Expression and Purification of Enzymatically Active Forms of the Human Lysyl Oxidase-like Protein 4*

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed β-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.

Lysyl oxidase (LOX) 1 is a copper-dependent amine oxidase that oxidizes primary amine substrates to reactive aldehydes. In most tissues, LOX is responsible for the lysine-derived crosslinks in collagen and elastin, which is the essential step for biogenesis of a fibrillar extracellular matrix (1). More recently, several new functions, as varied as tumor suppression, cellular senescence, developmental control, and chemotaxis, have been assigned to this amine oxidase (2)(3)(4). The importance of LOX has been further emphasized by the finding that abnormal LOX activity contributes to a number of different diseases, including atherosclerosis, aortic aneurysms, pulmonary fibrosis, and hepatic fibrosis (5)(6)(7)(8)(9).
In recent years, four novel LOX-like proteins, LOXL, LOXL2, LOXL3, and LOXL4, have been identified, each containing the characteristic domains of LOX in the carboxyl (C) terminus, such as the copper-binding domain, the lysyl-tyrosyl-quinone residues and the cytokine receptor-like domain (10 -14). The presence of a LOX family suggests that the multiple diverse functions currently attributed to a single enzyme, LOX, may be because of different LOX-like proteins. In addition to the conserved C-terminal domains, LOXL2, LOXL3 and LOXL4, unlike LOX and LOXL, contain four scavenger receptor cysteine rich (SRCR) domains in the amino (N) terminus, thus forming a subfamily within the LOX family. Based on the conservation of the C-terminal LOX domains, these SRCR domain-containing members were predicted to contain the amine oxidase function, but it has yet to be determined. SRCR domains are known to mediate ligand binding in a number of secreted and receptor proteins (15,16). The presence of SRCR domains within LOXL2, LOXL3, and LOXL4 indicates that these three proteins may play novel functions, possibly involving components of the extracellular matrix. The murine homologue of LOXL4, reported as LOXC, was initially identified as a transcript specifically expressed in the cartilage (17). However, Northern analyses of the human LOXL4 gene showed more ubiquitous expression in many tissues, but it was different from the other members of the LOX family (10,14), suggesting distinct tissue and substrate specificity of this protein. In this study, we report for the first time that LOXL4 functions as an active amine oxidase. The LOXL4 protein was expressed as His-tagged recombinant forms in inclusion bodies by using an Escherichia coli expression vector, extracted with 8 M urea, and renatured into enzymatically active forms toward a nonpeptidyl substrate, benzylamine. The amine oxidase activity of LOXL4 was sensitive to ␤-aminopropionitrile (BAPN), a well known specific inhibitor of LOX.

EXPERIMENTAL PROCEDURES
Construction of LOXL4 Expression Plasmids-The LOXL4 cDNAs were PCR-amplified from human placental total RNA (BD Biosciences) by using Pfu turbo polymerase (Stratagene), according to the manufacturer's suggestions. The forward primers, LOXL4-p1, LOXL4-p2, LOXL4-p3, LOXL4-p4, and LOXL4-p5, were designed to start the openreading frame of LOXL4 at Ala 2 , Val 159 , Val 311 , Val 421 , and Asp 570 , respectively. The sequences of oligonucleotide primers used for cloning LOXL4 cDNAs are described in Table I. A unique restriction site, either NheI or Hind III, was introduced in each primer for convenient subcloning. All of the recombinant proteins were designed to contain an additional Met-Ala-Ser sequence at the N terminus and a hexa-histidine tag at the C terminus. Thermocycling consisted of 30 cycles at 94°C for 60 s, 58°C for 60 s, and 72°C for 60 s, with a predenaturation at 94°C for 2 min, and a final extension at 72°C for 7 min. The PCR-amplified DNA fragments were gel-purified and then ligated into pET21a (Novagen) at NheI and Hind III restriction sites in frame with the C-terminal hexa-histidine tag. All of the resulting expression con-structs, pET21a-LOXL4-p1, pET21a-LOXL4-p2, pET21a-LOXL4-p3, pET21a-LOXL4-p4, and pET21a-LOXL4-p5, were confirmed to contain the desired LOXL4 sequences by DNA-sequencing analysis by using a Cycle Sequencing Ready Reaction kit (Applied Biosystems) according to the manufacturer's recommended method.
Expression and Purification of the LOXL4 Proteins-The E. coli strain BL21(DE3) (Novagen) was used for transformation of the pET21a-derived expression constructs of LOXL4. The transformants were grown in 1 liter of LB medium containing 100 g/ml of ampicillin at 37°C until the A 600 of culture reached 0.6. For induction, 1 mM of isopropyl-1-thio-␤-D-galactopyranoside (IPTG) was added. After 4 h of induction, the transformants were harvested by centrifugation at 7,000 ϫ g for 20 min and resuspended in 60 ml of lysis buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 1 mM PMSF, and 1 mg/ml of lysozyme). Triton X-100 (1%, w/v) and DNase (0.1 mg/ml) were added sequentially to the lysates at intervals of 30 min during inverted mixing, and then sonication was repeated twice at 70% efficiency with a sonicator (Sonics & Materials, Inc). The inclusion bodies were precipitated by centrifugation at 8,000 ϫ g for 20 min and homogenized in 60 ml of 8 M urea, 10 mM K 2 HPO 4 , pH 8.2, and 3 mM ␤-mercaptoethanol. The solubilized inclusion bodies were purified with the nickel-nitrilotriacetic acid agarose resins (Qiagen) according to the manufacturer's recommendations. All of the purification procedures were carried out at 4°C. The purity and sizes of the recombinant proteins were assessed by SDS-PAGE.
N-terminal Amino Acid Sequence Analysis-To confirm the N-terminal sequences of the recombinant LOXL4 proteins, the purified proteins were subjected to N-terminal analysis by Edman degradation. After purification by the nickel-chelating affinity chromatography, 5 g each of LOXL4-p2, LOXL4-p3, LOXL4-p4, and LOXL4-p5 were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences), and stained with Ponceau S. The visualized protein bands were excised and loaded into a CLC Capillary 492 sequencer (Applied Biosystems). The 10 N-terminal residues of each purified recombinant protein were analyzed by the automated N-terminal sequencing, and all of the recombinant LOXL4 proteins contained the expected sequences in the N terminus.
Refolding of the LOXL4 Proteins-For stepwise dialysis, the purified LOXL4 proteins were diluted to 100 g/ml in elution buffer (6 mM urea, 250 mM imidazol, and 10 mM K 2 HPO 4 , pH 8.2). The protein samples were dialyzed overnight first against a buffer of 10 mM K 2 HPO 4 , pH 9.6, 200 M CuCl 2 , and 2% sodium N-lauroylsarcosinate and then against a buffer of 10 mM K 2 HPO 4 , pH 9.6, and 5 M CuCl 2 . The proteins were further dialyzed twice against the phosphate buffer. The concentration of dialyzed protein samples was determined by the BCA method (18). After dialysis, the protein solutions were aliquoted and lyophilized in the presence of 10 mM trehalose using a Freeze dryer (Labconco).
Amine Oxidase Assays-Amine oxidase assays were performed with a UV spectrophotometer (Kontron Instruments). The assay reactions included 10 mM benzylamine and 100 g of a recombinant LOXL4 protein in 1 ml of the phosphate buffer. The reaction mixtures were incubated at 37°C with simultaneous detection at 250 nm. For the assays with BAPN, the LOXL4 protein was preincubated with 100 molar excess of BAPN in the phosphate buffer at 37°C for 1 h, and then 10 mM benzylamine was added to the reaction mixture for amine oxidase assays.

RESULTS AND DISCUSSION
Expression of the LOXL4 Proteins-In an effort to express and purify enzymatically active forms of LOXL4, we constructed a series of LOXL4 expression plasmids. The LOXL4 cDNAs of variable lengths were PCR-amplified and cloned into pET21a, an E. coli expression plasmid. All of the recombinant LOXL4 proteins were designed to contain the characteristic C-terminal domains of the LOX family, including the copperbinding domain, the CRL-domain, and the LTQ residues (Lys 638 and Tyr 674 ). However, the recombinant proteins were designed to start at different residues, either encoding the full-length polypeptide of LOXL4 (LOXL4-p1) or subsequently deleting the four SRCR domains (LOXL4-p2, LOXL4-p3, LOXL4-p4, and LOXL4-p5) (Fig. 1A).
Bone morphogenic protein-1, also known as procollagen Cproteinase, was shown to proteolytically cleave the LOX and LOXL precursor proteins at Ala/Gly-Asp, leaving an aspartic acid residue at the N terminus of each processed protein (19,20). A potential cleavage site of bone morphogenic protein-1 was also identified within LOXL4, at residues Ala 569 -Asp 570 . LOXL4-p5 was designed to correspond to this potentially processed form of LOXL4, deleting all of the SRCR domains in the N terminus (Fig. 1A).
Upon induction by 1 mM IPTG at 37°C, the recombinant LOXL4 proteins, except for LOXL4-p1, were expressed at high levels from the expression constructs (Fig. 1B). Fractionation of the cell lysates into different cellular compartments, such as cytoplasmic extracts, periplasmic extracts, and inclusion body fractions, revealed that most of the recombinant proteins were expressed in insoluble forms within inclusion bodies (data not shown). It was not clear yet why the LOXL4-p1 construct did not show any detectable expression, but it might be because of the presence of rare codons, such as CTA (Leu 16 ), CCC (Pro 19 and Pro 21 ), and AGG (Arg 23 ), in the N terminus of LOXL4, which are infrequently used in E. coli. Clusters of rare codons were shown to cause translation errors and reduction of the expression level in E. coli (21).
The apparent sizes of the expressed recombinant LOXL4 proteins were in good agreement with the deduced molecular masses; 62 kDa for LOXL4-p2, 50 kDa for LOXL4-p3, 38 kDa for LOXL4-p4, and 22 kDa for LOXL-p5 (Fig. 1, B and C). The hexa-histidine-tagged recombinant LOXL4-proteins were over 95% pure on SDS-PAGE gels (Fig. 1C). Automated N-terminal sequencing confirmed the presence of the designed N-terminal sequence in each LOXL4 recombinant protein, indicating that each protein band in the SDS-PAGE gels represents the desired length of the corresponding recombinant LOXL4 protein.
Amine Oxidase Activity of the Purified LOXL4 Proteins-In an attempt to refold the proteins denatured by urea during purification, we tried stepwise dialysis in the presence of Nlauroylsarcosinate and Cu 2ϩ , an oxidative catalyzer. In the first dialysis buffer, 2% (w/v) sodium N-lauroylsarcosinate and 200 M CuCl 2 were added. The second dialysis was carried out without any detergent but in the presence of 5 M CuCl 2 , and the protein samples were further dialyzed twice in the potassium phosphate buffer. The underlined letters indicate the unique restriction sites introduced for subcloning into pET21a. The backward primer was used for all the LOXL4 constructs.
These specific activities of the recombinant LOXL4 proteins were similar to or even higher than the previously reported activities of LOX, 0.019 units/mg and 0.02 Ϸ 0.04 units/mg, performed with the LOX protein purified from the bovine aorta (22,23). The differences in specific activity within the tested recombinant LOXL4 proteins were primarily due to the differences in molar concentration of the recombinant proteins in the assays. These assay results indicate that all of the rested recombinant proteins, including the potentially bone morphogenic protein-1 processed form of LOXL4, function as an active amine oxidase toward benzylamine and, moreover, that the conserved C-terminal domains of LOXL4 are sufficient to confer the amine oxidase function to this protein.
The SRCR Domains in LOXL4 -In our assays, the recombinant LOXL4 proteins containing one or more SRCR domains (LOXL4-p2, LOXL4-p3, and LOXL4-p4) showed BAPN-specific amine oxidase activity toward benzylamine (Table II). Although we have not yet tested the catalytic activity of these SRCR domain-containing recombinant proteins toward more physiological substrates, such as collagen and elastin, our results indicate that the presence of the SRCR domains does not interfere with the amine oxidase activity of LOXL4 at least in in vitro assays. The SRCR superfamily proteins are found either on the cell surface membranes or as secreted proteins. The SRCR domains within these proteins are known to be involved in protein-protein interactions for cell adhesion or cell signaling (15,16,24). The presence of SRCR domains within LOX4, therefore, may indicate possible existence of modulators that may affect the amine oxidase activity of LOXL4 through interaction with the SRCR domains. Identification of the interactive proteins of LOXL4 will be critical in further characterization of the functional significance of the SRCR domains in LOXL4.
In summary, the purpose of this study was to assess the amine oxidase activity of LOXL4. With purified recombinant forms of LOXL4, we have shown that LOXL4 functions as an amine oxidase. Furthermore, by using a series of deletion constructs, we confirmed that neither the presence nor the deletion of SRCR domains affects the amine oxidase activity of LOXL4. The availability of these enzymatically active forms of LOXL4 will pave the way for further functional characteriza-  tion of this latest member of amine oxidases, which should be critical in understanding the mechanistic bases for the diverse functions played by the LOX family members.