The exoskeleton collagens in Caenorhabditis elegans are modified by prolyl 4-hydroxylases with unique combinations of subunits

The collagen prolyl 4-hydroxylases (P4Hs, EC ) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are alpha(2)beta(2) tetramers, in which protein disulfide isomerase (PDI) serves as the beta subunit. Two conserved alpha subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single beta subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2)(2) mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2-/- and phy-1-/- (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln(121)-Ala(271) and Asp(1)-Leu(122) in PHY-1 and PHY-2, respectively, are critical for its assembly.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Lack of collagen type specificity for lysyl hydroxylase isoforms

Lysyl hydroxylase is the enzyme catalyzing the formation of hydroxylysyl residues in collagens. Large differences in the extent of hydroxylysyl residues are found among collagen types. Three lysyl hydroxylase isoenzymes (LH1, LH2, LH3) have recently been characterized from human and mouse tissues. Nothing is known about the distribution of these isoforms within cells or whether they exhibit collagen type specificity. We measured mRNA levels of the three isoforms, as well as the mRNAs of the main collagen types I, III, IV, and V and the alpha subunit of prolyl 4-hydroxylase, another enzyme involved in collagen biosynthesis, in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. Immunoblotting was utilized to confirm the results of Northern hybridization. The levels of mRNA of LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase showed significant correlations with each other. The LH3 mRNA levels did not correlate with those of LH1, LH2, or the alpa subunit of prolyl 4-hydroxylase, clearly indicating a difference in the regulation of LH3. No correlation was observed between LH isoforms and individual collagen types, indicating a lack of collagen type specificity for lysyl hydroxylase isoforms. Our observations suggest that LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase are coregulated together with total collagen synthesis but not with the specific collagen types and indicate that LH3 behaves differently from LH1 and LH2, implying a difference in their substrates. These observations set the basis for further studies to define the functions of lysyl hydroxylase isoforms.     

Hypoxia-inducible Factor-1 (HIF-1) but Not HIF-2 Is Essential for Hypoxic Induction of Collagen Prolyl 4-Hydroxylases in Primary Newborn Mouse Epiphyseal Growth Plate Chondrocytes

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α₂β₂ tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I–III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.     

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.     

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.