Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for 'scrambled' ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.     

Catalysis by protein disulphide-isomerase of the assembly of trimeric procollagen from procollagen polypeptide chains

Type-I procollagen,¹⁴C-biosynthetically labelled, was reduced under denaturing and non-denaturing conditions. Reoxidation to disulphide-linked trimers occurred with non-denatured chains in the presence of an oxidant system containing oxidized and reduced glutathione. Dimeric intermediates were not detected. This reoxidation was accelerated by homogeneous beef liver protein disulphide-isomerase.     

Thiol-protein disulphide oxidoreductases. Differences between protein disulphide-isomerase and glutathione-insulin transhydrogenase activities in ox liver.

1. Protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were assayed in parallel through a conventional purification of protein disulphide-isomerase from ox liver. 2. Throughout a series of purification steps (differential centrifugation, acetone extraction, (NH4)2SO4 precipitation and ion-exchange chromatography), the two activities appeared in the same fractions but were purified to different extents. 3. The final sample was 143-fold purified in protein disulphide-isomerase but only 10-fold purified in glutathione-insulin transhydrogenase; nevertheless the two activities in this preparation were not resolved by high-resolution isoelectric focusing and both showed pI4.65. 4. In a partially purified preparation containing both activities, glutathione-insulin transhydrogenase was far more sensitive to heat denaturation than was protein disulphide-isomerase; conversely protein disulphide-isomerase was more sensitive to inactivation by deoxycholate. 5. The data are inconsistent with a single enzyme being responsible for all the protein disulphide-isomerase and glutathione-insulin transhydrogenase activity of ox liver. It is suggested that several similiar thiol-protein disulphide oxidoreductases of overlapping specificities may better account for the data.     

Detection of protein disulphide-isomerase in sheep pancreas fractions

Conclusions The use of diethylpyrocarbonate to inhibit endogenous ribonuclease in sheep pancreas allows the detection of protein-disulphide-isomerase activity in homogenates, at specific activities of up to 4 units/g. This is higher than the specific activity in sheep liver homogenates (about 2 units/g) or in homogenates of other sheep tissues (16). It is thus evident that high levels of protein-disulphide-isomerase activity are present in sheep pancreas. This is consistent both with the postulated general role of protein disulphide-isomerase in protein biosynthesis (10,11) and with the in vitro action of the enzyme on its conventional substrate scrambled ribonuclease, since pancreas is the major site of ribonuclease synthesis.     

Collagen biosynthesis by human skin fibroblasts. III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. the results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased the amount of [3H]hydroxyproline synthesized. Ascorbic acid, however, did not increase the synthesis of 3H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and secretion of procollagen.     

Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII.

The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age-matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino-terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion.     

Mechanisms of stimulation of collagen synthesis during chick embryonic skin development.

To study how collagen synthesis is regulated in developing chick embryonic skin, hydroxyproline synthesis, incorporation of proline, and translational activity and content of collagen mRNA in 12-, 15-, and 18-day skins were determined and compared with each other. Hydroxyproline synthesis in the 18-day skins was markedly increased over that in the 12-day skins, whereas proline incorporation was moderately increased. The increase in collagen synthesis from day 15 to 18 was accompanied by increases in both the translational activity and the content of type I procollagen mRNA, with a selective increase in the lower-molecular-weight species of pro alpha 1 (I) collagen mRNA. In contrast, the stimulation of collagen synthesis from day 12 to day 15 did not parallel the levels of type I procollagen mRNA. These results suggest that the stimulation of collagen synthesis is regulated by collagen mRNA levels only in the later stage of development (from day 15 to day 18). Both the collagen synthesis and type I procollagen mRNA levels in the fibroblasts isolated on each corresponding day were constant. The difference in collagen synthesis under two different culture conditions suggests that cell-matrix interaction and/or some serum factors, including several growth factors, are essential for the marked stimulation of collagen synthesis observed in 12- to 18-day skin.     

Thiol-protein disulphide oxidoreductases. Assay of microsomal membrane-bound glutathione-insulin transhydrogenase and comparison with protein disulphide-isomerase.

1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.     

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihydroxybenzoate. Inhibition of prolyl hydroxylase activity as a mechanism of action

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of alpha-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4-hydroxylase, the enzyme catalyzing the synthesis of 4-hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydroxylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate markedly inhibited the synthesis of 4-hydroxyproline in normal cell cultures apparently as a result of reduced prolyl 4-hydroxylase activity, and the synthesis and secretion of both type I and type III procollagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type III procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxybenzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrating the potential applicability of ethyl-3,4-dihydroxybenzoate, or other structural alpha-ketoglutarate or ascorbate analogs, for treatment of fibrotic diseases.     

Subunit structure of wheat germ agglutinin

Cells isolated by enzymic digestion of embryonic tendon were incubated under N₂ so that they synthesized and accumulated the unhydroxylated form of procollagen which is known as protocollagen and which is largely comprised of pro-α chains linked by interchain disulfide bonds. The cells were then exposed to O₂ so that the intracellular protocollagen was hydroxylated and secreted as procollagen. When the hydroxylation was allowed to proceed at 31° or 34°, the procollagen secreted into the medium was triple-helical but its hydroxyproline content was less than two-thirds and its hydroxylysine content was less than half the control. Even when the hydroxylation was allowed to occur at 37°, the procollagen secreted by the cells was under-hydroxylated by about 15% in terms of its hydroxyproline content and about 45% in terms of its hydroxylysine content. The results may have consequences for collagen synthesis by tendons and similar tissues $\text{in vivo}$, since temporary anoxia in such tissues may well lead to the synthesis of a less stable procollagen or to fibers of decreased tensile strength.     

Bovine liver thiol-protein disulphide oxidoreductases. An alternative method for differential purification and resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) activities in bovine liver were studied in parallel during purification of 'thiol-protein disulphide oxidoreductase' by the procedure of Carmichael, Morin & Dixon [(1977) J Biol. Chem. 252, 7163-7167]. The two activities showed no quantitative co-purification and were partially resolved by (NH4)SO4 precipitation, indicating that distinct enzymes are present. 2. Protein disulphide-isomerase was purified by a relatively rapid method involving a combination of the early stages of the Carmichael procedure and covalent chromatography, with a new stepwise elution procedure. Ion-exchange chromatography yields a homogeneous preparation of mol.wt. 57 000. 3. The relationship between protein disulphide-isomerase, glutathione-insulin transhydrogenase and 'thiol-protein disulphide oxidoreductase' is discussed.     

Procollagen N-proteinase. Properties of the enzyme purified from chick embryo tendons

Procollagen N-proteinase was purified about 3700-fold from chick embryo tendons. Electrophoresis of the protein after iodination and denaturation suggested it was homogeneous. However, the native enzyme could not be examined by gel electrophoresis, and therefore homogeneity of the preparation was not conclusively established. Antibodies to the enzyme completely inhibited activity and gave a single precipitant line by double immuno-diffusion. The Km for a native procollagen substrate was 0.3-0.5 microM. The same protein after denaturation inhibited activity. The enzyme did not cleave type III procollagen from human fibroblasts or a type IV procollagen from a mouse sarcoma. Ca2+ was required for maximal enzymic activity. The data suggested a second metal requirement, but this was not identified. Reducing agents and metal chelators inhibited activity, but there was little if any inhibition from several inhibitors of other neutral metalloproteinases.     

Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction.

The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.     

Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.     

Palmitoyl ascorbate: selective augmentation of procollagen mRNA expression compared with L-ascorbate in human intestinal smooth muscle cells.

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.     

Primary and secondary effects of ascorbate on procollagen synthesis and protein synthesis by primary cultures of tendon fibroblasts.

The effects of ascorbic acid on collagen biosynthesis were studied in primary cultures of fibroblasts from chick embryo tendons. Addition of ascorbate to the cultures increased the rate of synthesis of procollagen hydroxyproline, but the effect was not explained by activation of prolyl hydroxylase as has been seen in other cell cultures. Instead the increase in the rate of hydroxyproline synthesis appeared to be the result of some direct cofactor effect of the vitamin. In the presence of ascorbate, most of the newly synthesized procollagen was hydroxylated and became triple helical. In the absence of ascorbate, the overall degree of hydroxylation in newly synthesized procollagen was reduced, but a small fraction of newly synthesized procollagen was near-maximally hydroxylated and became triple helical. When cultures were exposed to ascorbic acid for more than 6 h, there was an increase in rate of protein synthesis, rate of procollagen synthesis, and fraction of membrane-bound ribosomes. The increases in these parameters in the presence of ascorbate appeared to be a secondary effect produced by the accumulation of stable triple-helical procollagen in the culture system.     

Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-beta) enhanced procollagen synthesis by 45 +/- 3% (P < 0.001), and 38 +/- 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 +/- 8% (P < 0.01) and 72% +/- 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-beta-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.