Cell density regulates prolyl 4-hydroxylase activity independent of mRNA levels

In embryonic avian tendon, cell density regulates collagen production. This control is propagated through the alpha-subunit of prolyl 4-hydroxylase where protein levels were previously shown to rise fivefold with increasing cell density. In contrast, mRNA levels are now shown not to change by both Northern and RNAse protection assays. This lack of increase contrasts with previous reports as does the mRNA length: this is 50% larger as confirmed by sequencing the 3' end. Alternative sites for cell density regulation of the enzyme could rely on its sensitivity to sulfhydryl groups. Using a fluorescent sulfhydryl probe as well as a sulfhydryl inhibitor, one observes a strong cell density response, supporting the hypothesis that cellular redox potential could alter protein stability.     

A role for prolyl 3-hydroxylase 2 in post-translational modification of fibril-forming collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.     

Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.     

Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. We have found that anthraquinones with at least two hydroxy groups ortho to each other are potent inhibitors of this enzyme. Kinetic studies revealed that 2,7,8-trihydroxyanthraquinone (THA) competitively inhibited the co-substrate, 2-oxoglutarate, but was non-competitive with regard to ascorbate and was tentatively considered to be uncompetitive with regard to protocollagen. The inhibition by THA was greatly enhanced in the absence of added Fe2+ and was partially reversed by the addition of concentrations of Fe2+ in excess of the optimum for the enzymic reaction. Binding studies indicated that THA is an effective chelating agent for Fe2+. Several non-quinoidal compounds bearing the catechol moiety also inhibited the enzyme. The results suggest that THA inhibited prolyl 4-hydroxylase by binding to the enzyme at the site for 2-oxoglutarate possibly involving the Fe2+ atom, rather than by complexing with Fe2+ in free solution. The inhibition of prolyl 4-hydroxylase by THA exhibited strong positive co-operativity and may involve three distinct but non-independent binding sites.     

Direct and continuous assay for prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.     

Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase

Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4-hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme.     

Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines.

The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.     

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.     

Conformational preferences of substrates for human prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) catalyzes the posttranslational hydroxylation of (2 S)-proline (Pro) residues in procollagen strands. The resulting (2 S,4 R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. Even though its product (Hyp) differs from its substrate (Pro) by only a single oxygen atom, no product inhibition has been observed for P4H. Here, we examine the basis for the binding and turnover of substrates by human P4H. Synthetic peptides containing (2 S,4 R)-4-fluoroproline (Flp), (2 S,4 S)-4-fluoroproline (flp), (2 S)-4-ketoproline (Kep), (2 S)-4-thiaproline (Thp), and 3,5-methanoproline (Mtp) were evaluated as substrates for P4H. Peptides containing Pro, flp, and Thp were found to be excellent substrates for P4H, forming Hyp, Kep, and (2 S,4 R)-thiaoxoproline, respectively. Thus, P4H is tolerant to some substitutions on C-4 of the pyrrolidine ring. In contrast, peptides containing Flp, Kep, or Mtp did not even bind to the active site of P4H. Each proline analogue that does bind to P4H is also a substrate, indicating that discrimination occurs at the level of binding rather than turnover. As the iron(IV)-oxo species that forms in the active site of P4H is highly reactive, P4H has an imperative for forming a snug complex with its substrate and appears to do so. Most notably, those proline analogues with a greater preference for a C (gamma)- endo pucker and cis peptide bond were the ones recognized by P4H. As Hyp has a strong preference for C (gamma)- exo pucker and trans peptide bond, P4H appears to discriminate against the conformation of proline residues in a manner that diminishes product inhibition during collagen biosynthesis.     

Development of insulin sensitivity in rat skeletal muscle. Studies of glucose transporter and insulin receptor mRNA levels.

Expression of GLUT-4 and insulin receptor mRNAs was investigated in rat skeletal muscle by Northern hybridization. GLUT-4 mRNA was barely detectable in foetal muscle, was expressed at low levels by 1-8 days and at 2-3-fold higher levels during and after weaning (18-40 days). In contrast there was little change in insulin receptor mRNA levels prior to weaning and a reduction in mRNA abundance between 18 and 40 days. Weaning rats on to a diet rich in fat prevented the increase in GLUT-4 abundance seen between 15 and 29 days in animals weaned on a high-carbohydrate diet.     

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.     

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.     

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     

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.     

Separation of prolyl 3-hydroxylase and 4-hydroxylase activities and the 4-hydroxyproline requirement for synthesis of 3-hydroxyproline

Differences between prolyl 3-hydroxylase and prolyl 4-hydroxylase activities were found in their stimulation and inactivation by dithiothreitol and in their affinity to poly-L-proline linked to agarose. The two enzyme activities were separated by gel filtration, the results demonstrating that they are due to separate proteins. Comparison of [¹⁴C]proline-labelled protocollagen and the same protein when fully 4-hydroxylated as substrates indicated dependence of 3-hydroxyproline formation on the presence of 4-hydroxyproline. It is suggested that the main substrate sequence for 3-hydroxyproline synthesis is -Gly-Pro-4Hyp-Gly-.     

Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens

Recessive mutations that prevent 3-hydroxyproline formation in type I collagen have been shown to cause forms of osteogenesis imperfecta. In mammals, all A-clade collagen chains with a GPP sequence at the A1 site (P986), except α1(III), have 3Hyp at residue P986. Available avian, amphibian and reptilian type III collagen sequences from the genomic database (Ensembl) all differ in sequence motif from mammals at the A1 site. This suggests a potential evolutionary distinction in prolyl 3-hydroxylation between mammals and earlier vertebrates. Using peptide mass spectrometry, we confirmed that this 3Hyp site is fully occupied in α1(III) from an amphibian, Xenopus laevis, as it is in chicken. A thorough characterization of all predicted 3Hyp sites in collagen types I, II, III and V from chicken and xenopus revealed further differences in the pattern of occupancy of the A3 site (P707). In mammals only α2(I) and α2(V) chains had any 3Hyp at the A3 site, whereas in chicken all α-chains except α1(III) had A3 at least partially 3-hydroxylated. The A3 site was also partially 3-hydroxylated in xenopus α1(I). Minor differences in covalent cross-linking between chicken, xenopus and mammal type I and III collagens were also found as a potential index of evolving functional differences. The function of 3Hyp is still unknown but observed differences in site occupancy during vertebrate evolution are likely to give important clues.     

Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.