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. (+info)
Effects of pirfenidone on procollagen gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis.
A time course study was carried out to elucidate the mechanisms for antifibrotic effect of pirfenidone (PD). Hamsters were intratracheally (i.t.) instilled with saline (SA) or bleomycin (BL) (7.5 units/kg/5 ml). The animals were fed a diet containing 0.5% PD or the same control diet (CD) without the drug 2 days before and throughout the study. The animals were sacrificed at various times after instillation. The lung hydroxyproline level in BL + CD groups was gradually increased and peaked at 21 days to 181% of the SA + CD control. The BL + PD-treated groups showed a gradual decrease in their lung collagen content, showing a maximum reduction of 40% at day 21. The lung malondialdehyde levels of the BL + CD groups were increased by several-fold of the corresponding SA + CD groups at various times. The lung prolyl hydroxylase (PH) activities in the BL + CD groups were also increased by several-fold of the corresponding SA + CD groups at these time points. The hamsters in the BL + PD showed a gradual decrease in the lung malondialdehyde levels from 10 to 21days compared with their corresponding BL + CD groups. Treatment with PD also reduced the lung PH activities in the BL + PD groups compared with the corresponding BL + CD groups. However, PD failed to manifest any direct inhibitory effect on PH activity in vitro. BL treatment increased the lung procollagen I and III gene expressions in the BL + CD groups by several-fold at varying times compared with the corresponding SA + CD, and treatment with PD in the BL + PD groups significantly down-regulated the BL-induced overexpression of these genes. Studies evaluating the regulation of these genes at the transcriptional level revealed PD significantly reduced the transcription of PC I at 14 days. Our results indicate that the antifibrotic effect of PD was partly due to suppression of the BL-induced inflammatory events and partly due to down-regulation of BL-induced overexpression of lung procollagen I and III genes. (+info)
Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.
The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly. (+info)
mRNA levels for alpha-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle.
There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for alpha- and beta-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH alpha-subunit decreased by 49 and 55% (P < 0.01) in gastrocnemius muscle and by 41 and 39% (P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased (P < 0.05-0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26-56% (P < 0.05-0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy. (+info)
Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates.
4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins. We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase. (+info)
Increase of collagen synthesis and deposition in the arachnoid and the dura following subarachnoid hemorrhage in the rat.
Arachnoidal fibrosis following subarachnoid hemorrhage (SAH) has been suggested to play a pathogenic role in the development of late post-hemorrhagic hydrocephalus in humans. The purpose of this study was to investigate the rate of collagen synthesis in the arachnoid and the dura in the rat under normal conditions and to study the time schedule and the localization of the increased collagen synthesis following an experimental SAH. We found that the activity of prolyl 4-hydroxylase, a key enzyme in collagen synthesis, was 3-fold higher in the dura than that in the arachnoid and was similar to the activity in the skin. We then induced SAH in rats by injecting autologous arterial blood into cisterna magna. After SAH, we observed an increase in prolyl 4-hydroxylase activity of the arachnoid and the dura at 1 week. At this time point the enzyme activity in both tissues was 1.7-1.8-fold compared to that in the controls and after this time point the activities declined but remained slightly elevated at least till week 4. The rate of collagen synthesis was measured in vitro by labeling the tissues with [(3)H]proline. The rate increased to be 1.7-fold at 1 to 2 weeks after the SAH in both of the tissues. Immunohistochemically we observed a deposition of type I collagen in the meninges at 3 weeks after the SAH. SAH is followed by a transient increase in the rate of collagen synthesis in the arachnoid and, surprisingly, also the dura. Increased synthesis also resulted in an accumulation of type I collagen in the meningeal tissue, suggesting that the meninges are a potential site for fibrosis. The time schedule of these biochemical and histological events suggest that meningeal fibrosis may be involved in the pathogenesis of late post-hemorrhagic hydrocephalus. (+info)
Hsp47-dependent and -independent intracellular trafficking of type I collagen in corneal endothelial cells.
PURPOSE: Type I collagen is post-translationally regulated in corneal endothelial cells (CEC): CEC synthesize procollagen I and degrade it intracellularly. We investigated whether there is a Hsp47-independent pathway during intracellular trafficking of procollagen I. METHODS: Specific inhibitors were used to block intracellular transport of procollagen I and Hsp47. Immunocytochemical analysis was performed to determine the intracellular localization of the proteins of interest. RESULTS: When cells were treated with alpha,alpha'-dipyridyl, this specific inhibitor for collagen promoted retention in the endoplasmic reticulum (ER) of some of the underhydroxylated procollagen I, which was colocalized with Hsp47 in CEC. At the same time, another fraction of the alpha,alpha'-dipyridyl-induced underhydroxylated procollagen I was not located in the ER. When CEC were treated with brefeldin A, procollagen I and Hsp47 demonstrated a high degree of colocalization at the ER, whereas the inhibitor had less of an effect on the compartmentalization of procollagen I and prolyl 4-hydroxylase. When CEC were treated with either monensin or bafilomycin A1, procollagen I and Hsp47 were not colocalized: procollagen I was mostly localized at the Golgi area, while Hsp47 predominantly showed ER distribution. When colocalization of procollagen I and prolyl 4-hydroxylase was examined, a major population of procollagen I was not colocalized with prolyl 4-hydroxylase in the ER. CONCLUSIONS: These results indicate that some procollagen I and Hsp47 travel together from the ER to the cis-Golgi compartment and that a major population of procollagen I that may not be properly hydroxylated may be destroyed intracellularly via the Hsp47-independent pathway in CEC. (+info)
Microbial proline 4-hydroxylase screening and gene cloning.
Microbial proline 4-hydroxylases, which hydroxylate free L-proline to trans-4-hydroxy-L-proline, were screened in order to establish an industrial system for biotransformation of L-proline to trans-4-hydroxy-L-proline. Enzyme activities were detected in eight strains, including strains of Dactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library of Dactylosporangium sp. strain RH1 constructed in Escherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29, 715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the beta-galactosidase alpha-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13. 6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene. (+info)