to amino acids 51-56 of human urinary kallikrein and one oligonucleotide corresponding to amino acids 233-238 of human pancreatic kallikrein. The DNA sequence proved that, with the exception of two amino acid exchanges, kallikrein of the human salivary gland is identical with pancreatic kallikrein. Salivary gland and renal kallikrein was expressed in Escherichia coli from plasmid pKK223-3 under the control of the tac promoter. The protein was identified by Western-blot analysis and by demonstration of its specific proteolytic activity.

Secretion of antileucoprotease from a human lung tumor cell line.

Two human tumor cell lines were analyzed for the production of human antileucoprotease (ALP). One of them, a human squamous lung carcinoma cell line (HS-24) synthesized, as confirmed by Western blot analysis, high amounts of ALP in serum-free medium. The supernatant inhibited elastase, chymotrypsin and trypsin. Northern blot analysis with an 18-mer radiolabelled oligonucleotide, derived from an ALP specific cDNA clone, revealed a specific mRNA of about 700-800 nucleotides in HS-24 tumor cells. In contrast, a secondary human lung tumor cell line (SB-3), derived from the adrenal cortex, did not synthesize ALP when assayed under identical conditions. The supernatant inhibited only trypsin and chymotrypsin.

Molecular cloning and expression of cDNA for human antileukoprotease from cervix uterus.

We have isolated cDNA clones for the human antileukoprotease HUSI-I, an elastase inhibitor, from a library, containing cDNA inserts made from human cervix uterus. A library of 10 000 recombinants was screened using a mixture of 16 different oligodeoxyribonucleotides which correspond to amino acids 79-84 and one 20mer oligodeoxyribonucleotide corresponding to amino acids 19-26. Two overlapping cDNA clones, containing the entire coding sequence and part of the 5'- and 3'-untranslated region, were isolated. DNA sequence data showed that our clone corresponds with the available protein sequence data. For expression, the cDNA fragment was inserted in a derivative of plasmid pPLc236 and expressed under the control of lambda PL promoter. Expression of antileukoprotease was proven by Western blot analysis and inhibition of chymotrypsin.

The acid-stable proteinase inhibitor of human mucous secretions (HUSI-I, antileukoprotease). Complete amino acid sequence as revealed by protein and cDNA sequencing and structural homology to whey proteins and Red Sea turtle proteinase inhibitor.

The complete amino acid sequence of human antileukoprotease has been determined by direct sequencing of the inhibitory active protein isolated from seminal plasma (HUSI-I) and by sequence analysis of cDNA reverse-transcribed from mRNA prepared from cervical tissue. The inhibitor (Mr 11726) consists of 107 amino acid residues including 16 cysteines presumably forming disulfide bonds. The molecule comprises two consecutive domains which are homologous to each other, to the second domain of the basic protease inhibitor from Red Sea turtle (chelonianin) and to both domains of the whey proteins of rat and mouse. Both domains contain a pattern of cysteines known as the 'four-disulfide-core' that has also been found in wheat germ agglutinin and neurophysin.

Characterization of a human genomic DNA fragment coding for a myosin heavy chain.

A DNA segment from the human genome with information for myosin heavy chain (MHC) was isolated from a human genomic DNA library cloned in lambda Charon 4A phages. The isolation was accomplished by a myosin cDNA probe obtained from rabbit heart muscle mRNA (Sinha et al. 1982). The selected human DNA clone, designated lambda gMHC1, contains a genomic DNA fragment of about 14 kilobase pairs. The transcriptional polarity of this DNA was determined. The 5'-end of the gene is missing from the cloned fragment. This human gene exhibits sequence homology to MHC DNA of rabbit and chicken, but not to an MHC sequence of nematode. The isolated gene fragment is a member of the human MHC multi-gene family, which is presumed to consist of probably more than ten separate sarcomeric MHC genes per haploid genome.