Beta-subunit Of Prolyl 4-hydroxylase Research Articles

Collagen-specific chaperone, heat shock protein 47 kDa (HSP47)–pathway and expression patterns in cancer

Background: Human serpin gene SERPINH1 encodes for a non-inhibitory serpin shock protein 47kDa (HSP47) a client-specific chaperone, which is a hallmark in the collagen biosynthesis. Till today, there is no comprehensive study on the protein network for human HSP47. Thus the current study aimed at studying the pathway and expression patterns observed in collagen-specific chaperone, HSP47 in relation to cancer. Methodology: The study used online database STRING 10 (website: http://version10a.string-db.org (7)) in finding putative protein interaction partners of human HSP47. Also, database HMMER3 (www.hmmer.org/) with Pfam 32.0 (Sept 2018) dataset was used in identifying Pfam protein domains from proteins interacting with HSP47. The db DEPC 3.0, was used for evaluation of expression patterns of HSP47 in different cancer. Further three online resources namely, human protein atlas (HPA, https://www.proteinatlas.org/), genotype-tissue expression (GTEx https://gtexportal.org) and FANTOM5 project (http://fantom.gsc.riken.jp/5/) was used to examine HSP47 expression in normal human tissues. Results: Upon constructing protein interactive map of human HSP47, the study found that a set of molecular chaperones as interaction partners of HSP47, which included two copies each of cAMP response element binding (CREB) binding proteins, HSP27, HSP40, HSP70, HSP90, ubiquitin proteins and one copy each of cartilage associated protein (CRTAP), HSPH1, HSBP1, FK506-binding protein 4 (FKBP4), kruppel-like factor (KLF13), peptidyl-prolyl isomerase PIPB and Prolyl 4-hydroxylase beta subunit (P4HB). Conclusion: The study found a cocktail of different chaperones interacting with HSP47, originated at different time points from prokaryotes to eukaryotes. Overall, the study was successful in finding HSP47 expression patterns among several normal tissues using three different publicly available datasets. It also assessed the expression pattern of HSP47 in human cancer types. These findings will encourage further studies focusing on the role of HSP47 in human diseases.

Proteomic analysis of circulating monocytes in Chinese premenopausal females with extremely discordant bone mineral density.

Osteoporosis (OP) is a major public health problem, mainly characterized by low bone mineral density (BMD). Circulating monocytes (CMCs) may serve as progenitors of osteoclasts and produce a wide variety of factors important to bone metabolism. However, the specific action mechanism of CMCs in the pathogenesis of OP is far from clear. We performed a comparative protein expression profiling study of CMCs in Chinese premenopausal females with extremely discordant BMD, identified a total of 38 differentially expressed proteins, and confirmed with Western blotting five proteins: ras suppressor protein1 (RSU1), gelsolin (GSN), manganese-containing superoxide dismutase (SOD2), glutathione peroxidase 1(GPX1), and prolyl 4-hydroxylase beta subunit (P4HB). These proteins might affect CMCs' trans-endothelium, differentiation, and/or downstream osteoclast functions, thus contribute to differential osteoclastogenesis and finally lead to BMD variation. The findings promote our understanding of the role of CMCs in BMD determination, and provide an insight into the pathogenesis of human OP.

Proteomic Analysis of Testis Biopsies in Men Treated with Injectable Testosterone Undecanoate Alone or in Combination with Oral Levonorgestrel as Potential Male Contraceptive

Treatment with injectable testosterone undecanoate (TU) alone or in combination with oral levonorgestrel (LNG) resulted in marked decreases in sperm concentrations. In this study, we used proteomic analyses to examine the cellular/molecular events occurring in the human testis after TU or TU + LNG treatment. We conducted a global proteomic analysis of the human testicular biopsies before and at 2 weeks after TU alone or TU + LNG treatment. Proteins showing significant changes in expression were identified and analyzed. As a result, 17 and 46 protein spots were found with significant differential expression after the treatment with TU alone and TU + LNG, respectively. TU treatment changed the expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), proteasome inhibitor PI31 subunit (PSMF1), and superoxide dismutase [Mn] mitochondrial precursor (SOD2). These proteins inhibit "assembly", induce cell death, and promote compensatory "cell survival" in the testis. After TU + LNG treatment, "proliferation/cell survival" and "apoptosis/death" were the predominant responses in the testis. TU + LNG treatment inhibited the expression of Prolyl 4-hydroxylase beta subunit (P4HB) and Annexin A2 (Annexin II). These proteins are involved in apoptosis and cell proliferation, respectively. TU + LNG treatment also enhanced the expression of SOD2 and Parvalbumin alpha (Pvalb). These two proteins may protect testicular cells against apoptosis/death and promote cell survival. In conclusion, TU and TU + LNG treatments suppress spermatogenesis through different pathways by changing the expression of different proteins. hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb, are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.

Proteomic profiling of endorepellin angiostatic activity on human endothelial cells.

BackgroundEndorepellin, the C-terminal domain V of the heparan sulfate proteoglycan perlecan, exhibits powerful and targeted anti-angiogenic activity on endothelial cells. To identify proteins involved with endorepellin anti-angiogenic action, we performed an extensive comparative proteomic analysis between vehicle- and endorepellin-treated human endothelial cells.ResultsProteomic analysis of endorepellin influence on human umbilical vein endothelial cells identified five differentially expressed proteins, three of which (β-actin, calreticulin, and chaperonin/Hsp60) were down-regulated and two of which (vimentin and the β subunit of prolyl 4-hydroxylase also known as protein disulfide isomerase) were up-regulated in response to endorepellin treatment—and associated with a fold change (endorepellin/control) ≤ 0.75 and ≥ 2.00, and a statistically significant p-value as determined by Student's t test.ConclusionThe proteins identified represent potential target areas involved with endorepellin anti-angiogenic mechanism of action. Further elucidation as such will ultimately provide useful in utilizing endorepellin as an anti-angiogenic therapy in humans.

Cloning of a novel prolyl 4-hydroxylase subunit expressed in the fibrous cap of human atherosclerotic plaque.

The production of collagen is fundamental to atherosclerosis and critically dependent on posttranslational modification by prolyl 4-hydroxylase. We report the cloning of a novel prolyl 4-hydroxylase catalytic (alpha) subunit from human vascular smooth muscle cells. The peptide displayed conservation of critical residues for interacting with Fe2+ and 2-oxoglutarate, essential cosubstrates for prolyl 4-hydroxylase activity. Furthermore, when the recombinant protein was expressed in cells, it associated with the beta-subunit of prolyl 4-hydroxylase and could catalyze prolyl 4-hydroxylation of a collagen-like peptide. The tissue distribution was dissimilar from that of the 2 previously cloned alpha-subunits, suggesting a role beyond redundancy. Importantly, the novel gene was expressed in the fibrous cap of human carotid atherosclerotic lesions. The discovery of a novel prolyl 4-hydroxylase alpha-subunit, here termed the alpha(III)-subunit, suggests a new participant in collagen synthesis that, in view of the expression findings, may be relevant to atherosclerotic disease.

Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running.

The purpose of the study was to investigate pre-translational regulation of collagen expression after a single bout of exercise. We analysed steady-state messenger ribonucleic acid (mRNA) levels for collagen types I, III and IV, alpha- and beta-subunits of prolyl 4-hydroxylase and lysyl oxidase (enzymes modifying procollagen chains), and enzyme activity of prolyl 4-hydroxylase from rat soleus muscle (MS) and the red parts of quadriceps femoris muscle (MQF) after 12 h and after 1, 2, 4, 7 and 14 days of downhill (-13.5 degrees ) treadmill running at a speed of 17 m.min-1 for 130 min. Histological and biochemical assays revealed exercise-induced muscle damage in MQF but not MS. Steady-state mRNA levels for the alpha- and beta-subunits of prolyl 4-hydroxylase in MQF, lysyl oxidase in MS and MQF were increased 12 h after running, whereas prolyl 4-hydroxylase activity did not increase until 2 days after exercise. The mRNA levels for the fibrillar collagens (I and III) and basement membrane type IV collagen significantly increased 1 day and 12 h after exertion, respectively. Peak mRNA levels were observed 2-4 days after running, the increases being more pronounced in MQF than in MS. No significant changes were observed in types I or III collagen at the protein level. Strenuous downhill running thus causes an increase in gene expression for collagen types I and III and their post-translational modifying enzymes in skeletal muscle in a co-ordinated manner. These changes, together with the increased gene expression of type IV collagen, may represent the regenerative response of muscle extracellular matrix to exercise-induced injury and an adaptive response to running exertion.

Expression of an engineered form of recombinant procollagen in mouse milk.

We have examined the suitability of the mouse mammary gland for expression of novel recombinant procollagens that can be used for biomedical applications. We generated transgenic mouse lines containing cDNA constructs encoding recombinant procollagen, along with the alpha and beta subunits of prolyl 4-hydroxylase, an enzyme that modifies the collagen into a form that is stable at body temperature. The lines expressed relatively high levels (50-200 micrograms/ml) of recombinant procollagen in milk. As engineered, the recombinant procollagen was shortened and consisted of a pro alpha 2(I) chain capable of forming a triple-helical homotrimer not normally found in nature. Analysis of the product demonstrated that (1) the pro alpha chains formed disulphide-linked trimers, (2) the trimers contained a thermostable triple-helical domain, (3) the N-propeptides were aligned correctly, and (4) the expressed procollagen was not proteolytically processed to collagen in milk.

Immunohistochemical evaluation of cellular deposits on posterior chamber intraocular lenses.

We used immunohistochemistry to characterize cellular and proteinaceous deposits on the surfaces of explanted posterior chamber intraocular lenses (PC-IOLs). A total of 30 PC-IOLs were immunostained for the alpha- or beta-subunits of prolyl 4-hydroxylase, which is involved in collagen biosynthesis; cellular fibronectin; alpha B crystalline; and CD68, a macrophage marker, to characterize the cellular deposits that adhere to the IOL surfaces and to evaluate the distribution of cells involved in the deposition of extracellular matrix on IOLs. Cellular or proteinaceous deposits were observed on all 30 PC-IOLs. Cells that showed positive staining for alpha B crystallin were classified as lens epithelial cells; CD68-positive cells were considered to be of macrophagic origin. Positivity for cellular fibronectin, including the macrophages and related cells, appeared to be responsible for the accumulation of fibronectin on the surfaces of PC-IOLs. Prolyl 4-hydroxylase-positive cells were involved in the deposition of collagen on PC-IOLs. Immunohistochemical study revealed that macrophages, foreign-body giant cells, and lens epithelial cells adhered to explanted PC-IOLs. Such adherent cells are responsible for the deposition of extracellular matrix on the surfaces of PC-IOLs and may regulate the assembly of the extracellular matrix, influencing the biocompatibility of PC-IOLs.

Collagen synthesis in lens epithelial cells on capsules containing intraocular lens implants in rabbits: immunolocalization of prolyl 4-hydroxylase.

We evaluated ultrastructurally the presence and distribution of lens epithelial cells and extracellular matrix on the posterior capsule after implantation of a silicone or a polymethyl-methacrylate (PMMA) intraocular lens (IOL) in rabbits. The immunolocalization of prolyl 4-hydroxylase, an enzyme involved in procollagen synthesis, was also examined in such cells. Anesthetized adult albino rabbits (n = 12) underwent phacoemulsification and aspiration (PEA) of the crystalline lens and implantation of a silicone or a PMMA IOL into the capsular bag of one eye. They were killed by an intravenous overdose of pentobarbital sodium. The lens capsules were processed for transmission electron microscopy and for immunohistochemical detection of the beta-subunit of prolyl 4-hydroxylase. Ultrastructurally, lens epithelial cells on the posterior capsule evidenced an accumulation of collagenous extracellular matrix. Prolyl 4-hydroxylase immunoreactivity was detected in the cytoplasm of cells distributed on the capsules. Lens epithelial cells are involved in the production of collagenous matrix on lens capsules after PEA and implantation of IOL, resulting in a fibrotic process of the lens capsule.

Co-expression of fibroblastic, histiocytic and smooth muscle cell phenotypes on cultured adherent cells derived from human palatine tonsils: a morphological and immunocytochemical study.

Adherent cells derived from human palatine tonsils were isolated and cultivated. Exponentially growing adherent cells (TAC) were observed by phase-contrast microscopy and transmission electron microscopy. Immunocytochemical studies were also performed. TAC were composed of relatively monotonous cells with polygonal or spindle shapes and high proliferative activity. In addition to the development of rough endoplasmic reticulum and lysosomes, the TAC possessed a moderate amount of pinocytotic vesicles and a few microfilaments. All of the TAC strongly expressed fibroblastic markers and partial monocyte/macrophage markers, such as beta-subunit of prolyl 4-hydroxylase (DAKO-fibroblast), lysozyme, anti-alpha-1-antichymotrypsin (alpha ACT), and CD68 (KP-1, EBM/11). It was noted that, as the TAC were cultured for a longer period, they gradually increased the reactivity with the monoclonal antibody PG-M1. Furthermore, the TAC expressed myocytic phenotype, such as alpha-smooth muscle actin (alpha SMA) with various intensity. Moreover, as to extracellular matrix, TAC stained for collagen type I, collagen type III, laminin, and fibronectin. Collagen type IV was weakly positive. The results presented here showed that the TAC expressed three different phenotypes of fibroblasts, histiocytes and smooth muscle cells at the same time. The monoclonal antibody raised against the TAC reacted strongly with the subendothelial pericytes and/or smooth muscle cells in the extrafollicular area in human tonsils. The present results also suggested that the origin of the TAC was probably subendothelial pericytes and/or smooth muscle cells of the microvasculatures in the tonsil.

Hepatic retinopathy: morphological features of retinal glial (Müller) cells accompanying hepatic failure.

More than 80 years ago, Alzheimer described changes in the brains of patients who had suffered hepatic failure. Astrocytes are primarily affected; their nuclei become swollen, their intermediate filament protein composition is altered and their cytoplasm becomes vacuolated. Cells with these features are called Alzheimer type II astrocytes and these changes have been attributed to the toxic effects of elevated ammonia levels. The present study investigates whether the dominant glia of another part of the central nervous system, the Müller cells of the retina, undergo similar changes. Retinae of patients who had died with symptoms of hepatic failure were processed for histology, histochemistry, and immunocytochemistry. Cell nuclei were measured from brain astrocytes (insula cortex), Müller cells, and retinal bipolar neurons. Hepatic failure resulted in the enlargement of nuclei in astrocytes and Müller cells, and the enhanced expression in Müller cells of glial fibrillary acidic protein, cathepsin D, and the beta-subunit of prolyl 4-hydroxylase (glial-p55). In some retinae, signs of gliosis were also observed. We conclude that increased levels of serum ammonia resulting from hepatic insufficiency cause changes in Müller cells that are similar to those seen in brain astrocytes. We term this condition hepatic retinopathy.

Immunohistological detection of the beta subunit of prolyl 4-hydroxylase in rat and mini pig lungs with radiation-induced pulmonary fibrosis.

Polyclonal and monoclonal antibodies to the beta subunit of prolyl 4-hydroxylase, the protein disulphide isomerase, were used to compare the pulmonary cells in 13 normal and in 20 fibrotic rat and mini-pig lungs made fibrotic by X-ray irradiation, using the ABC immunoperoxidase technique. In normal lungs, prominent staining of Clara cells and type II pneumocytes and weaker reactivity with alveolar macrophages, fibroblasts, endothelial and smooth muscle cells were detectable. In pulmonary disease, in which interstitial fibrosis was the characteristic feature, the immunoreactivity was increased in both the epithelial and interstitial cells. Type I pneumocytes remained negative. In the early stages of disease (3 to 4 weeks after irradiation) when little morphological alteration was seen, capillary endothelial cells had already become immunoreactive. These results underline the complex involvement and interaction of different lung cell populations in the process of pulmonary fibrogenesis.

Immunohistochemical localization of the β subunit of prolyl 4-hydroxylase in human alveolar epithelial cells

The beta subunit of prolyl 4-hydroxylase, the protein-disulfide isomerase (PDJ), catalyzes the hydroxylation of proline residues of collagens and proteins with collagen-like structure, a step essential for the folding of the procollagen chains to form triple-helices. In the present study, we report the selective immunohistological localization of PDI in type II alveolar and bronchial epithelial cells. The detection of the hidden antigen with the monoclonal antibody 5B5 is usually not successful in paraffin sections but was possible after microwave pretreatment of tissue sections. In cases of severe lung injury (fibrosing alveolitis) enhanced immunoreactivity was found for this enzyme in epithelial, endothelial as well as interstitial cells and in alveolar macrophages. The results indicate a possible involvement of the pulmonary epithelial cells in the upregulation of collagen production during the process of fibrosis.

Efficient catalysis of disulphide bond rearrangements by protein disulphide isomerase.

Protein disulphide isomerase (PDI) is a highly abundant and ubiquitous eukaryotic protein that is essential for viability in yeast. Although PDI is thought to catalyse disulphide bond formation and isomerization during protein biosynthesis, PDI has been found previously to have only moderate effects (approximately 25-fold) on the rate of oxidative folding of proteins in vitro. In addition, PDI has been implicated in several apparently unrelated cellular functions. For example, PDI is the beta-subunit of prolyl 4-hydroxylase and is part of the triglyceride transfer complex. The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) is slow and inefficient in vitro. Here we report that PDI increases by a factor of 3,000-6,000 the rates of folding of kinetically trapped BPTI folding intermediates, in which native structure impedes disulphide bond formation. By contrast, PDI has only small effects on the rate of disulphide bond formation in intermediates that are oxidized readily in the absence of PDI. These results suggest that an important function of PDI is to catalyse disulphide bond formation and rearrangements within kinetically trapped, structured folding intermediates.

Predominant expression of the beta subunit of prolyl 4-hydroxylase (disulfide isomerase) in human extravillous trophoblasts.

Prolyl 4-hydroxylase is a heterodimeric enzyme that is crucial in the biosynthesis of collagen. The beta subunit of this enzyme is a multifunctional protein which is also known as protein-disulfide isomerase. Immunofluorescence and monoclonal antibody (Mab) 5B5 were used to localize the beta subunit in human extraembryonic tissues. The strongest sites of 5B5 reactivity were extravillous cytotrophoblasts in the basal plate, uteroplacental arteries and amniochorion, syncytiotrophoblast displayed variable weaker reactivity. Only a small fraction of placental 5B5 antigen was detected as a component of prolyl-4-hydroxylase by affinity chromatography on immobilized polyproline. The results indicate a difference in the expression of an endoplasmic reticulum marker between villous and extravillous trophoblast. The predominance of 5B5 antigen in extravillous trophoblast could be associated with an increased ability to synthesize collagen or other enzymatic reactions associated with prolyl 4-hydroxylase beta subunit.

Interaction of transcription factor Sp1 with the promoter of the gene for the multifunctional protein disulphide isomerase polypeptide.

Protein disulphide isomerase (PDI) is a unique polypeptide which resides in the lumen of the endoplasmic reticulum and also functions as the beta-subunit of prolyl 4-hydroxylase, as a cellular thyroid hormone-binding protein, as the smaller subunit of the microsomal triacylglycerol transfer protein complex, as a dehydroascorbate reductase and as a protein that binds various peptides in a specific manner. We have recently demonstrated that the promoter of the PDI gene contains six CCAAT boxes and other elements which are needed for efficient transcription. We now demonstrate that purified human recombinant transcription factor Sp1 interacts with two perfect GGGCGG sequences and three other GC-rich elements of the PDI promoter. Sp1 also appears to participate in the regulation of PDI gene expression, since overexpression of Sp1 stimulated PDI promoter activity in HeLa cells and mutations introduced into each of these Sp1-binding sites separately reduced the promoter strength, although even the largest decrease was only about 50%. These results support our view that expression of the gene for this polypeptide with multiple functions is secured by several regulatory elements, some of which are functionally redundant.

CDNA for R-cognin: homology with a multifunctional protein.

Retina cognin (R-cognin) is a developmentally regulated 50-kDa protein that was isolated from chicken embryo retina cell membranes. It mediates the adhesion and reaggregation in vitro of retina cells from chicken and mouse embryos, but not of cells from other tissues, and may be involved in neuronal differentiation. We report here the cloning of a cDNA for R-cognin. A chicken embryo retina cDNA library was constructed in lambda gt11 vector and was screened with polyclonal R-cognin antiserum, yielding several immunoreactive clones. Antiserum prepared to the R-cognin-beta-galactosidase fusion protein produced by one recombinant lysogen recognized the 50-kDa R-cognin protein derived from retina cell membranes. This antiserum inhibited the reaggregation of dissociated retina cells and immunostained chicken embryo retina tissue in a pattern similar to that obtained with R-cognin antiserum. In vitro translation of RNA from a cDNA subclone yielded a 50-kDa protein that was recognized by R-cognin antiserum on a Western blot. By these criteria we identify the cDNA clone as representative of the gene encoding R-cognin. This cDNA is nearly identical to a major portion of the cDNA for the multifunctional protein that is the beta subunit of prolyl 4-hydroxylase and has both protein disulfide isomerase activity and thyroid hormone-binding activity. These findings demonstrate that R-cognin differs from other cell adhesion molecules and suggest possible mechanisms for its action in cell adhesion and neuronal differentiation.

Characterization of a Partial cDNA for Lysyl Hydroxylase from Human Skin Fibroblasts; Lysyl Hydroxylase mRNAs Are Regulated Differently by Minoxidil Derivatives and Hydralazine

Lysyl hydroxylase (LH) is an essential enzyme in collagen biosynthesis that catalyzes the formation of hydroxylysine required for intermolecular crosslinking of collagen. We have isolated a partial (2.2-kb) cDNA for LH from human skin fibroblasts using PCR. DNA sequencing revealed 72% homology of the human coding sequence with the chick LH sequence at the nucleotide level and 76% homology predicted at the amino acid level. The LH cDNA hybridized strongly with two mRNA species of 2.4 and 3.4 kb on Northern blots of normal fibroblast RNA. Administration of minoxidil decreased both mRNA species without affecting levels of the mRNAs for the beta subunit of prolyl 4-hydroxylase (PH) or alpha 1(I) collagen. Two derivatives of minoxidil (3' hydroxyminoxidil and 4' hydroxyminoxidil) produced similar decreases in LH mRNAs. In contrast hydralazine increased the mRNAs for LH in parallel with its previously reported effect on the mRNA for the beta subunit of PH. This effect is accompanied by virtual elimination of the alpha 1(I) collagen mRNAs. These results on the action of minoxidil and hydralazine at the pretranslational level correlate well with their previously reported effect on enzyme activity and collagen biosynthesis and indicate that changes in steady-state mRNA levels can account directly for changes at the protein level. Moreover, the unique action of minoxidil in specifically decreasing LH mRNAs contrasts with the less specific stimulatory effects of hydralazine and suggests that these pharmaceuticals are regulating expression of LH at a pretranslational level by different mechanisms.

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)

Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.