Update Properties and Function of Lysyl Oxidase Herbert M. Kagan and Philip C. Trackman Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts
Lysyl oxidase catalyzes the oxidation of peptidyllysine to o-arninoadipic-d-semialdehyde, the precursor to the covalent crosslinkages that stabilize fibers of elastin and collagen. This enzyme contains both copper and a carbonyl cofactor consistent with an o-quinone. The proposed mechanism of action is derived from available kinetic and chemical data and also can account for mechanism-based inhibition of the enzyme by specific monoamines and diamines. Recent evidence for biosynthetic precursors and for the.regulation of lysyl oxidase in fibrotic and malignant diseases is discussed.
Elastin and collagen are examples of proteins whose formation and maturation are critically dependent upon post-translational modifications. Thus, collagen is post-translationally processed by several different catalysts to cleave signal peptides, hydroxylate proline and lysine residues, glycosylate hydroxylysines, cleave register peptides, and oxidize lysine residues. The latter modification, catalyzed by lysyl oxidase (EC 1.4.3.13), generates peptidyl o-aminoadipic-e-semialdehyde in collagen as well as elastin. The aldehyde residue can spontaneously condense with neighboring aldehydes or e-amino groups to form interchain or intrachain covalent crosslinkages, two of which are shown (Figure 1). Continuous, intermolecular condensations of this kind will then convert soluble monomers of elastin or collagen into insoluble fibers in the extracellular matrix. Thus, lysyl oxidase can potentially regulate the development and repair of the matrix in lung and other connective tissues. The present review will summarize findings appearing since a previous detailed review on the properties and regulation of this catalyst (1). Purification and Molecular Weight Prior to the finding that the bulk of tissue lysyl oxidase was rendered soluble by 4 to 6 M urea extraction buffers (2), the purification of this enzyme had been a difficult task that often yielded preparations significantly contaminated by other proteins (1). Varying molecular weights had also been reported, with values ranging from approximately 30 kD to greater than 100 kD (1). Urea-extractable enzyme has since (Received in original form May 6, 1991 and in revised form June 14, 1991) Address correspondence to: Herbert M. Kagan, Ph.D., Department of Biochemistry, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118. Abbreviations: l3-aminopropionitrile, BAPN; cis-l,2-diaminocyclohexane, cis-DACH; dissociation constant, K/; pyridoxal phosphate, PLP; pyrroloquinoline quinone (methoxatin), PQQ; trihydroxyphenylalanine, TOPA. Am. J. Respir, Cell Mol. BioI. Vol. 5. pp, 206-210, 1991
been highly purified from bovine aorta and lung (3, 4), human placenta (5), and rat lung (6), yielding Mr values of 30 to 32 kD in sodium dodecyl sulfate. Lysyl oxidase species identified by Western blotting of urea extracts of various human tissues also have molecular masses approximating 30 kD (7). A recently modified version of current purification methods induced polymerization of the enzyme by removing urea from the urea-extracted preparation. The polymerized enzyme was thereby retained at the top of a gel filtration column. After washing with urea-free buffer, column-bound enzyme was disaggregated and eluted with urea-supplemented buffer, resulting in a high degree of purification (8). Cofactors Studies with purified preparations of lysyl oxidase have confirmed that copper is essential to the expression of enzyme activity (1, 9, 10). The highly purified enzymes of chick bone (9) and bovine aorta (10) contained 1 g-atom of Cu per 32 kl), whereas the metal-free enzymes are catalytically inert. Although the enzyme also clearly requires a functional carbonyl cofactor, its chemical identity has not been unequivocally established. Early investigations involving the inhibition of collagen insolubilization by carbonyl reagents in vivo or using pyridoxine-deficient diets (1, 11) as well as spectral analyses of chick aorta lysyl oxidase (12) led to the hypothesis that pyridoxal phosphate (PLP) was the organic cofactor of lysyl oxidase. However, various analyses of the bovine aorta enzyme were completely negative for PLP (13). More recently, resonance Raman spectroscopy of an active site peptide of the bovine aortic enzyme implicated pyrroloquinoline quinone (methoxatin; PQQ) as the functional carbonyl (14), while it was also reported that PQQ was found in a proteolytic digest of human placentallysyl oxidase (15). Although PQQ had previously been reported to be the cofactor of one other mammalian enzyme, i.e., plasma amine oxidase (16, 17), a more recent analysis contradicted the presence of PQQ and, instead, provided strong support
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The search for specific and highly potent inhibitors of lysyl oxidase remains an important goal in view of the potential of such compounds as antifibrotic agents. Clearly, this catalyst would seem to be a reasonable chemotherapeutic target for such agents in view of its accessibility in the extracellular compartment and since excess collagen molecules are degraded if they are not insolubilized by crosslinking into accreting collagen fibers. A variety of in vivo studi~s. supp.ort the feasibility of this approach. For example, administration of {j-aminopropionitrile (BAPN) , an irreversible inhibitor of lysyl oxidase, has been shown to limit the fibrotic response following certain surgical procedures and to limit collagen deposition in models of lung fibrosis (1). As insights into the mechanism of action of lysyl oxidase have been gained, various molecular candidates have been tested as active site-directed inhibitors, some of which behave as mechanism-based, irreversible enzyme inhibitors. Examples include {j-haloethylamines and {j-nitroethylamine, which appear to undergo catalytic transformations leading to forms that stably derivatize and thus inactivate the enzyme (23). The dissociation constants (K,) of these agents are similar to that for BAPN (K, = 5 JlM), with K, values in the 6- to 10-JlM range. Irreversible inhibition also develops with BAPN (1), possibly because of similar enzyme processing of the bound inhibitor that le~ds to covalent ~~rivati~a tion. Benzylamines substituted In the para position WIth electron-withdrawing functions behave as ground-state inhibitors of lysyl oxidase presumably by forming enzymebound intermediates that are not completely processed to the aldehyde (24). More recently, it has been found that vicinal diamines such as cis-l,2-diaminocyclohexane (cis-DACH) and ethylenediamine are potent, irreversible inhibitors of lysyl oxidase (25). Analysis of the inhibition mechanism by these diamines indicates that they derivatize the o-quinone carbony1cofactor through both of their amino groups to form fully conjugated and thus highly stable six-membered rings composed of two carbons of an o-quinone cofactor and two carbons and two nitrogens of the diamine (25). Indeed, cisDACH proved to be the most potent irreversible inhibitor of lysyl oxidase found thus far (K, = 0.4 JlM) (25).
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Figure 1. Role oflysyl oxidase in crosslinkage formatio? AAS = o-aminoadipic-d-semialdehyde; deLNL = dehydroly.smonorleucine, a Schiff base crosslink; ACP = aldol condensation product crosslink. More complex crosslinks can arise from the bifunctional ACP and deLNL crosslinks in elastin and collagen.
for the presence of the quinone of peptidy1trihydroxypheny1alanine (TOPA) at the active site of this enzyme (18). A~ though correspondingly detailed analyses of the lysyl OXIdase cofactor have yet to be done, it is of some interest that PQQ-deficient rats develop growth anomalies and lathrytic symptoms, while the levels of lysyl oxidase protein and.activity were reduced (19). Feeding of PQQ to ~ese deficI~nt animals tended to reverse these effects (19), again suggestive of a role for this o-quinone in the function of lysyl oxidase. Mechanism of Action Steady-state kinetic analyses (20) as well as chemical approaches (21) indicate that lysyl oxidase fol~ow~ a ping-pong kinetic course with the order of substrate binding and product release as shown in Figure 2. A chemical mechanism following this kinetic pattern and that assumes a PQQ-like, o-quinone cofactor has been proposed in which the amine substrate is initially oxidized to the aldehyde by passage of two electrons derived from the a-carbon of the substrate into the enzyme-linked, carbonyl cofactor. The reduced enzyme species can then be reoxidized to the initial, catal~tically competent form upon the binding of oxygen to which two electrons are passed to form and release hydrogen peroxide (20, 21). The role of copper in this mechanism has yet to be ascertained. However, recent studies indicate that the metalfree apoenzyme cannot catalyze the first half-reaction in which the aldehyde is formed and released either in the presence or absence of oxygen (10). Thus, the presence of the metal ion is critical at least to the first half-reaction. It is possible that copper stabilizes a catalytically competent conformation and/or may align the carbonyl cofactor and the amine
Figure 2. Order of substrate binding and product release in lysyl oxidase catalysis. Possible enzyme intermediates are symbolically represented below the line.
Biosynthesis, Processing, and Secretion Insights into the nature ofbiosynthetic forms of lysyl oxidase and the amino acid sequence of the enzyme have recently
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
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skin fibroblasts was inhibited by monensin or nigericin, ionophores known to inhibit secretion of proteins that are processed through the Golgi pathway (29). These investigators also noted that matrix-bound enzyme activity decreased in parallel with that in the medium in monensin-treated cultures, suggesting that this bound form may be in equilibrium with the soluble catalyst in the medium.
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been gained by the sequencing of lysyl oxidase cDNA isolated from a rat aortic cDNA library (26), a summary representation of which is shown (Figure 3). This 2,678-bp cDNA consisted of 286 bp of partial 5'- and 1,159 bp of partial 3'-untranslated sequences flanking a 1,233-bp region coding for a 411-amino acid, 46.6-kD protein species. A putative signal peptide sequence exists at the N-terminal region of this species consistent with the secretory fate of lysyl oxidase. Proteolytic removal of the signal peptide by cleavage between Cys21 and Ala22 would yield a44.5-kD protein at the C-terminal region of which sequences corresponding to those of peptides isolated from the 32-kD active bovine aortic enzyme are found. The likelihood that such a larger species represents a lysyI oxidase precursor was supported by the specific immunoprecipitation of a 46- to 48-kD cellfree translation product of bovine smooth muscle cell mRNA by rabbit anti-Iysyl oxidase in this report (26) and by the finding of a 48-kD cell-free translation product of mRNA isolated from fibrotic rat liver (27). Moreover, monoclonal anti-human lysyl oxidase reacted with a 48-kD, enzymatically inactive peak resolved by gel exclusion chromatography of extracts of human placenta (28). The predicted amino acid sequence between residues 311 and 400 is consistent with known metal binding sequences in certain copper metalloproteins and occurs within the region accounting for the functional 32-kD enzyme. The putative precursor also contains Arg-Arg-Arg and Arg-Arg sequences at residues 62 through 64 and at 134 and 135, respectively. These polybasic sequences are potential proteolytic processing sites whereby cleavage at residues 62 through 64 would yield a 39.2-kD species, while cleavage at 134 and 135 would yield the 31.9kD enzyme (Figure 3). Using antibody raised against the purified 32-kD bovine aortic enzyme, studies in progress in our laboratory have identified protein bands at 46 to 48, 38, and 32 kD specifically immunoprecipitated from fractions of rat aorta smooth muscle cells and rat lung fibroblasts pulselabeled in culture, consistent with these predictions. Two Asn-Arg-Thr sequences also appear in the predicted sequence (Figure 3), indicating the potential for N-glycosylation at both Asn residues. Our current studies also support the conclusion that the 46-kD precursor is N-glycosylated. Several reports have noted that lysyl oxidase is a secreted protein, as reviewed (1). More recently, Kuivaniemi and colleagues (29) found that the secretion of enzyme by human
Localization in Tissues and Cells Enzyme was localized at the electron microscope level in the extracellular matrix of rat lung and aorta employing antilysyl oxidase enzyme and second antibody complexed with colloidal gold (30). The electron-dense particles were seen in association with the microfibrillar network surrounding elastic fibers and within the amorphous elastic fibers, themselves. However, using the same technique, others have reported relatively sparse association of gold deposits on matrix microfibrils but distinct deposits on and within elastin and collagen fibers of human placenta, skin, and aorta (31). The microfibrillar network appears in the extracellular space prior to the formation of elastic fibers and then appears to serve as a scaffolding within which amorphous elastin is deposited (32). These structures persist as a filamentous network largely on the perimeter and, to a lesser degree, within the developing elastic fiber. The apparent association oflysyl oxidase with this microfibrillar material implies that the enzyme may function at such sites to crosslink new tropoelastin units to the radially accreting elastic fiber. Possible mechanisms by which the appropriate intermolecular associations are achieved, to properly align tropoelastin, lysyl oxidase, and the microfibrillar scaffolding in extracellular space are yet to be discerned. As an additional component to be considered in such interactions, Fomieri and associates (33) reported that administration of lysyl oxidase inhibitors to developing chicks and rats resulted in the appearance of unusual, lateral aggregates of aortic elastic fibers that were permeated by glycosaminoglycans. It was postulated that polyanionic glycosaminoglycans might pair with charged s-amino groups oftropoelastin thus contributing to appropriate intermolecular relationships prior to the crosslinking process. In view of the evidence that lysyl oxidase functions as an extracellular enzyme, it is of interest that lysyI oxidase was localized along cytoskeletal structures within cultured human fibroblasts by fluorescence immunohistochemistry (34). The apparent predominance of the staining reaction at such loci warrants further investigation into the possible biologic significance of this observation. Lysyl Oxidase in Lung Disease The critical role played by lysyl oxidase in the development and homeostasis of the lung is reflected in the disruption of lung structure in chick and rat models of dietary copper deficiency (35, 36). Markedly decreased levels of lysyl oxidase seen in these models were consistent with the accompanying reduction in insoluble lung elastin, dilation of the airways, and increased level of soluble collagen (9, 35). Tinker and co-workers (37) demonstrated that copper-deficient diets result in both a decrease in crosslinking activity and an increase in elastin degradation in chick aorta while restoration of copper restores normal crosslinking and imparts resistance to elastolyis. Thus, the activity of lysyl oxi-
Update
dase appears essential to the accumulation as well as the prevention of loss of this connective tissue protein. Induction of lung fibrosis by exposure to agents such as bleomycin and cadmium is accompanied by large increases in lung lysyl oxidase activity (1). The elevated enzyme activity in cadmium-exposed rat lung was recently shown to reflect increased enzyme protein (6). The newly formed, cadmium-induced enzyme resolved into two major and additional minor peaks of activity upon elution from DEAETrisacryl by a salt gradient in 6 M urea (6), thus exhibiting the ionic heterogeneity that is characteristic of the highly purified enzymes of various bovine, chick, and human tissues (1). Although the basis of this phenomenon is unknown, this result suggests that it may occur in coincidence with enzyme synthesis. In contrast to the stimulation of lysyl oxidase in the lung by cadmium exposure, bone lysyl oxidase activity decreases in cadmium-fed chicks apparently because of the replacement of copper in the enzyme by cadmium (38). It is likely that the opposite effects on lysyl oxidase activities seen in lung and bone reflect the marked increase in metallothionein which occurs in the lung model, whereas this metal-scavenging protein apparently does not appear to be synthesized in response to cadmium in the bone (38). The activation of lung connective tissue protein formation in response to toxic agents could stem from factors released upon injury to lung cells or from fibrogenic factors introduced into the lung by infiltrating macrophages and neutrophils. One such mediator has been implicated in the development of fibrosis in silicotic rat lungs (39) in which lysyl oxidase and other enzymes of collagen biosynthesis are elevated (40). Thus, a 16-kD protein isolated from silicotic rat lungs and apparently derived from macrophages was found to stimulate collagen synthesis in granulation tissue cells at a concentration of 0.1 nM. Its effect on lysyl oxidase expression does not appear to have been determined. The first evidence for the regulation oflysyl oxidase at the genetic level in a fibrotic model stems from the 3-fold increase in functional lysyl oxidase mRNA activity seen in cell-free translations of fibrotic rat liver mRNA (27). Additional insights into the molecular levels of regulation should be forthcoming in view of the recent description of the sequence of lysyl oxidase cDNA (26). Genetic Disease As previously reviewed, levels oflysyl oxidase are markedly decreased in Menkes' disease and X-linked cutis laxa (1). A recent report describes an unusually severe case of Menkes' in which skin fibroblast lysyl oxidase levels were only 6 to 12 % of controls (41), a level that clearly can account for the severe connective tissue pathology associated with this disease. The enzyme deficiency in both of these diseases appears to be secondary to disturbances in copper metabolism, however. Other studies implying deficiencies oflysyl oxidase in osteogenesis imperfecta and Marfan's syndrome have recently been contradicted (42). Aortic lysyl oxidase activity is decreased by 30 to 50 % in the Brown Norway strain of rats that are genetically susceptible to disruption of the internal elastic lamina (43). Coupled with the apparent increase in aortic elastolytic activity in this strain, the decrease in lysyl oxidase could contribute to the manifestations of this
209
syndrome. It should be noted, however, that decreases of lysyl oxidase activity of these magnitudes may not result in deficient crosslinking, in view of the observation that the 64 % reduction of enzyme activity in the skin of glucocorticoid-treated rats was accompanied by an apparent increase in collagen crosslinking (44). Biologic Regulation of Lysyl Oxidase Lysyl oxidase activity responds to changes in levels of certain hormones, as illustrated by the estrogen-induced increase in mouse cervicallysyl oxidase activity (45). The addition of 10- to 100-nM levels of testosterone to the medium of cultured aortic smooth muscle cells increased cell layer lysyl oxidase activity by 5.5-fold (46). This effect is of interest in view of the greater tendency of males to develop arterial lesions that can become fibrotic with the progression of atherosclerosis. It has been commonly observed that the expression of lysyl oxidase by cells in culture is most active when proliferation has stopped or slowed, consistent with the most active stage of matrix production. It is of particular interest in this regard that lysyl oxidase activity as well as rates of collagen synthesis were markedly reduced in malignantly transformed human cell lines (47). A further intriguing aspect of the relationship between lysyl oxidase and tumorigenesis has recently emerged. Thus, the normal expression of gene rrg transcript in mouse NIH 3T3 cells is markedly downregulated by c-H-ras-induced transformation of these cells to the tumorigenic RS485 cell line (48). High levels of rrg expression were restored by interferon-induced reversion of RS485 cells to the nontumorigenic PR4 cell line. The specific importance of rrg to the reversion was emphasized by the fact that the normally persistent revertants developed the transformed phenotype when stably transfected with rrg antisense cDNA (48). Since the appearance of this report, it has been learned that rrg is 92 % homologous at the cDNA sequence level and ~ 98 % homologous at the predicted protein sequence level to rat aorta lysyl oxidase (49). It is not known at this writing whether the rrg protein is catalytically active or iflysyl oxidase catalysis is involved in the reversion ofthe tumorigenic state. However, these intriguing results do indicate that a lysyl oxidase-like protein may playa critical role in the maintenance of the nontumorigenic state in these fibroblast lines. Summary and Prospects Progress has been made on several aspects of the enzymology and biology oflysyl oxidase. While the precise chemical identity of the organic cofactor requires further investigation, this moiety exhibits the reactivity of an o-quinone and thus is consistent with a PQQ- or TOPA-like residue. The catalytic mechanism of this enzyme clearly parallels those proposed for other mammalian copper-dependent amine oxidases. As further details of the active site structures of these proteins become available, structural homologies in active site regions may also be found, although it should be noted that the sequence of the TOPA-bearing active site peptide of plasma amine oxidase is not seen in the lysyl oxidase protein sequence. Recent insights into the secretory mechanism and the biosynthetic precursor make it likely that detailed information about the biosynthesis and processing of this impor-
210
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
tant enzyme will soon be forthcoming. In addition, evidence pointing to an unexpected intracellular localization and the recent results implying a relationship between lysyl oxidase and tumorigenesis certainly raise important new prospects for future research. Acknowledgments: Work from the writers' laboratory reviewed here is supported by Grants R37-AM-18880, HL-13262, and HL-19717from the National Institutes of Health.
References 1. Kagan, H. M. 1986. Characterization and regulation of lysyl oxidase. In Biology of Extracellular Matrix: A Series. Regulation of Matrix Accumulation. Vol. 1. R. P. Mecham, editor. Academic Press, Orlando, FL. 321-398. 2. Narayanan, A. S., R. C. Siegel, and G. R. Martin. 1974. Stability and purification of lysyl oxidase. Arch. Biochem. Biophys. 162:231-237. 3. Kagan, H. M., K. A. Sullivan, T. A. Olsson, and A. L. Cronlund. 1979. Purification and properties of four species of lysyl oxidase from bovine aorta. Biochem. J. 177:203-214. 4. Cronlund, A. L., and H. M. Kagan. 1986. Comparison of lysyl oxidase of bovine aorta and lung. Connect. Tissue Res. 15: 173-185. 5. Kuivaniemi, H., E. R. Savolainen, and K. I. Kivirikko. 1984. Human placentallysyl oxidase. Purification, partial characterization and preparation of two specific antisera to the enzyme. J. Bioi. Chem. 259:69967003. 6. Alrnassian, B. J., P. C. Trackman, H. Iguchi, A. Boak, D. Calvaresi, and H. M. Kagan. 1990. Induction oflung lysyl oxidase activity and lysyl oxidase protein by exposure of rats to cadmium chloride: properties of the induced enzyme. Connect. Tissue Res. 25:197-208. 7. Kuivaniemi, H. 1985. Partial characterization of lysyl oxidase from several human tissues. Biochem. J. 230:639-641. 8. Shackleton, D. R., and D. J. S. Hulmes. 1990. Purification oflysyl oxidase from piglet skin by selective interaction with Sephacryl S-200. Biochem. J. 266:917-919. 9. Iguchi, H., and S. Sano. 1985. Cadmium- or zinc-binding to bone lysyl oxidase and copper replacement. Connect. Tissue Res. 14:129-139. 10. Gacheru, S. N., P. C. Trackman, M. A. Shah et al. 1990. Structural and catalytic properties of copper in Iysyl oxidase. J. Bioi. Chem. 265: 19022-19027. 11. Levene, C. I., M. P. O'Shea, and C. J. Carrington. 1988. Protein lysine 6-oxidase (lysyl oxidase) cofactor: methoxatin (PQQ) or pyridoxal? Int. J. Biochem. 20:1451-1456. 12. Bird, T. A., and C. I. Levene. 1982. Lysyl oxidase: evidence that pyridoxal phosphate is a cofactor. Biochem. Biophys. Res. Commun. 108:1172-1180. 13. Williamson, P. R., J. M. Kittler, J. W. Thanassi, and H. M. Kagan. 1986. Reactivity of a functional carbonyl moiety in bovine lysyl oxidase. Evidence against pyridoxal 5 '-phosphate. Biochem. J. 235 :597-605. 14. Myers, B. A., M. A. Dubick, R. D. Reynolds, and R. B. Rucker. 1985. Effect of vitamin B-6 (pyridoxine) deficiency on lung elastin cross-linking in perinatal and weanling rat pups. Biochem. J. 229:153-160. 15. Myers, B. A., M. A. Dubick, 1. E. Gerriets, K. M. Reiser, 1. A. Last, and R. B. Rucker. 1986. Lung collagen and elastin after ozone exposure in vitamine B6-deficient rats. Ioxicol. Lett. 30:55-61. 16. Ameyama, M., M. Hayashi, K. Matsushita, E. Shinagawa, and O. Adachi. 1984. Microbial production of pyrroloquinoline quinone. Agricultural and Biological Chemistry 48:561-565. 17. Lobenstein-Verbeek, C. L., 1. A. Jonjegan, 1. Frank, and 1. A. Duine. 1984. Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as a prosthetic group. FEBS Lett. 170:305-309. 18. Williamson, P. R., R. S. Moog, D. M. Dooley, and H. M. Kagan. 1986. Evidence for pyrroloquinoline quinone as the carbonyl cofactor in lysyl oxidase by absorption and resonance Raman spectroscopy. J. Biol. Chem. 261:16302-16305. 19. Van der Meer, R. A., and 1. A. Duine. 1986. Covalently bound pyrroloquinoline quinone is the organic prosthetic group in human placentallysyl oxidase. Biochem. J. 239:789-791. 20. Killgore. L, C. Smidt, L. Duich et al. 1989. Nutritional importance of pyrroloquinoline quinone. Science 245:850-852. 21. Janes, S. M., D. Mu, D. Wemmer et al. 1990. A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 248:981-987. 22. Williamson, P. R., andH. M. Kagan. 1986. Reactionpathwayofbovineaortic lysyl oxidase. J. BioI. Chem. 261:9477-9482. 23. Williamson, P. R., and H. M. Kagan. 1987. a-Proton abstraction and carbanion formation in the mechanism of action of lysyl oxidase. J. Bioi.
Chem. 262: 8196-8201. 24. Dooley, D. M., M. A. McGuirl, D. E. Brown, P. N. Turowski, W. S. McIntire, and P. F. Knowles. 1991. Evidence for a Cu(l)-semiquinone state in substrate-reduced amine oxidases. Nature 349:262-264. 25. Tang, S. S., D. E. Simpson, and H. M. Kagan. 1984. is-quinone-directed irreversible inhibitors of lysyl oxidase. J. Biol. Chem. 264:12963-12969. 26. Trackman, P. c., A. M. Pratt, A. Wolanski et al. 1990. Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence. Biochemistry 29:4863-4870. 27. Wakasaki, H., and A. Ooshima. 1990. Synthesis of lysyl oxidase in experimental hepatic fibrosis. Biochem. Biophys. Res. Commun. 166: 1201-1204. 28. Burbelo, P. D., A. Monckeberg, and C. 0. Chichester. 1986. Monoclonal antibodies to human lysyl oxidase. Collagen and Related Research 6:153-162. 29. Kuivaniemi, H., L. Ala-Kokko, and K. I. Kivirikko. 1986. Secretion oflysyl oxidase by cultured human skin fibroblasts and effectsof monensin, nigericin, tunicamycin and colchicine, Biochim. Biophys. Acta 883:326-334. 30. Kagan, H. M., C. A. Vaccaro, R. E. Bronson, S. S. Tang, and 1. S. Brody. 1986. Ultrastructural immunolocalization oflysyl oxidase in vascular connective tissue. J. Cell Bio!. 103:1121-1128. 31. Baccarani-Contri, M., D. Vincenzi, D. Quaglino, G. Mori, and I. PasqualiRonchetti. 1990. Localization of human placenta lysyl oxidase on human placenta, skin and aorta by immunoelectron-microscopy, Matrix 9:428-436. 32. Cleary, E. G. 1987. The microfibrillar component of elastic fibers. Morphology and biochemistry. In Connective Tissue Disease. Molecular Pathology ofthe Extracellular Matrix. 1. Uitto and A. J. Perejda, editors. Marcel Dekker, New York. 55-81. 33. Fomieri, c., M. Baccarani-Contri, D. Quaglino, and I. Pasquali-Ronchetti. 1987. Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas. J. Cell BioI. 105:1463-1469. 34. Wakasaki, H., and A. Ooshima. 1990. Immunohistochemical localization of lysyl oxidase with monoclonal antibodies. Lab. Invest. 63:377-384. 35. Harris, E. D. 1986. Biochemical defect in chick lung resulting from copper deficiency. J. Nutr. 116:252-258. 36. Dubick, M. A., C. L. Keen, and R. B. Rucker. 1985. Elastin metabolism during perinatal lung development in the copper-deficient rat: Exp. Lung Res. 8:227-241. 37. Tinker, D., N. Romero-Chapman, K. Resier, D. Hyde, and R. Rucker. 1990. Elastin metabolism during recovery from impaired crosslink formation. Arch. Biochem. Biophys. 278:326-332. 38. Hirano, S., N. Tsukamoto, and K. T. Suzuki. 1990. Biochemical changes in the rat lung and liver following intratracheal instillation of cadmium oxide. Toxicol. Lett. 50:97-105. 39. Aalto, M., E. Kulonen, and J. Pikkarainen. 1989. Isolation of silicadependent protein from rat lung with special reference to development of fibrosis. Br. J. Exp. Pathol. 70:167-182. 40. Poole, A., R. Myllyla, J. C. Wagner, and R. C. Brown. 1985. Collagen biosynthesis enzymes in lung tissue and serum of rats with experimental silicosis. Br. J. Exp. Pathol. 66:567-575. 41. Royce, P. M., and B. Steinmann. 1990. Markedly reduced activity oflysyl oxidase in skin and aorta from a patient with Menkes' disease showing unusually severe connective tissue manifestations. Pediat. Res. 28: 136-141. 42. Royce, P. M., and B. Steinmann. 1988. Lysyl oxidase in osteogenesis imperfecta and Marfan's syndrome. Collagen and Related Research 8: 183-184. 43. Osborne-Pellegrin, M. J., J. Farjanei, and W. Homebeck. 1'990. Role of elastase and lysyl oxidase activity in spontaneous rupture of internal elastic lamina in rats. 1990. Arteriosclerosis 10:1136-1146. 44. Counts, D. F., S. Shull, and K. Cutroneo. 1986. Skin lysyl oxidase is not rate limiting for collagen crosslinking in the glucocorticoid-treated rat. Connect. Tissue Res. 14:237-241. 45. Ozasa, H., T. Tominaga, and T. Takeda. 1986. Evidence of estrogen-like effect of dehydroepiandrosterone on lysyl oxidase activity in the mouse cervix. Acta Obstet. Gynecol. Scand. 65:543-545. 46. Bronson, R. E., S. D. Calaman, A. M. Traish, and H. M. Kagan. 1987. Stimulation of lysyl oxidase activity by testosterone and characterization of androgen receptors in cultured calf aorta smooth muscle cells. Biochem. J. 244:317-323. 47. Kuivaniemi, H., R. M. Korhonen, A. Vaheri, and K. I. Kivirikko. 1986. Deficient production of lysyl oxidase in cultures of malignantly transformed human cells. FEBS Lett. 195:261-264. 48. Contente, S., K. Kenyon, D. Rimoldi, and R. F. Friedman. 1990. Expression of gene rrg is associated with reversion of NIH 3T3 transformed by LTR-c-H-ras. Science 249:796-798. 49. Kenyon, K., S. Contente, P. C. Trackman, J. Tang, H. M. Kagan, and R. F. Friedman. 1991. A suppressor of the phenotypic expression of ras encodes lysyl oxidase. Science. In press.
Lysyl oxidase catalyzes the oxidation of peptidyllysine to o-arninoadipic-d-semialdehyde, the precursor to the covalent crosslinkages that stabilize fibers of elastin and collagen. This enzyme contains both copper and a carbonyl cofactor consistent with an o-quinone. The proposed mechanism of action is derived from available kinetic and chemical data and also can account for mechanism-based inhibition of the enzyme by specific monoamines and diamines. Recent evidence for biosynthetic precursors and for the.regulation of lysyl oxidase in fibrotic and malignant diseases is discussed.
Elastin and collagen are examples of proteins whose formation and maturation are critically dependent upon post-translational modifications. Thus, collagen is post-translationally processed by several different catalysts to cleave signal peptides, hydroxylate proline and lysine residues, glycosylate hydroxylysines, cleave register peptides, and oxidize lysine residues. The latter modification, catalyzed by lysyl oxidase (EC 1.4.3.13), generates peptidyl o-aminoadipic-e-semialdehyde in collagen as well as elastin. The aldehyde residue can spontaneously condense with neighboring aldehydes or e-amino groups to form interchain or intrachain covalent crosslinkages, two of which are shown (Figure 1). Continuous, intermolecular condensations of this kind will then convert soluble monomers of elastin or collagen into insoluble fibers in the extracellular matrix. Thus, lysyl oxidase can potentially regulate the development and repair of the matrix in lung and other connective tissues. The present review will summarize findings appearing since a previous detailed review on the properties and regulation of this catalyst (1). Purification and Molecular Weight Prior to the finding that the bulk of tissue lysyl oxidase was rendered soluble by 4 to 6 M urea extraction buffers (2), the purification of this enzyme had been a difficult task that often yielded preparations significantly contaminated by other proteins (1). Varying molecular weights had also been reported, with values ranging from approximately 30 kD to greater than 100 kD (1). Urea-extractable enzyme has since (Received in original form May 6, 1991 and in revised form June 14, 1991) Address correspondence to: Herbert M. Kagan, Ph.D., Department of Biochemistry, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118. Abbreviations: l3-aminopropionitrile, BAPN; cis-l,2-diaminocyclohexane, cis-DACH; dissociation constant, K/; pyridoxal phosphate, PLP; pyrroloquinoline quinone (methoxatin), PQQ; trihydroxyphenylalanine, TOPA. Am. J. Respir, Cell Mol. BioI. Vol. 5. pp, 206-210, 1991
been highly purified from bovine aorta and lung (3, 4), human placenta (5), and rat lung (6), yielding Mr values of 30 to 32 kD in sodium dodecyl sulfate. Lysyl oxidase species identified by Western blotting of urea extracts of various human tissues also have molecular masses approximating 30 kD (7). A recently modified version of current purification methods induced polymerization of the enzyme by removing urea from the urea-extracted preparation. The polymerized enzyme was thereby retained at the top of a gel filtration column. After washing with urea-free buffer, column-bound enzyme was disaggregated and eluted with urea-supplemented buffer, resulting in a high degree of purification (8). Cofactors Studies with purified preparations of lysyl oxidase have confirmed that copper is essential to the expression of enzyme activity (1, 9, 10). The highly purified enzymes of chick bone (9) and bovine aorta (10) contained 1 g-atom of Cu per 32 kl), whereas the metal-free enzymes are catalytically inert. Although the enzyme also clearly requires a functional carbonyl cofactor, its chemical identity has not been unequivocally established. Early investigations involving the inhibition of collagen insolubilization by carbonyl reagents in vivo or using pyridoxine-deficient diets (1, 11) as well as spectral analyses of chick aorta lysyl oxidase (12) led to the hypothesis that pyridoxal phosphate (PLP) was the organic cofactor of lysyl oxidase. However, various analyses of the bovine aorta enzyme were completely negative for PLP (13). More recently, resonance Raman spectroscopy of an active site peptide of the bovine aortic enzyme implicated pyrroloquinoline quinone (methoxatin; PQQ) as the functional carbonyl (14), while it was also reported that PQQ was found in a proteolytic digest of human placentallysyl oxidase (15). Although PQQ had previously been reported to be the cofactor of one other mammalian enzyme, i.e., plasma amine oxidase (16, 17), a more recent analysis contradicted the presence of PQQ and, instead, provided strong support
Update
207
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The search for specific and highly potent inhibitors of lysyl oxidase remains an important goal in view of the potential of such compounds as antifibrotic agents. Clearly, this catalyst would seem to be a reasonable chemotherapeutic target for such agents in view of its accessibility in the extracellular compartment and since excess collagen molecules are degraded if they are not insolubilized by crosslinking into accreting collagen fibers. A variety of in vivo studi~s. supp.ort the feasibility of this approach. For example, administration of {j-aminopropionitrile (BAPN) , an irreversible inhibitor of lysyl oxidase, has been shown to limit the fibrotic response following certain surgical procedures and to limit collagen deposition in models of lung fibrosis (1). As insights into the mechanism of action of lysyl oxidase have been gained, various molecular candidates have been tested as active site-directed inhibitors, some of which behave as mechanism-based, irreversible enzyme inhibitors. Examples include {j-haloethylamines and {j-nitroethylamine, which appear to undergo catalytic transformations leading to forms that stably derivatize and thus inactivate the enzyme (23). The dissociation constants (K,) of these agents are similar to that for BAPN (K, = 5 JlM), with K, values in the 6- to 10-JlM range. Irreversible inhibition also develops with BAPN (1), possibly because of similar enzyme processing of the bound inhibitor that le~ds to covalent ~~rivati~a tion. Benzylamines substituted In the para position WIth electron-withdrawing functions behave as ground-state inhibitors of lysyl oxidase presumably by forming enzymebound intermediates that are not completely processed to the aldehyde (24). More recently, it has been found that vicinal diamines such as cis-l,2-diaminocyclohexane (cis-DACH) and ethylenediamine are potent, irreversible inhibitors of lysyl oxidase (25). Analysis of the inhibition mechanism by these diamines indicates that they derivatize the o-quinone carbony1cofactor through both of their amino groups to form fully conjugated and thus highly stable six-membered rings composed of two carbons of an o-quinone cofactor and two carbons and two nitrogens of the diamine (25). Indeed, cisDACH proved to be the most potent irreversible inhibitor of lysyl oxidase found thus far (K, = 0.4 JlM) (25).
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Figure 1. Role oflysyl oxidase in crosslinkage formatio? AAS = o-aminoadipic-d-semialdehyde; deLNL = dehydroly.smonorleucine, a Schiff base crosslink; ACP = aldol condensation product crosslink. More complex crosslinks can arise from the bifunctional ACP and deLNL crosslinks in elastin and collagen.
for the presence of the quinone of peptidy1trihydroxypheny1alanine (TOPA) at the active site of this enzyme (18). A~ though correspondingly detailed analyses of the lysyl OXIdase cofactor have yet to be done, it is of some interest that PQQ-deficient rats develop growth anomalies and lathrytic symptoms, while the levels of lysyl oxidase protein and.activity were reduced (19). Feeding of PQQ to ~ese deficI~nt animals tended to reverse these effects (19), again suggestive of a role for this o-quinone in the function of lysyl oxidase. Mechanism of Action Steady-state kinetic analyses (20) as well as chemical approaches (21) indicate that lysyl oxidase fol~ow~ a ping-pong kinetic course with the order of substrate binding and product release as shown in Figure 2. A chemical mechanism following this kinetic pattern and that assumes a PQQ-like, o-quinone cofactor has been proposed in which the amine substrate is initially oxidized to the aldehyde by passage of two electrons derived from the a-carbon of the substrate into the enzyme-linked, carbonyl cofactor. The reduced enzyme species can then be reoxidized to the initial, catal~tically competent form upon the binding of oxygen to which two electrons are passed to form and release hydrogen peroxide (20, 21). The role of copper in this mechanism has yet to be ascertained. However, recent studies indicate that the metalfree apoenzyme cannot catalyze the first half-reaction in which the aldehyde is formed and released either in the presence or absence of oxygen (10). Thus, the presence of the metal ion is critical at least to the first half-reaction. It is possible that copper stabilizes a catalytically competent conformation and/or may align the carbonyl cofactor and the amine
Figure 2. Order of substrate binding and product release in lysyl oxidase catalysis. Possible enzyme intermediates are symbolically represented below the line.
Biosynthesis, Processing, and Secretion Insights into the nature ofbiosynthetic forms of lysyl oxidase and the amino acid sequence of the enzyme have recently
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skin fibroblasts was inhibited by monensin or nigericin, ionophores known to inhibit secretion of proteins that are processed through the Golgi pathway (29). These investigators also noted that matrix-bound enzyme activity decreased in parallel with that in the medium in monensin-treated cultures, suggesting that this bound form may be in equilibrium with the soluble catalyst in the medium.
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been gained by the sequencing of lysyl oxidase cDNA isolated from a rat aortic cDNA library (26), a summary representation of which is shown (Figure 3). This 2,678-bp cDNA consisted of 286 bp of partial 5'- and 1,159 bp of partial 3'-untranslated sequences flanking a 1,233-bp region coding for a 411-amino acid, 46.6-kD protein species. A putative signal peptide sequence exists at the N-terminal region of this species consistent with the secretory fate of lysyl oxidase. Proteolytic removal of the signal peptide by cleavage between Cys21 and Ala22 would yield a44.5-kD protein at the C-terminal region of which sequences corresponding to those of peptides isolated from the 32-kD active bovine aortic enzyme are found. The likelihood that such a larger species represents a lysyI oxidase precursor was supported by the specific immunoprecipitation of a 46- to 48-kD cellfree translation product of bovine smooth muscle cell mRNA by rabbit anti-Iysyl oxidase in this report (26) and by the finding of a 48-kD cell-free translation product of mRNA isolated from fibrotic rat liver (27). Moreover, monoclonal anti-human lysyl oxidase reacted with a 48-kD, enzymatically inactive peak resolved by gel exclusion chromatography of extracts of human placenta (28). The predicted amino acid sequence between residues 311 and 400 is consistent with known metal binding sequences in certain copper metalloproteins and occurs within the region accounting for the functional 32-kD enzyme. The putative precursor also contains Arg-Arg-Arg and Arg-Arg sequences at residues 62 through 64 and at 134 and 135, respectively. These polybasic sequences are potential proteolytic processing sites whereby cleavage at residues 62 through 64 would yield a 39.2-kD species, while cleavage at 134 and 135 would yield the 31.9kD enzyme (Figure 3). Using antibody raised against the purified 32-kD bovine aortic enzyme, studies in progress in our laboratory have identified protein bands at 46 to 48, 38, and 32 kD specifically immunoprecipitated from fractions of rat aorta smooth muscle cells and rat lung fibroblasts pulselabeled in culture, consistent with these predictions. Two Asn-Arg-Thr sequences also appear in the predicted sequence (Figure 3), indicating the potential for N-glycosylation at both Asn residues. Our current studies also support the conclusion that the 46-kD precursor is N-glycosylated. Several reports have noted that lysyl oxidase is a secreted protein, as reviewed (1). More recently, Kuivaniemi and colleagues (29) found that the secretion of enzyme by human
Localization in Tissues and Cells Enzyme was localized at the electron microscope level in the extracellular matrix of rat lung and aorta employing antilysyl oxidase enzyme and second antibody complexed with colloidal gold (30). The electron-dense particles were seen in association with the microfibrillar network surrounding elastic fibers and within the amorphous elastic fibers, themselves. However, using the same technique, others have reported relatively sparse association of gold deposits on matrix microfibrils but distinct deposits on and within elastin and collagen fibers of human placenta, skin, and aorta (31). The microfibrillar network appears in the extracellular space prior to the formation of elastic fibers and then appears to serve as a scaffolding within which amorphous elastin is deposited (32). These structures persist as a filamentous network largely on the perimeter and, to a lesser degree, within the developing elastic fiber. The apparent association oflysyl oxidase with this microfibrillar material implies that the enzyme may function at such sites to crosslink new tropoelastin units to the radially accreting elastic fiber. Possible mechanisms by which the appropriate intermolecular associations are achieved, to properly align tropoelastin, lysyl oxidase, and the microfibrillar scaffolding in extracellular space are yet to be discerned. As an additional component to be considered in such interactions, Fomieri and associates (33) reported that administration of lysyl oxidase inhibitors to developing chicks and rats resulted in the appearance of unusual, lateral aggregates of aortic elastic fibers that were permeated by glycosaminoglycans. It was postulated that polyanionic glycosaminoglycans might pair with charged s-amino groups oftropoelastin thus contributing to appropriate intermolecular relationships prior to the crosslinking process. In view of the evidence that lysyl oxidase functions as an extracellular enzyme, it is of interest that lysyI oxidase was localized along cytoskeletal structures within cultured human fibroblasts by fluorescence immunohistochemistry (34). The apparent predominance of the staining reaction at such loci warrants further investigation into the possible biologic significance of this observation. Lysyl Oxidase in Lung Disease The critical role played by lysyl oxidase in the development and homeostasis of the lung is reflected in the disruption of lung structure in chick and rat models of dietary copper deficiency (35, 36). Markedly decreased levels of lysyl oxidase seen in these models were consistent with the accompanying reduction in insoluble lung elastin, dilation of the airways, and increased level of soluble collagen (9, 35). Tinker and co-workers (37) demonstrated that copper-deficient diets result in both a decrease in crosslinking activity and an increase in elastin degradation in chick aorta while restoration of copper restores normal crosslinking and imparts resistance to elastolyis. Thus, the activity of lysyl oxi-
Update
dase appears essential to the accumulation as well as the prevention of loss of this connective tissue protein. Induction of lung fibrosis by exposure to agents such as bleomycin and cadmium is accompanied by large increases in lung lysyl oxidase activity (1). The elevated enzyme activity in cadmium-exposed rat lung was recently shown to reflect increased enzyme protein (6). The newly formed, cadmium-induced enzyme resolved into two major and additional minor peaks of activity upon elution from DEAETrisacryl by a salt gradient in 6 M urea (6), thus exhibiting the ionic heterogeneity that is characteristic of the highly purified enzymes of various bovine, chick, and human tissues (1). Although the basis of this phenomenon is unknown, this result suggests that it may occur in coincidence with enzyme synthesis. In contrast to the stimulation of lysyl oxidase in the lung by cadmium exposure, bone lysyl oxidase activity decreases in cadmium-fed chicks apparently because of the replacement of copper in the enzyme by cadmium (38). It is likely that the opposite effects on lysyl oxidase activities seen in lung and bone reflect the marked increase in metallothionein which occurs in the lung model, whereas this metal-scavenging protein apparently does not appear to be synthesized in response to cadmium in the bone (38). The activation of lung connective tissue protein formation in response to toxic agents could stem from factors released upon injury to lung cells or from fibrogenic factors introduced into the lung by infiltrating macrophages and neutrophils. One such mediator has been implicated in the development of fibrosis in silicotic rat lungs (39) in which lysyl oxidase and other enzymes of collagen biosynthesis are elevated (40). Thus, a 16-kD protein isolated from silicotic rat lungs and apparently derived from macrophages was found to stimulate collagen synthesis in granulation tissue cells at a concentration of 0.1 nM. Its effect on lysyl oxidase expression does not appear to have been determined. The first evidence for the regulation oflysyl oxidase at the genetic level in a fibrotic model stems from the 3-fold increase in functional lysyl oxidase mRNA activity seen in cell-free translations of fibrotic rat liver mRNA (27). Additional insights into the molecular levels of regulation should be forthcoming in view of the recent description of the sequence of lysyl oxidase cDNA (26). Genetic Disease As previously reviewed, levels oflysyl oxidase are markedly decreased in Menkes' disease and X-linked cutis laxa (1). A recent report describes an unusually severe case of Menkes' in which skin fibroblast lysyl oxidase levels were only 6 to 12 % of controls (41), a level that clearly can account for the severe connective tissue pathology associated with this disease. The enzyme deficiency in both of these diseases appears to be secondary to disturbances in copper metabolism, however. Other studies implying deficiencies oflysyl oxidase in osteogenesis imperfecta and Marfan's syndrome have recently been contradicted (42). Aortic lysyl oxidase activity is decreased by 30 to 50 % in the Brown Norway strain of rats that are genetically susceptible to disruption of the internal elastic lamina (43). Coupled with the apparent increase in aortic elastolytic activity in this strain, the decrease in lysyl oxidase could contribute to the manifestations of this
209
syndrome. It should be noted, however, that decreases of lysyl oxidase activity of these magnitudes may not result in deficient crosslinking, in view of the observation that the 64 % reduction of enzyme activity in the skin of glucocorticoid-treated rats was accompanied by an apparent increase in collagen crosslinking (44). Biologic Regulation of Lysyl Oxidase Lysyl oxidase activity responds to changes in levels of certain hormones, as illustrated by the estrogen-induced increase in mouse cervicallysyl oxidase activity (45). The addition of 10- to 100-nM levels of testosterone to the medium of cultured aortic smooth muscle cells increased cell layer lysyl oxidase activity by 5.5-fold (46). This effect is of interest in view of the greater tendency of males to develop arterial lesions that can become fibrotic with the progression of atherosclerosis. It has been commonly observed that the expression of lysyl oxidase by cells in culture is most active when proliferation has stopped or slowed, consistent with the most active stage of matrix production. It is of particular interest in this regard that lysyl oxidase activity as well as rates of collagen synthesis were markedly reduced in malignantly transformed human cell lines (47). A further intriguing aspect of the relationship between lysyl oxidase and tumorigenesis has recently emerged. Thus, the normal expression of gene rrg transcript in mouse NIH 3T3 cells is markedly downregulated by c-H-ras-induced transformation of these cells to the tumorigenic RS485 cell line (48). High levels of rrg expression were restored by interferon-induced reversion of RS485 cells to the nontumorigenic PR4 cell line. The specific importance of rrg to the reversion was emphasized by the fact that the normally persistent revertants developed the transformed phenotype when stably transfected with rrg antisense cDNA (48). Since the appearance of this report, it has been learned that rrg is 92 % homologous at the cDNA sequence level and ~ 98 % homologous at the predicted protein sequence level to rat aorta lysyl oxidase (49). It is not known at this writing whether the rrg protein is catalytically active or iflysyl oxidase catalysis is involved in the reversion ofthe tumorigenic state. However, these intriguing results do indicate that a lysyl oxidase-like protein may playa critical role in the maintenance of the nontumorigenic state in these fibroblast lines. Summary and Prospects Progress has been made on several aspects of the enzymology and biology oflysyl oxidase. While the precise chemical identity of the organic cofactor requires further investigation, this moiety exhibits the reactivity of an o-quinone and thus is consistent with a PQQ- or TOPA-like residue. The catalytic mechanism of this enzyme clearly parallels those proposed for other mammalian copper-dependent amine oxidases. As further details of the active site structures of these proteins become available, structural homologies in active site regions may also be found, although it should be noted that the sequence of the TOPA-bearing active site peptide of plasma amine oxidase is not seen in the lysyl oxidase protein sequence. Recent insights into the secretory mechanism and the biosynthetic precursor make it likely that detailed information about the biosynthesis and processing of this impor-
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tant enzyme will soon be forthcoming. In addition, evidence pointing to an unexpected intracellular localization and the recent results implying a relationship between lysyl oxidase and tumorigenesis certainly raise important new prospects for future research. Acknowledgments: Work from the writers' laboratory reviewed here is supported by Grants R37-AM-18880, HL-13262, and HL-19717from the National Institutes of Health.
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