High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli.

The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments.     

Production of human prolyl 4-hydroxylase in Escherichia coli

Prolyl 4-hydroxylase (P4H) catalyzes the post-translational hydroxylation of proline residues in collagen strands. The enzyme is an alpha2beta2 tetramer in which the alpha subunits contain the catalytic active sites and the beta subunits (protein disulfide isomerase) maintain the alpha subunits in a soluble and active conformation. Heterologous production of the native alpha2beta2 tetramer is challenging and had not been reported previously in a prokaryotic system. Here, we describe the production of active human P4H tetramer in Escherichia coli from a single bicistronic vector. P4H production requires the relatively oxidizing cytosol of Origami B(DE3) cells. Induction of the wild-type alpha(I) cDNA in these cells leads to the production of a truncated alpha subunit (residues 235-534), which assembles with the beta subunit. This truncated P4H is an active enzyme, but has a high Km value for long substrates. Replacing the Met235 codon with one for leucine removes an alternative start codon and enables production of full-length alpha subunit and assembly of the native alpha2beta2 tetramer in E. coli cells to yield 2 mg of purified P4H per liter of culture (0.2 mg/g of cell paste). We also report a direct, automated assay of proline hydroxylation using high-performance liquid chromatography. We anticipate that these advances will facilitate structure-function analyses of P4H.     

Domains b' and a' of protein disulfide isomerase fulfill the minimum requirement for function as a subunit of prolyl 4-hydroxylase. The N-terminal domains a and b enhances this function and can be substituted in part by those of ERp57

Protein disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a', plus an acidic C-terminal extension, c. PDI carries out multiple functions, acting as the beta subunit in the animal prolyl 4-hydroxylases and in the microsomal triglyceride transfer protein and independently acting as a protein folding catalyst. We report here that the minimum sequence requirement for the assembly of an active prolyl 4-hydroxylase alpha(2)beta(2) tetramer in insect cell coexpression experiments is fulfilled by the PDI domain construct b'a' but that the sequential addition of the b and a domains greatly increases the level of enzyme activity obtained. In the assembly of active prolyl 4-hydroxylase tetramers, the a and b domains of PDI, but not b' and a', can in part be substituted by the corresponding domains of ERp57, a PDI isoform that functions naturally in association with the lectins calnexin and calreticulin. The a' domain of PDI could not be substituted by the PDI a domain, suggesting that both b' and a' domains contain regions critical for prolyl 4-hydroxylase assembly. All PDI domain constructs and PDI/ERp57 hybrids that contain the b' domain can bind the 14-amino acid peptide Delta-somatostatin, as measured by cross-linking; however, binding of the misfolded protein "scrambled" RNase required the addition of domains ab or a' of PDI. The human prolyl 4-hydroxylase alpha subunit has at least two isoforms, alpha(I) and alpha(II), which form with the PDI polypeptide the (alpha(I))(2)beta(2) and (alpha(II))(2)beta(2) tetramers. We report here that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides tested were more effectively associated with the alpha(II) subunit than the alpha(I) subunit.     

Three binding sites in protein-disulfide isomerase cooperate in collagen prolyl 4-hydroxylase tetramer assembly

Protein-disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a'. It is a ubiquitous protein folding catalyst that in addition functions as the beta-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) alpha(2)beta(2) tetramers. We report here that point mutations in the primary peptide substrate binding site in the b' domain of PDI did not inhibit C-P4H assembly. Based on sequence conservation, additional putative binding sites were identified in the a and a' domains. Mutations in these sites significantly reduced C-P4H tetramer assembly, with the a domain mutations generally having the greater effect. When the a or a' domain mutations were combined with the b' domain mutation I272W tetramer assembly was further reduced, and more than 95% of the assembly was abolished when mutations in the three domains were combined. The data indicate that binding sites in three PDI domains, a, b', and a', contribute to efficient C-P4H tetramer assembly. The relative contributions of these sites were found to differ between Caenorhabditis elegans C-P4H alphabeta dimer and human alpha(2)beta(2) tetramer formation.     

Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

Background  

We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α₂β₂ tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.     

The peptide-substrate-binding domain of human collagen prolyl 4-hydroxylases. Backbone assignments,secondary structure,and binding of proline-rich peptides

The collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. The vertebrate enzymes are alpha 2 beta 2 tetramers in which protein-disulfide isomerase serves as the beta subunit. Two isoforms of the catalytic alpha subunit have been identified and shown to form [alpha(I)]2 beta 2 and [alpha(II)]2 beta 2 tetramers, the type I and type II C-P4Hs, respectively. The peptide-substrate-binding domain of type I C-P4H has been shown to be located between residues 138 and 244 in the 517-residue alpha(I) subunit and to be distinct from the catalytic domain that is located in the C-terminal region. We report here that a recombinant human C-P4H alpha(I) polypeptide Phe144-Ser244 forms a folded domain consisting of five alpha helices and one short beta strand. This structure is quite different from those of other proline-rich peptide-binding modules, which consist mainly of beta strands. Binding of the peptide (Pro-Pro-Gly)2 to this domain caused major chemical shifts in many backbone amide resonances, the residues showing the largest shifts being mainly hydrophobic, including three tyrosines. The Kd values determined by surface plasmon resonance and isothermal titration calorimetry for the binding of several synthetic peptides to the alpha(I) and the corresponding alpha(II) domain were very similar to the Km and Ki values for these peptides as substrates and inhibitors of the type I and type II C-P4H tetramers. The Kd values of the alpha(I) and alpha(II) domains for (Gly-Pro-4Hyp)5 were much higher than those for (Pro-Pro-Gly)5, indicating a marked decrease in the affinity of hydroxylated peptides for the domain. Many characteristic features of the binding of peptides to the type I and type II C-P4H tetramers can thus be explained by the properties of binding to this domain rather than the catalytic domain.     

Prolyl 4-Hydroxlase Activity Is Essential for Development and Cuticle Formation in the Human Infective Parasitic Nematode Brugia malayi

Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.     

Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.     

Egg shell collagen formation in Caenorhabditis elegans involves a novel prolyl 4-hydroxylase expressed in spermatheca and embryos and possessing many unique properties

The collagen prolyl 4-hydroxylases (EC ) play a critical role in the synthesis of all collagens. The enzymes from all vertebrate species studied are alpha(2)beta(2) tetramers, in which the beta subunit is identical to protein disulfide isomerase (PDI). Two isoforms of the catalytic alpha subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. We report here on the cloning and characterization of a third C. elegans alpha subunit isoform, PHY-3. It is much shorter than the previously characterized vertebrate and C. elegans alpha subunits and shows 23-30% amino acid sequence identity to PHY-1 and PHY-2 within the catalytic C-terminal region. Recombinant PHY-3 coexpressed in insect cells with a C. elegans PDI isoform that does not associate with PHY-1 was found to be an active prolyl 4-hydroxylase. The phy-3 gene consists of five exons, and its expression pattern differs distinctly from the hypodermally expressed phy-1 and phy-2 in that it is expressed in embryos, late larval stages, and adult nematodes, expression in the latter being restricted to the spermatheca. Nematodes homozygous for a phy-3 deletion are phenotypically of the wild type and fertile, but the 4-hydroxyproline content of phy-3(-/-) early embryos was reduced by about 90%. PHY-3 is thus likely to be involved in the synthesis of collagens in early embryos, probably of those in the egg shell.     

Cloning of the alpha subunit of prolyl 4-hydroxylase from Drosophila and expression and characterization of the corresponding enzyme tetramer with some unique properties

Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, whereas the Caenorhabditis elegans enzyme is an alphabeta dimer, the beta subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha subunit and 31% identity to the C. elegans alpha subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1. 9-kilobase mRNA coding for this alpha subunit was present in Drosophila larvae.     

Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits.

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are alpha(2)beta(2) tetramers, in which the beta subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the Caenorhabditis elegans catalytic alpha subunit isoforms PHY-1 and PHY-2 and the beta subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)(2) tetramer is the major form, while PHY-1/PDI-2 and PHY-2/PDI-2 dimers are also assembled but less efficiently. Cloning and characterization of the orthologous subunits from the closely related nematode Caenorhabditis briggsae revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the C. briggsae and C. elegans subunits. In addition to a PHY-1/PHY-2(PDI-2)(2) tetramer and a PHY-1/PDI-2 dimer, an active (PHY-2)(2)(PDI-2)(2) tetramer was formed in C. briggsae instead of a PHY-2/PDI-2 dimer. Site-directed mutagenesis studies and generation of inter-species hybrid polypeptides showed that the N-terminal halves of the Caenorhabditis PHY-2 polypeptides determine their assembly properties. Genetic disruption of C. briggsae phy-1 (Cb-dpy-18) via a Mos1 insertion resulted in a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding C. elegans mutants (Ce-dpy-18). C. briggsae phy-2 RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in phy-1 mutants. Genetic complementation of the C. briggsae and C. elegans phy-1 mutants was achieved by injection of a wild type phy-1 gene from either species.     

Collagen prolyl 4-hydroxylase tetramers and dimers show identical decreases in Km values for peptide substrates with increasing chain length: mutation of one of the two catalytic sites in the tetramer inactivates the enzyme by more than half

The collagen prolyl 4-hydroxylases (collagen P4Hs, EC 1.14.11.2) play a key role in the synthesis of the extracellular matrix. The vertebrate enzymes are alpha(2)beta(2) tetramers, the beta subunit being identical to protein disulfide isomerase (PDI). The main Caenorhabditis elegans collagen P4H form is an unusual PHY-1/PHY-2/(PDI)(2) mixed tetramer consisting of two types of catalytic alpha subunit, but the PHY-1 and PHY-2 polypeptides also form active PHY/PDI dimers. The lengths of peptide substrates have a major effect on their interaction with the P4H tetramers, the K(m) values decreasing markedly with increasing chain length. This phenomenon has been explained in terms of processive binding of the two catalytic subunits to long peptides. We determined here the K(m) values of a collagen P4H having two catalytic sites, the C. elegans mixed tetramer, and a form having only one such site, the PHY-1/PDI dimer, for peptides of varying lengths. All the K(m) values of the PHY-1/PDI dimer were found to be about 1.5-2.5 times those of the tetramer, but increasing peptide length led to identical decreases in the values of both enzyme forms. The K(m) for a nonhydroxylated collagen fragment with 33 -X-Y-Gly-triplets but only 11 -X-Pro-Gly-triplets was found to correspond to the number of the former rather than the latter. To study the individual roles of the two catalytic sites in a tetramer, we produced mutant PHY-1/PHY-2/(PDI)(2) tetramers in which binding of the Fe(2+) ion or 2-oxoglutarate to one of the two catalytic sites was prevented. The activities of the mutant tetramers decreased to markedly less than 50% of that of the wild type, being about 5-10% and 20-30% with the enzymes having one of the two Fe(2+)-binding sites or 2-oxoglutarate-binding sites inactivated, respectively, while the K(m) values for these cosubstrates or peptide substrates were not affected. Our data thus indicate that although collagen P4Hs do not act on peptide substrates by a processive mechanism, prevention of hydroxylation at one of the two catalytic sites in the tetramer impairs the function of the other catalytic site.     

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)     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase.

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.     

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.