Evidence for structural similarities in the multiple forms of aortic and cartilage lysyl oxidase and a catalytically quiescent aortic protein

Lysyl oxidase purified from urea extracts of various connective tissues resolves into multiple catalytically functional species upon chromatography on DEAE-cellulose in 6 M urea. The four enzyme species of bovine aorta retain their original chromatographic behavior on DEAE with time of storage and after purification to homogeneity by gel exclusion chromatography. The peptide maps of each aortic enzyme partially digested by STaphylococcus aureus V8 protease are very similar to each other, as are the peptide maps of complete tryptic digests of each enzyme. Such similarity also exists between the peptide maps of the aortic enzyme and the urea-extractable lysyl oxidase of bovine cartilage, as well as with the peptide maps of a catalytically quiescent protein resolved from the aortic enzyme by gel exclusion chromatography. The substrate activity profiles of the multiple aortic enzyme species are also extremely similar. Although the origin of the enzyme multiplicity remains to be established, there is evident structural and catalytic similarities between the enzyme forms.     

Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5''-phosphate.

Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.     

Lysyl oxidase: evidence that pyridoxal phosphate is a cofactor

Both crude and partially purified preparations of embryonic chick aortic lysyl oxidase tend to gradually lose enzymic activity when illuminated, or when urea is removed by dialysis. Full activity is restored to such preparations by dialysis versus low concentrations of pyridoxal 5′-phosphate prior to assay. Upon treatment with potassium cyanide or semicarbazide, purified embryonic chick aortic lysyl oxidase gives rise to fluorescent derivatives. The fluorescence spectrum of the semicarbazide adduct closely resembles that of pyridoxal phosphate semicarbazone. A preliminary ultraviolet/visible spectrum of bovine aortic lysyl oxidase is also presented; this shows features which add to the existing evidence that lysyl oxidase contains an essential pyridoxal phosphate cofactor.     

Purification and properties of four species of lysyl oxidase from bovine aorta

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.     

Effects of L-ascorbic acid on lysyl oxidase in the formation of collagen cross-links

To clarify the role of L-ascorbic acid (AsA) in the formation of pyridinoline, we examined the effects of AsA in vitro using soluble collagen and partially purified lysyl oxidase from bovine aorta. The concentration of dehydrodihydroxylysinonorleucine decreased when AsA was added in the early stage of pyridinoline formation. However, when AsA was added in a later stage of pyridinoline formation, the concentration of pyridinoline was not affected. These findings indicated that AsA was involved in the initial enzymatic reaction in pyridinoline synthesis. We purified lysyl oxidase to confirm its association of AsA. AsA inhibited the enzyme activity. Erythorbic acid and 3,4-dihydroxybenzoate suppressed the enzyme activity as well as AsA did. The inhibition by AsA of the lysyl oxidase activity arose from characteristics of AsA structure. AsA might be important in the regulation of the oxidative reaction of lysine.     

Immunological characterization of lysyl hydroxylase, an enzyme of collagen synthesis

Antibodies to pure lysyl hydroxylase from whole chick embryos were prepared in rabbits and used for immunological characterization of this enzyme of collagen biosynthesis. In double immunodiffusion a single precipitation line was seen between the antiserum and crude or pure chick-embryo lysyl hydroxylase. The antiserum effectively inhibited chick-embryo lysyl hydroxylase activity, whether measured with the biologically prepared protocollagen substrate or a synthetic peptide consisting of only 12 amino acids. This suggests that the antigenic determinant was located near the active site of the enzyme molecule. Essentially identical amounts of the antiserum were required for 40% inhibition of the same amount of lysyl hydroxylase activity units from different chick-embryo tissues synthesizing various genetically distinct collagen types. In double immunodiffusion a single precipitation line of complete identity was found between the antiserum and the purified enzyme from whole chick embryos and the crude enzymes from chick-embryo tendon, cartilage and kidneys. These results do not support the hypothesis that lysyl hydroxylase has collagen-type-specific or tissue-specific isoenzymes with markedly different specific activities or immunological properties. The antibodies to chick-embryo lysyl hydroxylase showed a considerable degree of species specificity when examined either by activity-inhibition assay or by double immuno-diffusion. Nevertheless, a distinct, although weak, cross-reactivity was found between the chick-embryo enzyme and those from all mammalian tissues tested. The antiserum showed no cross-reactivity against prolyl 3-hydroxylase, hydroxylysyl galactosyl-transferase or galactosylhydroxylysyl glucosyltransferase in activity-inhibition assays, whereas a distinct cross-reactivity was found against prolyl 4-hydroxylase. Furthermore, antiserum to pure prolyl 4-hydroxylase inhibited lysyl hydroxylase activity. These findings suggest that there are structural similarities between these two enzymes, possibly close to or at their active sites.     

Expression of lysyl oxidase from cDNA constructs in mammalian cells: The propeptide region is not essential to the folding and secretion of the functional enzyme

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32–2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including β-aminopropionitrile, phenylhydrazine, ethylenediamine, α,α′-dipyridyl, and diethyl-dithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase purified from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase. © 1995 Wiley-Liss, Inc.     

Structural and catalytic properties of copper in lysyl oxidase

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.     

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.     

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.     

A micromethod for the purification of lysyl oxidase

A simple, efficient, and rapid procedure for the purification of lysyl oxidase is described. This method utilizes an affinity scheme involving powdered elastin-hydroxyethylmethacrylate hydrogels and high-performance liquid chromatography and permits the study of enzyme from sources which contain limited amounts of enzyme, such as aortic smooth muscle cells in culture.     

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.     

Assessment of lysyl oxidase variants by urea gel electrophoresis: evidence against disulfide isomers as bases of the enzyme heterogeneity

Methods for the copurification and rapid assessment of the protein profiles corresponding to the multiple variants of bovine aortic lysyl oxidase are described. The individual variants do not resolve from each other by electrophoresis in sodium dodecyl sulfate but are resolved by gel electrophoresis in 8 M urea, thus providing a new method for their detection independent of enzyme assay. Alkylation of the purified mixture of the variants with iodoacetamide after reduction with dithiothreitol identified three disulfides per 32,000-Da monomer. Urea gel electrophoresis revealed that the heterogeneity of lysyl oxidase persists after reduction and alkylation, indicating that disulfide isomers are not the bases of the enzyme heterogeneity.     

Human placental lysyl oxidase. Purification, partial characterization, and preparation of two specific antisera to the enzyme

Lysyl oxidase from human placentas gave four catalytically active forms on DEAE-cellulose chromatography in 6 M urea. The first tow of these were combined to form pool I and the remaining two to form pool II. Pool I was purified to homogeneity, while the final pool II enzyme usually had one minor contaminant. The molecular weight of both enzyme pools was identical, being about 30,000 by gel filtration in 6 M urea and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No distinct differences were found between the two pools in amino acid composition, specific activity, or the use of various substrates. Two antisera were prepared, one to the total enzyme protein (pools I and II) and the other to pool I. Both antisera inhibited and precipitated crude placental lysyl oxidase, the two enzyme pools, and crude human skin fibroblast enzyme, there being no differences between the various enzyme forms. Both antisera also stained the two enzyme pools in immunoblotting of denatured proteins. The data suggest that there are no major catalytic, molecular, or immunological differences between the multiple forms of human lysyl oxidase. An antiserum prepared to any of the enzyme forms can, therefore, probably be used to study the total enzyme protein.     

Histone H1 is a substrate for lysyl oxidase and contains endogenous sodium borotritide-reducible residues

Incubation of purified bovine aortic lysyl oxidase with rat liver or calf thymus H1 histone results in the catalytic formation of hydrogen peroxide, indicating the substrate potential of H1 for this connective tissue enzyme. Sodium borotritide-reducible residues consistent with aminoadipic semialdehyde and the lysinonorleucine crosslinkage were generated in H1 by incubation with lysyl oxidase. H1 histone also contains endogenous reducible functions including an unidentified prominent tritiated peak eluting near tyrosine as well as other lesser peaks, one of which is consistent with lysinonorleucine.     

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.     

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 coupled with catalase in egg shell membrane.

The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2).