Mechanism for the regulation of post-translational modifications of procollagens synthesized by matrix-free cells from chick embryos.
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Three possible mechanisms are considered to account for the variations of post-translational modifications in different collagen types. 1) The cells have different amounts of post-translational modifying enzymes, 2) the rate of prolylhydroxylation of different procollagen types is varied, and 3) the rate of chain association of pro-alpha chains of different collagen types is modulated. In an attempt to examine the three possibilities, we have determined the activities of prolyl hydroxylase and lysyl hydroxylase, and we have examined the kinetics of the secretion of procollagens and the kinetics of pro-gamma chain formation of different procollagen types in matrix-free cells isolated from tissues of 17-day-old chick embryos. Type II collagen synthesized by cartilage cells contains more hydroxylysine than type I collagen synthesized by tendon and cornea cells. It was found, however, that cartilage cells contain significantly less lysyl hydroxylase than tendon and cornea cells. In contrast, we found only a small difference in the amount of prolyl hydroxylase in tendon, cornea, and cartilage cells. The secretion of type I procollagen by tendon and cornea cells can be described by two first order processes. In contrast, the secretion of type II procollagen by cartilage cells, type IV procollagen by lens cells, and type V procollagen by cornea cells can be described by single first order processes. Examination of the formation of pro-gamma components of procollagen types I and II revealed that it occurs via intermediate dimers of two pro-alpha chains. The formation or pro-gamma(I) chains in tendon and cornea cells is about three times faster than the formation of pro-gamma(II) chains in cartilage cells. These results are consistent with the hypothesis that the rate of association of pro-alpha chains regulates the synthesis of procollagens with different degrees of post-translational modifications. (+info)
Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase.
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The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles. (+info)
The source of oxygen in the reaction catalysed by collagen lysyl hydroxylase.
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The synthetic peptides (Pro-Pro-Gly)5 and (Ile-Lys-Gly)5-Phe were hydroxylated with collagen prolyl hydroxylase and lysyl hydroxylase in an 18O2 atmosphere. The oxygen atoms in the hydroxy groups of hydroxyproline and hydroxylysine were 87% and 6.5% respectively derived from the atmospheric 18O2. The results are consistent with those reported previously for proline hydroxylation in vivo [Fujimoto & Tamiya (1962) Biochem. J. 84, 333-335; Prockop, Kaplan & Udenfriend (1962) Biochem. Biophys. Res. Commun. 9, 192-196; Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501; Prockop, Kaplan & Udenfriend (1963) Arch. Biochem. Biophys. 101, 499-503] and in vitro [Cardinale, Rhoads & Udenfriend (1971) Biochem. Biophys. Res. Commun. 43, 537-543] and for lysine hydroxylation in vivo [Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501]. In view of the similarities of these two oxygenase-type hydroxylation reactions the participation of intermediates is proposed, the oxygen atoms of which are exchangeable with those of water. The atmospheric oxygen atoms incorporated into the intermediate must be equilibrated with water oxygen atoms in the slower lysyl hydroxylase reaction. (+info)
Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein.
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Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol. (+info)
Catalytic properties of lysyl hydroxylase from cells synthesizing genetically different collagen types.
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Crude preparations of lysyl hydroxylase were extracted from chick-embryo tendons synthesizing exclusively type I collagen, chick-embryo sterna synthesizing exclusively type II collagen and HT-1080 sarcoma cells synthesizing exclusively type IV collagen. No differences were found in the Km values for Fe2+, 2-oxoglutarate and ascorbate between these three enzymes preparations. Similarly no differences were found in the Km values for type I and type II protocollagens and the rate at which type IV protocollagen is hydroxylated between these enzyme preparations. The extent to which type I protocollagen could be hydroxylated by the three enzymes was likewise identical. These data strongly argue against the existence of collagen-type-specific lysyl hydroxylase isoenzymes. (+info)
Tranilast, a selective inhibitor of collagen synthesis in human skin fibroblasts.
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Effects of tranilast, N-(3,4-dimethoxycinnamoyl)anthranilic acid, on collagen synthesis in cultured human skin fibroblasts were studied. Tranilast was found to inhibit collagen synthesis in a dose-dependent manner to a maximum of 55% at 300 microM during 48 h of treatment; the synthesis of type I and type III collagens was equally affected. Administered simultaneously or subsequently, tranilast reduced the stimulatory effect of transforming growth factor beta 1 (2.5 ng/ml) on collagen synthesis without affecting the accompanying stimulation of noncollagen protein synthesis. It did not affect prolyl or lysyl hydroxylase activity in vitro and in cells. The content of pro alpha 1(I) collagen mRNA was decreased 60% by tranilast. Tranilast prevented the TGF beta 1-mediated increase in pro alpha 1(I) collagen mRNA. These results indicate that tranilast specifically inhibits collagen production at a pretranslational level by interfering with TGF beta 1 effects. Tranilast also inhibited collagen synthesis in scleroderma fibroblasts to the same extent and in keloid fibroblasts to a greater extent than in normal fibroblasts, attesting to its therapeutic potential as an antifibrotic drug. (+info)
Alu-Alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ehlers-Danlos syndrome.
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The type VI variant of the Ehlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hypotonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS. (+info)
Lysyl hydroxylase, a collagen processing enzyme, exemplifies a novel class of luminally-oriented peripheral membrane proteins in the endoplasmic reticulum.
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Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of the two previously characterized ER-specific retention motifs (KDEL or the double lysine motif) in its primary structure. We now show that LH, nevertheless, resides in the lumen of the ER. In immunofluorescence experiments, LH co-localizes with a KDEL-containing protein, protein disulfide isomerase (PDI), and also co-sediments with it after fractionation of subcellular organelles by sucrose density gradient centrifugation. In addition, LH seems to be stress-inducible. In one respect, however, LH differs from PDI and other known luminal proteins in the organelle. It is found in situ only in association with the ER membranes. Our cell fractionation and Triton X-114 phase separation experiments suggest that it binds to the membranes via weak electrostatic interactions. LH can thus be regarded as a first luminally-oriented "peripheral membrane" protein which has been characterized in the ER. The results suggest a novel possibility by which ER lumen can acquire its specific protein components from the bulk flow. (+info)