Introduction

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that regulates formation of lysine-derived cross-links in elastin and collagen fibers. LOX catalyzes oxidative deamination of ε-amino groups of peptidyl lysine residues to α-aminoadipic δ-semialdehyde groups which, in turn, undergo spontaneous condensation to form the intra- and inter-molecular cross-linkages in collagen and elastin fibers [1]. In addition to the cross-linking activity toward extracellular matrix proteins, several novel functional roles of LOX have been identified in tumor suppression, hypoxia-induced metastasis, and chemotaxis [2–4].

The lysyl oxidase-like 2 gene (LOXL2) is a member of the LOX family that is composed of five paralogues (LOX, LOXL, LOXL2, LOXL3, and LOXL4) in humans. Each LOX family protein contains a copper-binding motif, lysyl-tyrosyl-quinone (LTQ) residues, and a cytokine receptor-like (CRL) domain in its highly conserved carboxyl (C)-terminus [5–8]. In contrast to the characteristic C-terminal domains, the LOX family members show sequence divergence in their amino (N)-terminal regions. In particular, LOXL2, LOXL3, and LOXL4 contain four scavenger receptor cysteine-rich (SRCR) domains in their N-terminal regions [6–8]. The functional role of the SRCR domains in these three LOX paralogues has not yet been characterized; however, SRCR domains are involved in protein–protein interactions in several secreted or receptor proteins [9]. While the regulation of the LOX family gene expression has been well characterized in various cells and tissues [10–13], the specific mechanisms by which these members exert the enzymatic activity are yet to be further elucidated.

LOXL2 was first isolated as an overexpressed gene in senescent fibroblasts [6], and LOXL2 upregulation has been reported more often in invasive metastatic tumors than noninvasive tumors [14, 15]. Further, LOXL2 has been shown to mediate induction of epithelial-mesenchymal transition by repression of E-cadherin expression, suggesting a functional role for LOXL2 in tumor progression [16]. Based on conservation of the C-terminal domains, LOXL2 was predicted to function as an amine oxidase. However, the amine oxidase activity of LOXL2 has not yet been thoroughly studied. In order to assess the amine oxidase activity of LOXL2, we expressed the LOXL2 protein as a series of recombinant forms, with deletions in the SRCR domains. We then tested amine oxidase activity of the purified recombinant proteins toward physiological substrates of LOX, including elastin and several different types of collagen. All of the recombinant LOXL2 proteins, in the presence or absence of the SRCR domains, showed significant amine oxidase activity toward collagen and elastin in in vitro peroxidase-coupled fluorometric assays. However, the amine oxidase activity of the recombinant LOXL2 proteins was not significantly inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX, suggesting that LOX and LOXL2 may be subject to potentially different biochemical mechanisms for modulation of the amine oxidase activity.

Materials and methods

Construction of LOXL2 expression plasmids

A full-length LOXL2 cDNA was PCR-amplified from human placental total RNA (BD Biosciences) with Pfu turbo polymerase (Stratagene), according to the manufacturer’s suggestions. Subsequently, LOXL2 cDNAs, with a series of deletions in the SRCR domains, were generated by PCR amplification of the full-length LOXL2 cDNA using the following primers:

  • LOXL2-fl forward primer: 5′-GCGGCTAGCATGGAGAGGCCTCTGTGC-3′

  • LOXL2-p1 forward primer: 5′-GCGGCTAGCGTGCGACTGAGAGGCGG-3′

  • LOXL2-p2 forward primer: 5′-GCGGCTAGCCTGCGCCTGAACGGCG-3′

  • LOXL2-p3 forward primer: 5′-GCGGCTAGCGATGTCAACAGCAACAAAG-3′

  • Reverse primer: 5′-GCGAAGCTTCTGCGGGGACAGCTGGTTG-3′

Underlined letters in the primer sequences indicate a unique NheI or HindIII site introduced into the primers for convenient subcloning. The reverse primer was commonly used for construction of all LOXL2 expression plasmids. Thermocycling consisted of 30 cycles at 94°C for 45 s, 58°C for 45 s, and 72°C for 60 s, with predenaturation at 94°C for 2 min and final extension at 72°C for 7 min. PCR-amplified DNA fragments were gel-purified and then ligated into pET21a (Novagen) at NheI and HindIII restriction sites in frame with the C-terminal hexa-histidine tag. All resulting expression constructs were confirmed by DNA-sequencing analysis to contain the desired LOXL2 sequences.

Expression and purification of the recombinant LOXL2 proteins

Expression, purification, and refolding of recombinant LOXL2 proteins were carried out as we previously described for other LOX family proteins [17, 18]. Briefly, the Escherichia coli strain BL21 (DE3) (Novagen) was transformed with pET21a-derived expression constructs of LOXL2. After induction with 1 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG), the transformed bacterial cells were lysed in a buffer containing 50 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 1 mM PMSF, 1% lysozyme, Triton X-100, and 0.1 mg/ml DNase. Inclusion bodies were isolated by centrifugation and homogenized in a buffer containing 6 M urea, 10 mM K2HPO4, pH 8.2, and 3 mM β-mercaptoethanol. Solubilized inclusion bodies were then purified using Ni-NTA agarose resins (Qiagen) according to the manufacturer’s suggestions. For refolding of the recombinant LOXL2 proteins denatured by urea during the purification steps, stepwise dialysis was performed in a buffer of 10 mM K2HPO4, pH 9.6, 200 μM CuCl2, and 2% sodium N-lauroylsarcosinate, and then in a buffer of 10 mM K2HPO4, pH 9.6 and 5 μM CuCl2. The protein samples were further dialyzed twice in 10 mM K2HPO4, pH 9.6. After the final dialysis, the protein solutions were lyophilized in the presence of 10 mM trehalose using a freeze dryer (Labconco). All purification procedures were carried out at 4°C. Purity and sizes of the recombinant proteins were assessed using SDS-PAGE.

Amine oxidase assays of the recombinant LOXL2 proteins

Amine oxidase activity of the recombinant LOXL2 proteins was assessed using a peroxidase-coupled fluorometric assay with the Amplex red hydrogen peroxide assay kit (Molecular Probes), as previously described [19]. Each reaction contained 5 μg of a purified LOXL2 protein (0.48 μM for LOXL2-p1, 0.61 μM for LOXL2-p2, and 0.74 μM for LOXL2-p3) and 20 pmol of a substrate in a reaction volume of 200 μl. Parallel assays were performed in the absence or presence of 1 mM BAPN. Bovine neck ligament elastin (Sigma), calf skin collagen type I (Sigma), calf skin collagen type III, and human placenta collagen type IV were used as substrates for the assays. Fluorescence was measured using a fluorescence spectrophotometer (Molecular Devices) with excitation and emission wavelengths of 571 and 585 nm, respectively.

Results and discussion

Expression and purification of the recombinant LOXL2 proteins

The recombinant LOXL2 proteins, LOXL2-fl, LOXL2-p1, LOXL2-p2, and LOXL2-p3 were designed to start at different residues, with a serial deletion of the repeated SRCR domains located in the N-terminus, beginning at Met1, Val326, Leu435, and Asp498, respectively. The characteristic C-terminal region containing the copper-binding motif, LTQ residues (Lys653 and Tyr689), and the CRL domain was included in all recombinant LOXL2 proteins (Fig. 1a). After induction with 1 mM IPTG, all recombinant LOXL2 proteins, with the exception of LOXL2-fl, showed high levels of expression. The apparent sizes of the hexa-histidine tagged recombinant LOXL2 proteins were in good agreement with the deduced molecular mass; 52 kDa for LOXL2-p1, 41 kDa for LOXL2-p2, and 34 kDa for LOXL2-p3 (Fig. 1b). Recombinant LOXL2-proteins were more than 95% pure on SDS-PAGE gels. The reason that the LOXL2-fl protein was not expressed at detectable levels is currently unknown, but is possibly due to the presence of rare codons clustered in the N-terminal region of LOXL2. Clusters of rare codons have been shown to frequently impede translation of heterologous genes in E. coli [20]. The rare codons, found in the N-terminal region of LOXL2, include CCC (Pro21, Pro31, Pro34, Pro47, Pro50, Pro120), AGG (Arg3, Arg65, Arg162, Arg240, Arg254), and CGG (Arg71, Arg98, Arg187, Arg253).

Fig. 1
figure 1

Expression and purification of LOXL2 in E. coli. a Schematic diagrams of the recombinant LOXL2 proteins. All recombinant LOXL2 proteins were tagged with an additional Met-Ala-Ser sequence at the N-terminus and six histidine residues at the C-terminus. b purification of the recombinant LOXL2 proteins. Each lane contains approximately 5 μg of a purified recombinant LOXL2 protein. Lane M, protein standard markers; lane 1, LOXL2-p1; lane 2, LOXL2-p2; lane 3, LOXL2-p3

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Effect of SRCR domains on amine oxidase activity of LOXL2

In an attempt to convert the purified recombinant LOXL2 proteins into enzymatically active forms, the purified proteins were subjected to stepwise dialysis in the presence of N-lauroylsarcosinate and Cu2+. Following refolding, the recombinant LOXL2 proteins were assessed for amine oxidase activity toward collagen and elastin using an in vitro peroxidase-coupled fluorometric assay. Elastin and collagen types I, III, and IV were used as substrates for the amine oxidase assays. All recombinant LOXL2 proteins showed significantly increased levels of fluorescence over negative controls in the amine oxidase assays, with or without the SRCR domains in the N-terminus (Fig. 2). Further, the recombinant LOXL2 proteins showed no noticeable differences in substrate specificity toward collagen and elastin substrates, indicating that deletion of the SRCR domains did not affect amine oxidase activity of LOXL2 (Fig. 2). Previously, we showed that the conserved C-terminal domains of the LOX family proteins were sufficient to confer amine oxidase activity [17]. We also reported that neither deletion nor truncation of the N-terminal SRCR domains affected amine oxidase activity LOXL4, a close paralogue of LOXL2 [18]. Our current findings with the recombinant LOXL2 proteins provide further support that deletion of the SRCR domains does not interfere with amine oxidase activity of the LOX family proteins toward physiological substrates such as collagen and elastin, at least in in vitro assays.

Fig. 2
figure 2

Amine oxidase activity of the LOXL2 proteins toward collagen and elastin. Reactions that did not contain any of the recombinant LOXL2 proteins but the substrates were used as negative controls for both BAPN (−) and (+) reactions. After subtraction of the negative control value from the value of each reaction, each relative activity was expressed as the fluorescence value per pmol of the recombinant LOXL2 used for the assay. Amine oxidase activity assays were repeated in quadruplicate. Standard deviations are indicated on the graph

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The SRCR superfamily consists mainly of secreted or membrane proteins often associated with the innate immune system. The SRCR domain is a highly conserved domain of approximately 110 residues, containing six (group A) or eight (group B) cysteines that are presumably important to tertiary structural conformation [21]. LOXL2 contains four repeated copies of the group A SRCR domain in its N-terminus. In several different types of SRCR superfamily proteins, including CD6, the Mac-2 binding protein, and the macrophage receptor with collagenous structure (MARCO), SRCR domains have been shown to play essential roles in interaction with ligands or other regulatory proteins for cell signaling or adhesion [22–24]. Therefore, in more in vivo circumstances, it may be possible that the repeated SRCR domains modulate the amine oxidase activity of LOXL2, possibly through interacting with regulatory ligands or proteins that may exist in the extracellular matrix. Detailed functional analysis on interactive property of LOXL2 will be helpful for further characterizing the role of SRCR domains in regulation of the amine oxidase activity of LOXL2.

Effect of BAPN on amine oxidase activity of LOXL2

In our in vitro assays with physiological substrates, BAPN did not inhibit the amine oxidase activity of all recombinant LOXL2 proteins at 1 mM that corresponded to a greater than 1000 M excess of the recombinant LOXL2 proteins (Fig. 2). BAPN is a well known irreversible inhibitor of LOX [25]. It was recently reported that amine oxidase activity of LOXL2 could not be inhibited by BAPN, although the assays were performed in less direct ways without any purified LOXL2 protein [26]. Our current results with the recombinant LOXL2 proteins provide more straightforward evidence that amine oxidase activity of LOXL2 is not susceptible to inhibition by BAPN, suggesting that the structural conformation and binding affinity of LOXL2 may be different from those of LOX.

BAPN was initially identified as a lathyrogen found in plants of the genus Lathyrus, and its administration to animals resulted in increased fragility of connective tissues [25]. Our results indicate that LOXL2 may not be involved in the lathyrism caused by BAPN, implying a different functional role of LOXL2 in maintenance of the structural integrity of connective tissues. BAPN contains the same CH2–CH2–NH2 group as lysyl residues in the substrates of LOX, such as collagen and elastin. It has been previously proposed that BAPN covalently binds to LOX in a substrate-like manner, irreversibly inhibiting the amine oxidase activity [27]. However, the active or catalytic site that BAPN binds to has not yet been identified in LOX, while cofactor sites, such as the copper-binding motif and LTQ residues, have been well characterized in the conserved C-terminal region. Further detailed structural analysis will be helpful for elucidation of enzymatic differences between LOX and LOXL2.