Beta-subunit Of Prolyl 4-hydroxylase Research Articles

Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.

Promoter of the gene for the multifunctional protein disulfide isomerase polypeptide. Functional significance of the six CCAAT boxes and other promoter elements.

Protein disulfide isomerase (PDI) is a highly unusual multifunctional polypeptide that is identical to the beta-subunit of prolyl 4-hydroxylase, a cellular thyroid hormone-binding protein and a subunit of the microsomal triglyceride transfer protein complex, and very similar to a polypeptide functioning in vitro as a glycosylation site binding protein of oligosaccharyl transferase. The human PDI gene possesses several putative transcriptional control elements, including the highly unusual presence of six CCAAT boxes between -108 and -378 of the 5'-flanking region. We report here on a promoter analysis of this gene. Eleven PDI promoter elements recognized by DNA-binding proteins present in HeLa cell and HT-1080 cell nuclear extracts were identified by DNase I footprinting analysis within the first 630 nucleotides of the 5'-flanking region. Interestingly, these included all six CCAAT elements. Functional 5' deletion analysis suggested that only two or three of the CCAAT elements may contribute significantly to the promoter activity in HeLa cells. Mutations introduced into each of the CCAAT boxes separately indicated, however, that all six appear to contribute to the promoter strength, the largest decreases (approximately 50%) being seen with mutations in the second or fifth CCAAT box. These data thus suggested that efficient expression of the multifunctional PDI polypeptide is secured by multiple CCAAT elements, some of which appear to be functionally redundant. The 5' deletion analysis further suggested that a region between -623 and -518 may contain additional positively and negatively acting elements.

Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli. This multifunctional polypeptide has two independently acting catalytic sites for the isomerase activity.

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a highly unusual multifunctional polypeptide, being identical to the beta subunit of prolyl 4-hydroxylase, a cellular thyroid hormone binding protein and a component of the microsomal triglyceride transfer protein complex, and highly similar to a polypeptide acting in vitro as a glycosylation site binding protein. It has two -Cys-Gly-His-Cys- sequences which, it has been proposed, act as catalytic sites for the isomerase activity, but few data have been available to indicate whether one or both of them do indeed act as catalytic sites and whether the two presumed catalytic sites act independently or cooperatively. We report here on the expression of human PDI in Escherichia coli with three different signal sequences. All three polypeptide variants were secreted into the periplasmic space as fully active enzymes. Oligonucleotide-directed mutagenesis was used to convert either one or both of the -Cys-Gly-His-Cys- sequences to -Ser-Gly-His-Cys-. The PDI activity of both polypeptides containing a single modified sequence was about 50% of that of the wild-type polypeptide, whereas the polypeptide with two modified sequences had no isomerase activity. It is thus concluded that both -Cys-Gly-His-Cys- sequences act as catalytic sites for the isomerase activity, and the two catalytic sites appear to operate independently of one another.

Expression and methylation of the beta-subunit gene of prolyl 4-hydroxylase: in erythrocytes, tendon and cornea of chick embryos.

It has recently been demonstrated that the beta-subunit of prolyl 4-hydroxylase (E.C. 1.14.11.2) is the same gene product as protein disulfide isomerase (PDI) and cellular thyroid hormone binding protein (THP). Therefore, it is very likely that the beta-subunit of the prolyl 4-hydroxylase gene serves as a house keeping gene in most cell types. In the present study, we examined the distribution of the chicken beta-subunit of prolyl 4-hydroxylase/protein disulfide isomerase (CPH beta/PDI) in erythrocytes, corneas and tendons of 13-, 17-, and 19-day-old chick embryos by immunohistochemistry using antibodies against CPH beta/PDI. Our data indicate that erythrocytes do not express the CPH beta/PDI gene whereas tendon cells express CPH beta/PDI at all developmental stages examined. The basal cells of corneal epithelium express CPH beta/PDI, but the superficial cell layers of stratified corneas of 19-day-old chick embryos do not. The expression of the CPH beta/PDI gene is also confirmed by in situ hybridization with cDNA encoding CPH beta/PDI. The results indicate that the expression of CPH beta/PDI in cornea is probably developmentally regulated. It has been suggested that methylation of genomic DNA is one of many possible regulatory mechanisms for gene expression. In order to examine whether methylation of genomic DNA may play any role in the expression of the beta-subunit gene, genomic DNA was isolated from corneas, tendons, and erythrocytes of individual 13-, 17-, and 19-day-old chick embryos. DNA samples were digested with Sma I and Eco RI, or Pst I and Sma I and followed by either Msp I, Hpa II, or Hha I and were then subjected to Southern hybridization with 32P-labeled genomic DNA fragments of CPH beta/PDI. Our results indicate that the CPH beta/PDI gene is methylated at the Hha I site in the 4th exon in erythrocytes whereas the same sites in tendon and cornea are hypomethylated. Examination of 5'-end flanking sequences of exon 1 of the CPH beta/PDI gene with the methylation sensitive endonucleases, Hha I and Hpa II did not reveal any difference in erythrocyte, cornea and tendon cells. Thus, our results indicated that DNA methylation may not play an important role in the expression of CPH beta/PDI.

A novel 53-kDa polypeptide from chicken embryo.

We have isolated from chicken embryos a novel 53-kDa protein possessing properties which are similar, but not identical to the 55-kDa PDI polypeptide from chicken embryos. The novel 53-kDa polypeptide copurifies with PDI, but is separated by ion-exchange chromatography. The novel 53-kDa polypeptide cross-reacts strongly with antibodies specific for bovine PDI and cross-reacts to varying degrees with six different preparations of antibodies specific for chicken PDI which is identical to the beta-subunit of chicken prolyl 4-hydroxylase. Anti-bovine PDI immunoglobulins selected by the purified 53-kDa polypeptide react with bovine PDI but not with the beta-subunit of prolyl 4-hydroxylase, suggesting that the 53-kDa polypeptide shares epitopes with bovine PDI but not with the chicken prolyl 4-hydroxylase beta-subunit. Amino acid compositional analysis of the purified polypeptide yielded unique data when compared to PDI and other PDI-like polypeptides. Edman degradation from the N terminus of the 53-kDa polypeptide yields a sequence very different from the N terminus of PDI. This sequence is unique when compared to all entries in available databases. A 20-residue sequence of an internal cyanogen bromide fragment of the 53-kDa polypeptide gives a nearly identical match with human beta-endorphin. The 53-kDa polypeptide is capable of cleaving the disulphides of insulin under conditions where PDI is active. The periodic acid-Schiff assay failed to detect bound carbohydrate. These observations support evidence for a family of PDI-like proteins in chicken embryo and suggest that PDI activity is not confined to only one protein.

Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.

Regional assignment of the human gene coding for a multifunctional polypeptide (P4HB) acting as the beta-subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase to 17q25.

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens and related proteins by hydroxylating proline residues in peptide linkages. The beta-subunit of prolyl 4-hydroxylase (P4HB) is a highly unusual multifunctional polypeptide that is identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and is highly similar to a glycosylation site-binding polypeptide of oligosaccharyl transferase. We report here the regional assignment of the gene for this multifunctional polypeptide. In situ hybridization mapped the gene to 17q25. Southern blot analyses of restricted DNA from a chromosome-mediated gene transfer transfectant panel suggested that the P4HB gene is located distal to the gene for thymidine kinase, either between the genes for thymidine kinase and galactokinase or on the telomeric side of both these genes.

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.

Increases in mRNA concentrations of the alpha and beta subunits of prolyl 4-hydroxylase accompany increased gene expression of type IV collagen during differentiation of mouse F9 cells.

Mouse F9 teratocarcinoma stem cells differentiate in monolayer cultures in the presence of retinoic acid, dibutyryl cAMP, and isobutyl methylxanthine. This differentiation is associated with a marked increase in the synthesis rates and mRNA concentrations of basement membrane proteins such as type IV collagen. We report here that the differentiation also involves an increase of up to 50-fold in the concentrations of the mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase, the enzyme required for the cotranslational and post-translational hydroxylation of proline residues in collagens. The time courses and magnitudes of increases in these two mRNA concentrations were similar to those observed in the same experiments for the mRNA of the alpha chain of type IV collagen. In the differentiated F9 cells the concentration of the alpha subunit mRNA was about 30% of the beta subunit mRNA concentration. Northern blot analyses indicated that the sizes of the alpha and beta subunit mRNAs in the differentiated mouse F9 cells are similar to those in human skin fibroblasts. The F9 cell differentiation system appears to provide a useful model for studies on the regulation of prolyl 4-hydroxylase synthesis.

The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens. The beta subunit is known to be identical with the enzyme protein disulphide-isomerase and to possess disulphide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer. We here report that lysyl hydroxylase, a homodimer, and algal prolyl 4-hydroxylase, a monomer, do not contain detectable protein disulphide-isomerase activity. Since the hydroxylase reaction mechanisms are similar, the data suggest that the protein disulphide-isomerase activity of the vertebrate prolyl 4-hydroxylase beta subunit is unlikely to be involved in the catalytic mechanism of the hydroxylation reaction.

Human Deoxycytidine Kinase: Sequence of cDNA Clones and Analysis of Expression in Cell Lines with and without Enzyme Activity

Abstract Deoxycytidine kinase (dC kinase) is the rate-limiting enzyme in the anabolism of important anticancer and retroviral nucleoside derivatives. Its activity is often decreased in resistance to these drugs. To analyze the structure, function, and control of this clinically important enzyme we isolated 15 cDNA clones for human deoxycytidine kinase from lambda gt11 thymus and Molt 4 libraries. Four clones were sequenced. The largest clone is 2.9 kilobases and codes for a 626-amino acid open reading frame. The DNA and deduced amino acid sequence of the human dC kinase clones are homologous with a previously unidentified murine cDNA clone p3.4J (EMBL:MM34j) reported to be related to granulocyte-macrophage colony-stimulating factor. Deoxycytidine kinase also has cysteine-rich regions that are homologous with thioredoxin, the beta subunit of prolyl 4-hydroxylase, phosphoinositide-specific phospholipase C, thyroid hormone-binding protein, and protein disulfide isomerase. No differences were seen in the amount and size of deoxycytidine kinase protein and mRNA between CCRF/CEM and L1210 leukemic cell lines that express and do not express enzyme activity. Genomic restriction fragments were similar between the active and inactive CCRF/CEM cell lines. These data suggest that the cells deficient in dC kinase activity have a small defect in the structural gene.

Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts.

Prolyl 4-hydroxylase [procollagen-proline, 2-oxyglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2], an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the alpha-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the beta-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Glu-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha-subunit does not have this C-terminal sequence, and thus one function of the beta-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Interestingly, three of the cDNA clones for the alpha-subunit contained a 64-nucleotide sequence homologous but not identical to the corresponding 64-nucleotide sequence found in four other cDNA clones. Nuclease S1 mapping experiments demonstrated that this difference was due to the existence of two types of mRNA present in approximately equal amounts. Southern blot analyses of human genomic DNA with a cDNA probe for the alpha-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

Characterization of the human gene for a polypeptide that acts both as the beta subunit of prolyl 4-hydroxylase and as protein disulfide isomerase.

A single polypeptide acts as the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase and may also function as a cellular thyroid hormone binding protein. We report here that the human gene for this polypeptide is about 18 kilobase pairs and consists of 11 exons. The two thioredoxin-like regions are coded by exons 1-2 and 8-9, respectively. The codons for the two presumed active sites of protein disulfide isomerase, each a Cys-Gly-His-Cys sequence, are located 12 base pairs from the beginning of exons 2 and 9. The last 3 amino acids coded by exons 1 and 8 and the first 9 amino acids coded by exons 2 and 9, including a broken codon for Tyr, are identical in the respective exon-intron junctions. These regions are also highly homologous to the active sites of bacterial thioredoxins. The data suggest that evolution of this gene has involved exon shuffling and duplication of a two-exon unit, in which the internal exon-intron junctions have been entirely conserved. The region between exons 1-2 and 8-9 appears to contain other duplications. The 5' flanking sequences contain a TATA box, six CCAAT boxes, and other elements which may be involved in regulation of the cellular amounts of this polypeptide.

Protein-disulphide isomerase and prolyl isomerase act differently and independently as catalysts of protein folding.

Two enzymes are now known that catalyse slow steps in protein folding. Peptidyl-prolyl cis-trans isomerase catalyses the cis-trans isomerization of Xaa-Pro peptide bonds in oligopeptides and during the refolding of several proteins. The other enzyme, protein-disulphide isomerase, accelerates the reactivation of reduced proteins, presumably by catalysis of thiol-disulphide exchange reactions. Recent evidence indicates that the beta-subunit of prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, is identical with disulphide isomerase. On the basis of this important finding, it was suggested that disulphide isomerase accelerates protein folding, not by 'reshuffling' incorrect disulphide bonds, but in the same way as prolyl isomerase by catalysing proline isomerization which is known to be important for the folding of collagen and other proteins. Here we show that the catalytic activities of these two enzymes are different. Disulphide isomerase accelerates the reformation of native disulphide bonds during protein reoxidation. We find no evidence that this enzyme can catalyse the isomerization of proline peptide bonds, a reaction efficiently accelerated by prolyl isomerase. When both enzymes are present simultaneously during protein folding, they act independently of one another.

A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase.

A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.

Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.