Development of a peroxidase-coupled fluorometric assay for lysyl oxidase

Lysyl oxidase catalyzes the oxidation of peptidyl lysine in elastin and collagen and also acts upon nonpeptidyl amines, although the enzyme becomes slowly inactivated while processing nonpeptidyl substrates. In spite of this complexity, it has been possible to devise a continuously monitored peroxidase-coupled fluorometric assay for the oxidation of simple amines by lysyl oxidase. In the present study, optimal assay conditions have been explored and found to include assay temperatures of 50 to 60°C, the presence of urea in the assay, and the use of diaminopentane as substrate. Although the assay is subject to interference by contaminating macromolecules in enzyme fractions, a linear assay response to enzyme concentration is obtained with highly purified lysyl oxidase with a limiting sensitivity of 0.3 μg of enzyme per assay.     

Multiple modes of catalysis-dependent inhibition and inactivation of aortic lysyl oxidase

Lysyl oxidase purified from bovine aorta can oxidize simple alkyl mono- and diamine substrates yielding the respective aldehyde, H2O2, and ammonia as products. The oxidation of such substrates is limited to approximately 100 catalytic turnovers per enzyme molecule since lysyl oxidase is syncatalytically and irreversibly inactivated in the course of oxidation of these amines. The present study reveals that addition of oxidant scavengers protects significantly against inactivation of lysyl oxidase and that the ammonia product is a reversible competitive inhibitor of amine oxidation. Further, the enzyme becomes covalently labeled by the amine substrate or its enzyme-processed derivative during catalysis. Thus, lysyl oxidase appears subject to multiple modes of catalysis-dependent inhibition or inactivation. Syncatalytic inactivation of lysyl oxidase might represent a means of restricting the activity of this enzyme toward its elastin and collagen substrates in vivo.     

Lysyl oxidase activates the transcription activity of human collagene III promoter. Possible involvement of Ku antigen

Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis.     

Lysyl oxidase and P-ATPase-7A expression during embryonic development in the rat

Lysyl oxidase activity is critical for the assembly and cross-linking of extracellular matrix proteins, such as collagen and elastin. Moreover, lysyl oxidase activity is sensitive to changes in copper status and genetic perturbations in copper transport, e.g., mutations in the P-type ATPase gene, ATP7A, associated with cellular copper transport. Lysyl oxidase may also serve as a vehicle for copper transport from extracellular matrix cells. Herein, we demonstrate that sufficient lysyl oxidase functional activity is present in the rat embryo at gestation day (GD) 9 to be detected in conventional enzyme assays. Estimation of embryonic lysyl oxidase functional activity, however, required partial purification in order to remove inhibitors. From GD 9 to GD 15, lysyl oxidase activity was relatively constant when expressed per unit of protein or DNA. In contrast, the steady-state levels of lysyl oxidase and ATP7A mRNA, measured by RT-PCR and expressed relative to total RNA and cyclophilin mRNA, increased approximately fourfold from GD 9 to 15. The pattern of temporal expression for ATP7A was consistent with its possible role in copper delivery to lysyl oxidase.     

Inhibition by heparin of the oxidation of lysine in collagen by lysyl oxidase

The generation of covalent cross-linkages in collagen is initiated by the deamination by lysyl oxidase of specific lysine residues in this connective tissue protein. Since lysyl oxidase activity is influenced by ionic ligands bound to its protein substrates, the effect of heparin, an anionic glycosaminoglycan known to bind to collagen, was explored by using collagen and elastin substrates and highly purified lysyl oxidase. Concentrations of heparin up to 1 mg mL-1 had little effect on the enzymatic rate of oxidation if it was added prior to the addition of enzyme to a preformed fibrillar collagen substrate or to an insoluble elastin substrate. However, collagen oxidation was inhibited by 85% if this glycosaminoglycan was present at 0.4 mg mL-1 during collagen fibril formation before addition of the enzyme. Similarly, the rate and extent of collagen fibrillogenesis in the absence of lysyl oxidase were each markedly inhibited in the presence of 0.4 mg mL-1 heparin. Heparin also inhibited the extent of tight binding of lysyl oxidase to preformed fibrils by about 40% under conditions where enzyme activity against preformed fibrils was hardly affected. These results suggest that heparin may modulate the oxidation and thus the insolubilization of extracellular collagen fibers, possibly under conditions where elastin fiber synthesis is not affected, and that the tight binding of lysyl oxidase to collagen is not completely related to the expression of enzyme activity toward this substrate. These results also have mechanistic implications for the retarding effect of heparin on postoperative wound healing.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

Properties of highly purified lysyl oxidase from embryonic chick cartilage.

Lysyl oxidase the enzyme which oxidately deaminates lysine residues in collagen and elastin, was purified from embryonic chick cartialge by employing an affinity column of lathyritic rat skin collagen coupled to Sepharose, followed by separation on DEAE-cellulose. An enzyme preparation was obtained which was pure as shown by polyacrylamide gel electrophoresis. The specific activity was 1800-fold higher than that of the original extract. The pure enzyme utilized both collagen and elastin substrate. Furthermore, the ratios of enzyme activity with elastin substrate versus that with collagen substrate were the same at all stages of purity. Only one protein band was found after polyacrylamide gel electrophoresis of the pure lysyl oxidase in sodium dodecyl sulfate and mercaptoethanol. The molecular weight was estimated to be 28000. It was found that the enzyme contained a large number of cysteine and tyrosine residues. Evidence was obtained for molecular heterogeneity of lysyl oxidase. The enzyme eluted from DEAE-cellulsoe in at least four distinct regions. When the peaks were rechromatographed separately, they eluted at salt concentrations similar to those of the original chromatogram. However, the substrate specificity and the electrophoretic mobility on polyacrylamide gel were the same for all enzyme fractions.     

The propeptide domain of lysyl oxidase induces phenotypic reversion of ras-transformed cells

Lysyl oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, lysyl oxidase reverts ras-mediated transformation, and lysyl oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces lysyl oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of lysyl oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. beta-aminopropionitrile, the selective inhibitor of lysyl oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense lysyl oxidase demonstrated that lysyl oxidase gene expression mediated phenotypic reversion. Since lysyl oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the lysyl oxidase propeptide, and not the lysyl oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Akt-dependent induction of NF-kappaB activity. Thus, the lysyl oxidase propeptide, which is released during extracellular proteolytic processing of pro-lysyl oxidase, functions to inhibit ras-dependent cell transformation.     

Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1

 Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors, and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix. In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete distance from the lesion not exceeding 350 μm from the inflammatory cells. Staining was associated with mesenchymal cells with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the regulation of extracellular matrix accumulation. Accepted: 19 December 1998     

Synthesis of lysyl oxidase in experimental hepatic fibrosis.

The synthesis of lysyl oxidase, which initiates the cross-linking of collagen and elastin, was investigated in carbon tetrachloride (CCl4) induced fibrotic liver of rat. Lysyl oxidase activity of the fibrotic liver was 4 times greater than that of normal liver. mRNAs from the livers of normal and CCl4-treated rats were prepared for in vitro protein synthesis and the products were analyzed by immunoprecipitation with a monoclonal antibody against lysyl oxidase. The mRNAs from the fibrotic liver gave more than 3 times higher level of messenger copies for lysyl oxidase than did mRNAs from normal liver. The molecular weight of the nascent lysyl oxidase was 48,000.     

Reduced lysyl oxidase activity in skin fibroblasts from patients with Menkes'' syndrome.

Lysyl oxidase activity against both collagen and elastin substrates has been examined in the culture medium of skin fibroblasts derived from unrelated patients with Menkes' syndrome and from control subjects. The medium of three Menkes' fibroblast lines showed 3--30% of the activity present in the medium of control fibroblasts, against a purified collagen substrate. Lysyl oxidase activity in the culture medium of two of the Menkes' fibroblast lines was also examined by using a crude aortic-elastin substrate and was similarly decreased in comparison with that in the medium of control fibroblasts. Lysyl oxidase activity in the medium of a fourth fibroblast line, derived from a foetus with Menkes' syndrome, was 42% of that in the medium of control fibroblasts derived from a 1-day-old baby against a collagen substrate, and 26% of that in control fibroblast medium against an elastin substrate. The copper content of the cell layers of the Menkes' fibroblast cultures was elevated in comparison with normal fibroblast cultures, as has previously been reported to be characteristic of such cells. It is suggested that the decrease in lysyl oxidase activity would help to explain the connective tissue defects observed in Menkes' syndrome, and that this reduction, in conjunction with the elevated concentrations of cellular copper, would support the hypothesis that a functional intracellular copper deficiency exists in Menkes' syndrome.     

Induction of human monocyte motility by lysyl oxidase

Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10⁻¹⁰ M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with β-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant reponse to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen.     

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.     

The biological function of the R2a regulatory gene for alkaline phosphatase in Escherichia coli

Previous attempts to purify lysyl oxidase have been frustrated by the failure to recover activity during ion exchange or affinity chromatography. We have found that lysyl oxidase from chick cartilage shows marked stability in buffers containing urea and in these solutions can be recovered in high yield from DKAE-cellulose and collagen-derivatized Sepharose. The purified enzyme was active against both collagen and elastin substrates but devoid of monoamine oxidase activity. An absolute requirement for oxygen for activity was found.     

Modulation of lysyl oxidase substrate specificity by the oleate anion

Extracts of bovine ligamentum nuchae have been assayed for lysyl oxidase activity using as substrates soluble elastin and soluble collagen labeled with tritiated lysine. The assays were performed in the presence and absence of sodium oleate. At 0.8 mM, oleate decreased activity with elastin more than 50% and enhanced activity with collagen to approximately 200% that of controls without oleate. The results show that this hydrophobic anion modulates lysyl oxidase specificity in crude extracts and suggests a mechanism for modifying activity in tissues.     

Opposing effects of ascorbate on collagen and elastin deposition in the neonatal rat aorta

Ascorbic acid plays an important role in connective tissue metabolism, where, among other effects, it acts as a reducing factor in the reactions catalyzed by prolyl and lysyl hydroxylases. In vitro, ascorbic acid has been shown to have a positive influence on collagen synthesis at pre- and/or post-translational levels and a negative effect on elastin production. In the present work, the effects of vitamin C on extracellular matrix deposition have been studied in vivo. Stereological analysis on electron micrographs showed, compared to age-matched controls, a 50 to 60% increase of collagen deposition in the media and in the adventitia of the aorta of rats treated for 30 days from the 18th day of life with 10% ascorbate in their drinking water. By contrast, elastin volume density was significantly reduced by the treatment at all ages examined. These morphological data were supported by in situ hybridization observations showing enhanced collagen type I mRNA and reduced elastin mRNA expression upon treatment. Although vitamin C did not inhibit lysyl oxidase activity in vivo, being only slightly higher than in controls, enzyme activity was significantly reduced, when high doses of ascorbate were added in vitro. Lysyl oxidase activity may be a function of enhanced collagen metabolism rather than a direct effect of the vitamin on the enzyme activity. These data indicate that ascorbate exerts opposite effects on the deposition of two major components of the extracellular matrix in vivo, at least during periods of rapid growth.     

Lysyl oxidase: a pituitary hormone-dependent enzyme.

Aldehyde-deficient non-crosslinked collagen obtained from lathyritic rats and collagen from penicillamine-treated rats, which is not deficient in aldehydes but the crosslinking of which is also inhibited, were implanted into the peritoneal cavity of hypophysectomized rats using the diffusion chamber technique. The enzyme lysyl oxidase which catalyses the aldehyde formation in certain lysyl residues of collagen and elastin was extracted from the skin of hypophysectomized rats. The activity of the enzyme was determined following its incubation with an L-[4,5-3H] lysine-labeled elastin substrate prepared from aortas of 17-day-old chick embryos. The result showed that the aldehyde deficient collagen did not crosslink while in the hypophysectomized animal indicating the lack of active lysyl oxidase in the rats. The enzyme activity in the skin of hypophysectomized animals was markedly reduced as compared with the controls indicating directly the dependance of lysyl oxidase activity on pituitary gland hormones.     

Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells.

Lysyl oxidase, an extracellular amine oxidase, controls the maturation of collagen and elastin. We examined the regulation of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium (RPE) cells in relation to the changes in subretinal fluid transport and phenotype of RPE cells. The level of the mRNA in cells grown on microporous membranes was markedly increased by application of hyperosmotic mannitol solution on the apical side (191% of control), implying that RPE cells express more lysyl oxidase in the condition which may cause the accumulation of subretinal fluid. Platelet-derived growth factor increased the mRNA level in subconfluent cells in culture (137% of control) and basic fibroblast growth factor decreased it (79% of control). In addition, exposure of cells to retinoic acid alone or in combination with dibutyryl cAMP for 22 days markedly decreased the level of lysyl oxidase mRNA (52 or 35% of control) while increasing the level of mRNA of N-acetylglucosaminidase (NAG), a marker enzyme for lysosomes (162 or 142% of control). Moreover, the level of lysyl oxidase mRNA in cells grown on microporous membranes was lower than that in cells grown on plastic dishes, while the level of NAG mRNA in the former cells was higher than that in the latter. Taken together, the expression of lysyl oxidase seemed to increase during proliferation of RPE cells and decrease toward differentiation. beta-Aminopropionitrile, an inhibitor of lysyl oxidase, significantly inhibited the contraction of collagen gels by fetal calf serum, suggesting that lysyl oxidase may be involved in pathogenesis caused by RPE cells.