Correlation of the steady-state RNA levels among the alpha-subunit of prolyl 4-hydroxylase and the alpha 1 and alpha 2 chains of type I collagen during growth of chicken embryo tendon fibroblasts.

The relative steady-state levels of RNAs encoding type I collagen and prolyl 4-hydroxylase were examined in exponentially growing primary cultures of chicken embryo tendon fibroblasts. The RNA levels of the alpha 1 and alpha 2 chains of type I collagen were maximal when the fibroblasts reached the confluent state. The RNA levels of the alpha-subunit of prolyl 4-hydroxylase were also maximal at confluency and rose and fell with the RNA levels of the two collagen chains. The RNA levels of the beta-subunit of prolyl 4-hydroxylase did not correlate with the changes observed for the alpha-subunit or for either chain of type I collagen. The RNA levels of the beta-subunit were slightly higher than the RNA levels of the alpha-subunit. These results support our hypothesis that the synthesis of the alpha-subunit and thus the association of newly synthesized alpha-subunits with pre-existing beta-subunits is the rate-limiting factor in determining prolyl 4-hydroxylase activity in cultured cells.     

Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells

We established stably transfected insect cell lines containing cDNAs encoding the alpha and beta subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain.     

Assignment of the gene coding for the alpha-subunit of prolyl 4-hydroxylase to human chromosome region 10q21.3-23.1.

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages and plays a crucial role in the synthesis of these proteins. The gene for the beta-subunit of prolyl 4-hydroxylase has recently been mapped to the long arm of human chromosome 17, at band 17q25. We report here chromosomal localization of the gene for the catalytically and regulatorily important alpha-subunit of human prolyl 4-hydroxylase. Analysis of 24 rodent x human cell hybrids by Southern blotting with cDNA probes for the human alpha-subunit indicated complete cosegregation of the gene for the alpha-subunit with human chromosome 10. A cell hybrid containing only part of chromosome 10 mapped the gene to 10q11----qter. In situ hybridization mapped the gene to 10q21.3-23.1. The gene for the alpha-subunit is thus not physically linked to that for the beta-subunit of the enzyme.     

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.     

A simple procedure for the isolation of protein disulphide-isomerase.

A two-step procedure is described for the purification of protein disulphide-isomerase (PDI). This procedure is based on the previous finding that the beta-subunit of the prolyl 4-hydroxylase tetramer (alpha 2 beta 2) is identical with PDI [Koivu, Myllylä, Helaakoski, Pihlajaniemi, Tasanen & Kivirikko (1987) J. Biol. Chem. 262, 6447-6449; Pihlajaniemi, Helaakoski, Tasanen, Myllylä, Huhtala, Koivu & Kivirikko (1987) EMBO J. 6, 643-649]. The procedure involves purification of the prolyl 4-hydroxylase tetramer by a simple affinity chromatography and subsequent isolation of the beta-subunit from the dissociated tetramer by ion-exchange chromatography.     

Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells.

Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolyl 3-hydroxylase 1, enzyme characterization and identification of a novel family of enzymes

The collagen prolyl hydroxylases are enzymes that are required for proper collagen biosynthesis, folding, and assembly. They reside within the endoplasmic reticulum and belong to the group of 2-oxoglutarate and iron-dependent dioxygenases. Although prolyl 4-hydroxylase has been characterized as an alpha2beta2 tetramer in which protein disulfide isomerase is the beta subunit with two different alpha subunit isoforms, little is known about the enzyme prolyl 3-hydroxylase (P3H). It was initially characterized and shown to have an enzymatic activity distinct from that of prolyl 4-hydroxylase, but no amino acid sequences or genes were ever reported for the mammalian enzyme. Here we report the characterization of a novel prolyl 3-hydroxylase enzyme isolated from embryonic chicks. The primary structure of the enzyme, which we now call P3H1, demonstrates that P3H1 is a member of a family of prolyl 3-hydroxylases, which share the conserved residues present in the active site of prolyl 4-hydroxylase and lysyl hydroxylase. P3H1 is the chick homologue of mammalian leprecan or growth suppressor 1. Two other P3H family members are the genes previously called MLAT4 and GRCB. In this study we demonstrate prolyl 3-hydroxylase activity of the purified enzyme P3H1 on a full-length procollagen substrate. We also show it to specifically interact with denatured collagen and to exist in a tight complex with other endoplasmic reticulum-resident proteins. Immunohistochemistry with a monoclonal antibody specific for chick P3H1 localizes P3H1 specifically to tissues that express fibrillar collagens, suggesting that other P3H family members may be responsible for modifying basement membrane collagens.     

Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caenorhabditis elegans

The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The beta subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase alpha-subunit (PHY; EC 1.14.11.2)- and beta-subunit (PDI; EC 5.3.4.1)-encoding genes with respect to their role in collagen modification and formation of the C. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combined phy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterized dpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.     

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 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.     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.     

Monoclonal antibodies to human prolyl 4-hydroxylase

Monoclonal antibodies against human prolyl 4-hydroxylase (EC 1.14.11.2), an intracellular enzyme of collagen biosynthesis, were produced by fusing spleen cells from BALB/c mice hyperimmunized with human prolyl 4-hydroxylase and mouse myeloma cells (P3/NS 1/1-AG 4-1). Hybridomas from 14 different primary microtiter-plate well cultures produced antibodies to human prolyl 4-hydroxylase; six of them with the highest antibody titer were cloned and antibodies produced by one clone from each of the six lines were further characterized. All of the six cloned hybrids produced antibodies of the IgG class as detected by immunodiffusion. The enzyme antigen used in the present study was a tetramer composed of two pairs of different subunit proteins, alpha and beta. Only one clone which produced antibodies to the alpha subunit was obtained, the other five antibodies being directed against the beta subunit. All the antibodies reacted with the tetramer form of the enzyme. Species cross-reactivity of the antibodies was tested using cultured human, mouse and chick fibroblasts and purified prolyl 4-hydroxylase from chick and mouse sources. None of the antibodies cross-reacted with chick or mouse fibroblasts, as determined by immunofluorescence, whereas one antibody reacted with purified chick and mouse prolyl 4-hydroxylase when examined by the western blotting technique. This antibody caused a strong inhibition of human prolyl 4-hydroxylase activity, but the other five antibodies had negligible inhibitory effect on the activity of the enzyme.     

Structural characterization of the chicken SF-1/Ad4BP gene

SF-1/Ad4BP was identified as a master regulator controlling steroidogenic P-450 genes and belongs to the steroid hormone receptor superfamily. It is expressed in the adrenal cortex, gonads, and pituitary gonadotroph. Targeted disruption of the mouse SF-1/Ad4BP gene showed that it plays a critical role in the development of the steroidogenic tissues and pituitary gonadotroph. We have recently cloned the chicken SF-1/Ad4BP cDNA and have now cloned the chicken SF-1/Ad4BP gene and analyzed its promoter activity. This gene consists of seven exons as well as mammalian counterparts and spans about 15 kb. In mice, the gene encodes another protein, ELP, but we could not find the open reading frame of ELP in the chicken SF-1/Ad4BP gene. The promoter of this gene included five putative cis elements (E, CCAAT, GC and TATA boxes and a GA-rich element), although no TATA box has been found in mammalian counterparts. The E and CCAAT boxes moderately affected promoter activity and the GA-rich element and TATA box were essential for the expression of the chicken SF-1/Ad4BP gene.     

Hydralazine differentially increases mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase whereas it decreases pro alpha 1(I) collagen mRNAs in human skin fibroblasts.

We have used specific oligonucleotide probes to measure the effect of hydralazine on mRNA levels of the alpha and beta subunits of prolyl 4-hydroxylase (PH), a key post-translational modifying enzyme in collagen biosynthesis. Hydralazine exerts a paradoxical effect on collagen biosynthesis in cultured fibroblasts. Cells exposed to hydralazine synthesize substantially reduced amounts of collagen, which is severely deficient in hydroxyproline. Surprisingly, however, the level of prolyl hydroxylase activity assayed in extracts of treated cells is markedly increased, suggesting overproduction of the enzyme. Hybridization analysis indicated that in untreated cells the concentration of the alpha PH subunit mRNA was about 20-25% of the beta PH subunit mRNA concentration. Hydralazine treatment increased the mRNAs for both alpha and beta subunits of PH by three- to fourfold. A differential induction of these mRNAs was observed, however. The alpha subunit mRNA was maximally increased within 24 h, whereas the beta subunit mRNA was increased more slowly, reaching a maximum at 72 h. In contrast, the 5.8 and 4.8-kb mRNAs for pro alpha 1(I) collagen were virtually eliminated by 72 h. This study demonstrates that the increased prolyl hydroxylase activity is a direct result of hydralazine-mediated increases in steady state mRNA content for the alpha and beta subunits of this enzyme. Moreover, the earlier induction of alpha PH mRNA may provide the first evidence at the mRNA level that regulation of PH activity occurs mainly through regulation of the alpha subunit of PH. In addition, the decrease in collagen synthesis by hydralazine appears to result directly from suppression of both species of mRNA for pro alpha 1(I) collagen.     

Characterization of the 5' flanking region of the rat Na+/K(+)-ATPase beta 1 subunit gene.

We have isolated the 5' end of the rat Na+/K(+)-ATPase beta 1 subunit gene. A genomic fragment containing 817 bp of the 5' flanking sequence, exon 1 and 479 bp of intron 1 was sequenced. The 5' flanking region contains a potential TATA box and several putative CAAT and GC boxes. Potential binding sites for thyroid and glucocorticoid receptors were identified together with multiple sequence motifs which exhibit homology to calcium and serum responsive elements.     

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.