Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3^′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu²⁺. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.     

Intracellular distribution of the lysyl oxidase propeptide in osteoblastic cells

Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule network. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation. microtubules; confocal immunofluorescence microscopy; extracellular matrix; osteoblast differentiation     

Intracellular localization of the matrix enzyme lysyl oxidase in polarized epithelial cells.

Considerable evidence supports novel functions for lysyl oxidase (LOX) beyond its traditional role in initiating cross-linkages in collagen and elastin within the extracellular matrix. These novel roles are particularly relevant during the transition of malignant epithelial cells towards a migratory and invasive phenotype. However, knowledge on cellular and matrix functions of LOX has been generated almost exclusively in mesenchymal cell types. But it is becoming increasingly evident that these cell types are not adequate to address these novel and highly significant roles for LOX in epithelial tissues. In this initial report, we demonstrate that active LOX is expressed by polarized MDCK II kidney and MCF-10A breast epithelial cells. Furthermore, we show evidence for the presence of mature LOX in the cytoplasm and establish these cell lines as models for epithelial LOX studies.     

The role of lysyl oxidase and collagen crosslinking during sea urchin development

Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.     

The Lysyl Oxidase Inhibitor, β-Aminopropionitrile,Diminishes the Metastatic Colonization Potential of Circulating Breast Cancer Cells

Lysyl oxidase (LOX), an extracellular matrix remodeling enzyme, appears to have a role in promoting breast cancer cell motility and invasiveness. In addition, increased LOX expression has been correlated with decreases in both metastases-free, and overall survival in breast cancer patients. With this background, we studied the ability of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, to regulate the metastatic colonization potential of the human breast cancer cell line, MDA-MB-231. BAPN was administered daily to mice starting either 1 day prior, on the same day as, or 7 days after intracardiac injection of luciferase expressing MDA-MB-231-Luc2 cells. Development of metastases was monitored by in vivo bioluminescence imaging, and tumor-induced osteolysis was assessed by micro-computed tomography (μCT). We found that BAPN administration was able to reduce the frequency of metastases. Thus, when BAPN treatment was initiated the day before, or on the same day as the intra-cardiac injection of tumor cells, the number of metastases was decreased by 44%, and 27%, and whole-body photon emission rates (reflective of total tumor burden) were diminished by 78%, and 45%, respectively. In contrast, BAPN had no effect on the growth of established metastases. Our findings suggest that LOX activity is required during extravasation and/or initial tissue colonization by circulating MDA-MB-231 cells, lending support to the idea that LOX inhibition might be useful in metastasis prevention.     

Lysyl oxidase oxidizes cell membrane proteins and enhances the chemotactic response of vascular smooth muscle cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.     

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.     

Beta-substituted ethylamine derivatives as suicide inhibitors of lysyl oxidase

Lysyl oxidase initiates the covalent cross-linking of elastin and collagen, converting lysyl residues in these proteins to peptidyl aldehyde residues. The present study explored structural and electron withdrawing features required to generate mechanism-based inhibitors of this enzyme with antifibrotic potential. It was found that the electron withdrawing nitrile moiety of beta-aminopropionitrile (BAPN), a naturally occurring syncatalytic inhibitor of lysyl oxidase, can be replaced by chlorine, bromine, or the nitro function to yield 2-haloamines or nitroethylamine compounds which also act as mechanism-based irreversible inhibitors of this enzyme. BAPN and 2-bromo- and 2-chloroethylamine exhibit similar KI values of 6-10 microM. However, the enzyme becomes irreversibly inactivated significantly faster by either of the 2-haloamines than by BAPN. 2-Nitroethylamine has by far the poorest affinity for the enzyme and inactivates much more slowly than the other amines of this series, consistent with interference with optimal enzyme-inhibitor interactions by the anionic nitro group. Unlike BAPN, 2-bromoethylamine is processed to a detectable aldehyde product upon incubation with enzyme, showing a partition ratio of 1.2 mol of acetaldehyde formed per mol of 2-bromo-ethylamine which becomes covalently incorporated in the enzyme. The results are consistent with the processing of 2-bromo-ethylamine to an enzyme-ethyleneamine Schiff base subject to hydrolysis to acetaldehyde or to covalent attack at carbon 2 by an enzyme nucleophile. Thus, beta-haloamines represent a new series of suicide inhibitors of lysyl oxidase which can inactivate the enzyme faster than BAPN and hence may have antifibrotic potential.     

Effect of beta-aminopropionitrile and ascorbate on fibroblast migration

Ascorbate and beta-aminopropionitrile (BAPN) have direct, but diverse affects on collagen matrix production. Ascorbate is necessary for the intracellular hydroxylation of prolyl and lysyl residues during collagen biosynthesis whereas BAPN inhibits the enzyme lysyl oxidase in the extracellular space thus preventing collagen crosslink formation. To study the influence of these two agents on fibroplasia, an in vitro model was used to analyze fibroblast migration, proliferation, and collagen synthesis. Biopsies of chicken tendon were covered with a fibrin clot to simulate an in vivo wound environment, and then they were exposed to either ascorbate or BAPN for up to 7 days. Fibroblast migration into the fibrin clot was measured using a Zeiss Mopp II planimeter, DNA synthesis by 125IUDR incorporation, and collagen synthesis by [3H]proline incorporation into collagenase-digestible protein. Tendon biopsies treated daily with fresh ascorbate (0.1 mM) had significantly greater fibroblast migration than controls without ascorbate (P less than 0.05). Cellular proliferation, collagen synthesis, and total protein synthesis were not significantly altered by ascorbate treatment. In contrast, BAPN inhibited fibroblast migration in a dose-dependent fashion without inhibiting proliferation (0.25 and 0.5 mM), collagen, and noncollagen protein synthesis. Therefore, the effect of BAPN on migration does not appear to be due to generalized cytotoxicity. These combined studies suggest that compounds such as ascorbate and BAPN which can modify collagen may also modify fibroblast migration.     

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX‐PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full‐length LOX cDNA (pre‐pro‐LOX), the N‐glycosylation null pre‐[N/Q]pro‐LOX cDNA and the deletion mutant pre‐LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N‐terminal propeptide sequence. The results show that glycosylation of the LOX‐PP is not required for secretion and extracellular processing of pro‐LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX‐PP for pro‐LOX exit from the ER and is the first to highlight the influence of LOX‐PP glycosylation on LOX enzyme activity. J. Cell. Biochem. 111: 1231–1243, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of lysyl oxidase (LOX) on corpus cavernous fibrosis caused by ischaemic priapism

Penile fibrosis caused by ischemic priapism (IP) adversely affects patients’ erectile function. We explored the role of lysyl oxidase (LOX) in rat and human penes after ischemic priapism (IP) to verify the effects of anti‐LOX in relieving penile fibrosis and preventing erectile dysfunction caused by IP in rats. Seventy‐two rats were randomly divided into six groups: control group, control + β‐aminopropionitrile (BAPN) group, 9 hrs group, 9 hrs + BAPN group, 24 hrs group, and 24 hrs + BAPN group. β‐aminopropionitrile (BAPN), a specific inhibitor of LOX, was administered in the drinking water. At 1 week and 4 weeks, half of the rats in each group were randomly selected for the experiment. Compared to the control group, the erectile function of IP rats was significantly decreased while the expression of LOX in the corpus cavernosum was significantly up‐regulated in both 9 and 24 hrs group. Proliferated fibroblasts, decreased corpus cavernosum smooth muscle cells/collagen ratios, destroyed endothelial continuity, deposited abnormal collagen and disorganized fibers were observed in IP rats. The relative content of collage I and III was not obviously different among the groups. β‐aminopropionitrile (BAPN) could effectively improve the structure and erectile function of the penis, and enhance recovery. The data in this study suggests that LOX may play an important role in the fibrosis of corpus cavernosum after IP and anti‐LOX may be a novel target for patients suffering with IP.     

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.     

Down-regulation of lysyl oxidase-induced tumorigenic transformation in NRK-49F cells characterized by constitutive activation of ras proto-oncogene

Several investigations have suggested a putative tumor suppressor role for lysyl oxidase because it is down-regulated in many human and oncogene-induced tumors. To address this issue we down-regulated the enzyme in normal rat kidney fibroblasts by stable transfection of its cDNA in an antisense orientation. The selected clones revealed an absence of lysyl oxidase and dramatic phenotypic changes, interpretable as signs of transformation. The antisense lysyl oxidase clones showed, indeed, loose attachment to the plate and anchorage-independent growth and were highly tumorigenic in nude mice. Moreover, we found an impaired response of the PDGF and IGF-1 receptors to their ligands. In particular, the transformed cells showed a down-regulation of both PDGF receptors and expressed the 105-kDa isoform of the IGF-1 beta receptor, which was not present in the normal control cells. The lack of response to PDGF-BB has been described as a feature of many ras-transformed phenotypes. Therefore, we looked at the status of the p21(ras). Indeed, we found a significantly higher level of active p21(ras) both during steady-state growth and prolonged starvation. Our data reveal new evidence for a tumor suppressor activity of lysyl oxidase, highlighting its particular role in controlling Ras activation and growth factor dependence.     

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

Secretion of lysyl oxidase by cultured human skin fibroblasts and effects of monensin, nigericin, tunicamycin and colchicine

Lysyl oxidase is an extracellular enzyme that initiates crosslink formation in the major connective tissue proteins, the collagens and elastin. This enzyme activity accumulated in a fresh medium of cultured human skin fibroblasts for at least 24 h, but the accumulation was distinctly non-linear after the first 12 h. Most of the total enzyme activity was present in the medium, the activity found in the cell layer representing about 30% of the total activity at 4 h, and about 10-15% at 24 h. The bulk of the cell-layer-associated activity appeared to be extracellular, as more than half was lost upon trypsinization. Culturing of the cells for 8 h in the presence of either monensin or nigericin, ionophores known to inhibit the secretion of many proteins at the level of the Golgi complex, markedly reduced the accumulation of lysyl oxidase activity in the medium. Monensin was particularly effective, as it produced a distinct inhibition even at a 10 nM concentration, reaching 50% at 30 nM. Both ionophores also reduced enzyme activity in the cell layer, whereas no definite decrease was seen in the activity of the trypsinized cells. The effect of monensin was evidently not due to any general toxicity on the part of the drug, since even a 500 nM concentration gave no inhibition of the incorporation of [3H]leucine into total protein. Tunicamycin also reduced lysyl oxidase activity in the medium and to a lesser extent in the cell layer, but the effective dose, 1-10 micrograms/ml, also inhibited the incorporation of [3H]leucine into total protein. The reduced enzyme activity may therefore not be due to a direct effect of tunicamycin on the glycosylation of the lysyl oxidase protein itself but may be mediated through other actions of the drug. Colchicine caused no inhibition in lysyl oxidase activity secretion even at a 10 microM concentration, although it has been reported to inhibit collagen secretion at doses more than one order of magnitude lower.     

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells

Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.     

Effect of Lysyl Oxidase Inhibition on Angiotensin II-Induced Arterial Hypertension,Remodeling, and Stiffness

It is well accepted that angiotensin II (Ang II) induces altered vascular stiffness through responses including both structural and material remodeling. Concurrent with remodeling is the induction of the enzyme lysyl oxidase (LOX) through which ECM proteins are cross-linked. The study objective was to determine the effect of LOX mediated cross-linking on vascular mechanical properties. Three-month old mice were chronically treated with Ang II with or without the LOX blocker, β -aminopropionitrile (BAPN), for 14 days. Pulse wave velocity (PWV) from Doppler measurements of the aortic flow wave was used to quantify in vivo vascular stiffness in terms of an effective Young’s modulus. The increase in effective Young’s modulus with Ang II administration was abolished with the addition of BAPN, suggesting that the material properties are a major controlling element in vascular stiffness. BAPN inhibited the Ang II induced collagen cross-link formation by 2-fold and PWV by 44% (P<0.05). Consistent with this observation, morphometric analysis showed that BAPN did not affect the Ang II mediated increase in medial thickness but significantly reduced the adventitial thickness. Since the hypertensive state contributes to the measured in vivo PWV stiffness, we removed the Ang II infusion pumps on Day 14 and achieved normal arterial blood pressures. With pump removal we observed a decrease of the PWV in the Ang II group to 25% above that of the control values (P=0.002), with a complete return to control values in the Ang II plus BAPN group. In conclusion, we have shown that the increase in vascular stiffness with 14 day Ang II administration results from a combination of hypertension-induced wall strain, adventitial wall thickening and Ang II mediated LOX ECM cross-linking, which is a major material source of vascular stiffening, and that the increased PWV was significantly inhibited with co-administration of BAPN.