HHS Public Access Author manuscript Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. A u t Published in final edited form as: h o Arch Biochem Biophys. 2014 March 15; 546: 19–32. doi:10.1016/j.abb.2013.12.022. r M a n u Human Copper-Dependent Amine Oxidases s c r ip t Joel Finney, Hee-Jung Moon, Trey Ronnebaum, Mason Lantz, and Minae Mure* Department of Chemistry, The University of Kansas, Lawrence, KS 66045, USA Abstract Copper amine oxidases (CAOs) are a class of enzymes that contain Cu2+ and a tyrosine-derived A quinone cofactor, catalyze the conversion of a primary amine functional group to an aldehyde, and u t generate hydrogen peroxide and ammonia as byproducts. These enzymes can be classified into h o two non-homologous families: 2,4,5-trihydroxyphenylalanine quinone (TPQ)-dependent CAOs r M and the lysine tyrosylquinone (LTQ)-dependent lysyl oxidase (LOX) family of proteins. In this a n review, we will focus on recent developments in the field of research concerning human CAOs and u s the LOX family of proteins. The aberrant expression of these enzymes is linked to inflammation, c rip fibrosis, tumor metastasis/invasion and other diseases. Consequently, there is a critical need to t understand the functions of these proteins at the molecular level, so that strategies targeting these enzymes can be developed to combat human diseases. Keywords copper amine oxidase; lysyl oxidase; quinoprotein A u t h o r INTRODUCTION AND REACTION MECHANISM OF TYROSINE-DERIVED M a n QUINONE COFACTORS u s c Copper amine oxidases (CAOs) are copper- and quinone-dependent enzymes that catalyze r ip the oxidative deamination of primary amine functional groups to aldehydes, concomitantly t producing hydrogen peroxide and ammonia. Currently, they are grouped into two nonhomologous subgroups based on the nature of their organic cofactors, namely 2,4,5- trihydroxyphenylalanine quinone (TPQ)-dependent CAOs and the lysine tyrosylquinone (LTQ)-dependent lysyl oxidase (LOX) family of proteins (1). A number of reviews of CAOs and LOXs are available (1-11), including an excellent recent review by Klema and Wilmot A that focuses on structural biology studies of the mechanisms of TPQ biogenesis and catalysis u th of amine oxidation in the TPQ-containing bacterial and yeast CAOs (12). In the present o r review, we will first briefly summarize the current understandings of the mechanisms of 1) M TPQ and LTQ biogenesis and 2) amine oxidation by CAOs and LOX. We will also discuss a n u their commonly used in vitro inhibitors. We will then highlight recent research developments s c concerning human CAOs and the human LOX family of proteins, with an emphasis on their r ip proposed roles in disease and health defects. t *To whom correspondence should be addressed: Minae Mure, Department of Chemistry, The University of Kansas, 1251 Wescoe Hall Drive, 2054 Malott Hall, Lawrence, KS 66045, USA, Tel.: (785) 864-2901, Fax: (785) 864-5396; [email protected]. Finney et al. Page 2 Tyrosine-derived Quinone Cofactors: TPQ and LTQ A u TPQ and LTQ (Figure 1) were discovered by Klinman and coworkers as the respective t h organic cofactors of a CAO isolated from bovine plasma and a LOX isolated from bovine o r calf aorta (13,14). Both cofactors are post-translationally derived from a conserved active- M a site tyrosine residue via an autocatalytic mechanism requiring only Cu2+ and O2 (15,16). n u Dopaquinone (DPQ) is proposed to be the common intermediate during the biogenesis of s c TPQ and LTQ, where the 1,4-addition of either water or the ε-amino side chain of a peptidyl r ip lysine residue to DPQ yields TPQ or LTQ, respectively (13,14) (Figure 2). A careful t inspection of the reaction product of TPQ biogenesis in the presence of H 18O and 18O by 2 2 resonance Raman spectroscopy revealed that the C2 oxygen of TPQ is from solvent water, rather than O (17). In the same study, substantial electron delocalization between the C2 2 and C4 oxygens of the TPQ cofactor was observed, whereas the C5=O bond had more carbonyl character. These results support a solution study demonstrating that the A delocalization directs the addition of substrate amine at the C5 carbonyl group (18). u t h o X-ray snapshot analysis of TPQ biogenesis revealed that the precursor tyrosine and the r M biogenesis intermediates (i.e. DPQ and the trihydroxybenzene form, i.e. TPQ ) are all red a n ligated to Cu2+ (i.e. “on-copper” forms) at their O4 oxygen atoms (19). In the last O - u 2 s oxidation step of TPQ to TPQ, the TPQ ring finally moves away from the Cu2+ binding c red r ip site and becomes trapped in a hydrophobic wedge-like cavity in the active site; this is the t “off-copper” conformation (Figure 3)(described in greater detail under Reaction Mechanism). The conformational change of TPQ is critical for optimal catalytic activity of CAOs, since the on-copper form of TPQ is unable to interact with substrate amines (7,20,21). The factor that drives TPQ to move off Cu2+ in the final step of biogenesis remains to be elucidated. A u In contrast to TPQ, the details of the LTQ biogenesis mechanism (Figure 2) have not been t h o explored, mainly due to the unavailability of diffracting crystals suitable for X-ray r M crystallography. However, to gain some insight in the intermediacy of DPQ in the biogenesis a of TPQ and LTQ, a lysine residue was incorporated into the active site of a bacterial CAO by n u site-directed mutagenesis, replacing the conserved Asp residue located at the far end of the s c r wedge (22). In this mutant, an LTQ-like quinone was produced instead of TPQ, where the ip t covalent bond between the lysine side chain and DPQ was confirmed by X-ray crystallography (Figure 4). These results not only support the hypothesized common intermediacy of DPQ in the biogenesis of TPQ or LTQ (7,23), but also suggest that at room temperature the DPQ intermediate has sufficient motional flexibility to swing out of the Cu2+ site and interact with the ε-amino group of the lysine side chain in the wedge (Figure 4). A u t h Reaction Mechanism of CAOs and LOX in Amine Oxidation o r M The reaction mechanism of CAOs in the oxidation of primary amines follows a classical a ping-pong mechanism involving covalent intermediates formed between TPQ and amines, as n u s well as oxidoreduction reactions of the TPQ cofactor (Figure 5) (7,8,12). A conserved Asp c r residue acts as an active site base to remove an α-proton from the first covalent intermediate ip t between TPQ and the substrate amine (i.e. a substrate Schiff base), and also serves as a Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 3 proton sink to regulate the protonation state of the substrate and the TPQ-derived reaction A intermediates, which are essential for optimal catalytic activity (24-26) (Figure 6). The u th protonation state of the reaction intermediates is also carefully controlled by the two o r conserved Asn and Asp residues (flanking the conserved precursor Tyr residue in the Asn- M Tyr-Asp/Glu consensus sequence)(27,28) and water molecules in the active site (29). The a n reaction mechanism of amine oxidation by LOX is expected to be similar to CAOs, where an u s unidentified active site residue with pK ~ 7.6 is thought to be the catalytic base for a LOX c a r ip isolated from bovine aorta (30). t TPQ is expected to have some motional flexibility in the active site, since it is connected to the peptide backbone by a single covalent bond. In the on-copper conformation, the O4 of TPQ ligates to the active site Cu2+ and the active carbonyl group at C5 of TPQ faces away from the substrate entry channel and the active site base (Asp)(Figure 3). Therefore, the on- copper TPQ form of CAOs is catalytically inactive. To prevent this, the mobility of the TPQ A u cofactor and TPQ-derived intermediates is carefully modulated in the active sites of CAOs t h o by hydrogen bonding interactions among the O4 of TPQ, a conserved Tyr in the active site, a r M conserved Asp (the active site base), and the surrounding hydrophobic wedge-like cavity a (Figure 3). These interactions maintain optimal activity by preventing the O4 of the TPQ n u s ring from directly ligating to copper (i.e. by retaining TPQ in the off-copper conformation) c r (7,26,31-33). In contrast to TPQ, the LTQ cofactor of LOX is covalently linked to the ip t peptide backbone at two positions, and is consequently fixed in one conformation. Known Inhibitors of CAOs and LOX CAOs and LOX can be inhibited irreversibly by hydrazine derivatives that form a hydrazone adduct with the active carbonyl group of TPQ and LTQ, mimicking the Schiff base reaction intermediates in the catalytic cycle (Figure 7). The most commonly used in vitro inhibitors A u for these proteins are phenylhydrazine and its derivatives, such as 4-phenylhydrazine, 2,4- t h o phenylhydrazine and 2-hydrazinopyridine. r M a CAOs can also be inhibited by semicarbazide, which forms a semicarbazone adduct with the n u TPQ cofactor; therefore, CAOs are often classified as semicarbazide-sensitive amine s c oxidases (SSAOs) to distinguish them from other amine oxidases, such as monoamine r ip oxidases A and B (maoA and maoB). However, it should be noted that semicarbazide also t inhibits LOX from bovine aorta (IC = 30 μM), which is similar to CAOs from bovine and 50 human plasma (IC = 50, 100 μM, respectively) ( 34). Additionally, semicarbazide-induced 50 inactivation of LOX has been shown to induce abnormality in arterial structure and function in mice (35). These experiments indicate that the members of the LOX family of proteins are also likely to be SSAOs. A u th For the LOX family of proteins, β-aminopropionitrile (BAPN) is one of the most commonly o r used small molecule inhibitors for in vitro and in vivo experiments. BAPN is considered a M LOX-specific inhibitor (K = 6 μM)( 36) because it does not inhibit CAOs or flavin- a i n dependent maoA or maoB (34). The IC for LOX isolated from chick embryo and bovine u 50 s c aorta were reported to be 10 mM and 25 μM, respectively ( 37,38), while the IC50 for LOX- r ip like 2 (LOXL2, a member of the LOX family of proteins) produced in murine myeloma cells t was reported to be 3-5 μM ( 39). For both LOX and LOXL2, the mode of inhibition is Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 4 competitive. However, there has been some controversy over the specificity of BAPN toward A the LOX family of proteins, as a few groups have reported that BAPN does not inhibit u th LOXL2 in cell culture (39-42). o r M Sequences of Human CAOs and the LOX Family of Proteins a n u Humans have four genes encoding CAOs: AOC1 (diamine oxidase), AOC2 (retina-specific s c amine oxidase), AOC3 (vascular adhesion protein-1, VAP-1), and AOC4 (a pseudo-gene, r ip truncated in the active site). The translated sequences of AOC2 and AOC3 share 65% t identity, but AOC1 only shares ~38% identity with either AOC2 or AOC3 (43). The Tyr precursor for TPQ, the active-site base (Asp), three His for the copper-binding site, and a Tyr residue that has a hydrogen bond interaction with the TPQ cofactor are all conserved (44-46). AOC1 and AOC2 contain predicted secretion signals at their N-termini, while AOC3 does not contain a secretion signal, but has a helical transmembrane (type II) domain. A u t Humans also possess five genes encoding the LOX family of proteins: lox (LOX), loxl1 h o (lysyl oxidase-like 1, LOXL1), loxl2 (lysyl oxidase-like 2, LOXL2), loxl3 (lysyl oxidase- r M like 3, LOXL3), and loxl4 (lysyl oxidase-like 4, LOXL4). The LOX family of proteins can a n be grouped into two subgroups based on the nature of their N-terminal domains: LOX and u s LOXL1 contain a highly basic peptide at their N-termini (termed the propeptide), whereas c rip LOXL2, LOXL3 and LOXL4 each contain four scavenger receptor cysteine-rich (SRCR) t domains (Figure 8)(2). There is a conserved bone morphogenetic protein-1 cleavage site between the propeptide and the LOX catalytic domain of LOX and LOXL1 (47), but this site is not conserved in LOXL2, LOXL3 and LOXL4. Moreover, the C-terminal LOX catalytic domains of LOX and LOXL1 share 77% identity and 88% homology, while the C-terminal LOX catalytic domains of LOXL2, LOXL3 and LOXL4 share 71-72% identity and 84-88% homology. The LOX catalytic domains of the two subgroups share 51-54% identity and A u 64-68% homology. The precursor residues of the LTQ cofactor (Lys and Tyr) and the t h o predicted Cu2+-binding site (His-X-His-X-His) are conserved in all five family members. r M Additionally, all LOX family members possess an N-terminal secretion signal, but lack a n predicted transmembrane domains; therefore, they are generally considered to be secreted u s proteins. Whereas CAOs are known to be homodimers (reviewed in (12)), the oligomeric c r status of the LOX-family of proteins has not been characterized. ip t AMINE OXIDASE (COPPER-CONTAINING) FAMILY Amine oxidase, copper-containing 1, AOC1 (EC1.4.3.22) AOC1 was first described as a histaminase in 1929 (48), and is synonymous with diamine oxidase (DAO1), kidney amine oxidase (KAO), amiloride-sensitive amine oxidase precursor, A u and amiloride-binding protein (ABP1). AOC1 is mainly expressed in the kidney, placenta, t h o intestine, thymus, and seminal vesicles (49), and is proposed to be released from the kidney r M and intestinal epithelial cells through basolateral vesicles at the plasma membrane in a response to an external stimulus, such as heparin (50). AOC1 is the main enzyme n u responsible for metabolism of ingested histamine, and is implicated in histamine intolerance s c r (51). Additionally, AOC1 is highly expressed in the placenta during a healthy pregnancy ip t (1000-fold higher than in other organs), and low AOC1 activity has been linked to high-risk Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 5 pregnancies (52). A recent mice study indicated that AOC1 plays a critical role in A homeostasis of histamine and putrescine levels (preferred substrates of AOC1, see below), u th which is essential for decidualization (i.e. remodeling of the endometrium in preparation for o r embryo implantation) and embryo implantation itself (53). In that study, the expression of M AOC1 was shown to be under the control of estrogen via CCAAT/enhancer-binding protein. a n u s The biochemistry of AOC1 has been studied mostly using recombinant protein produced in c r insect cells (49). The preferred substrates for AOC1 are histamine (K = 2.8 ± 0.07 μM), 1- ip m t methylhistamine (K = 3.4 ± 0.3 μM), and putrescine ( K = 20 ± 1 μM) (Table 1). Longer m m polyamines, such as benzylamine (a common in vitro substrate for serum CAOs and LOX) and spermidine, are poor substrates for AOC1 (Table 1). The X-ray crystal structure of AOC1 was solved at 1.8 Å resolution (43,54), using a template model (AOC3, PDB entry 2c10 (55)) and a sequence alignment of AOC1 and A u AOC3 using CHAINSAW (56). AOC1 is a homodimer of two 85-kDa subunits, and the t h crystal structure revealed the presence of an intermolecular disulfide bridge linking Cys736 o r of the A and B subunits. This intermolecular disulfide bridge has been detected in the crystal M a structures of AOC3 and a plant CAO, but is absent in bacterial and yeast CAOs. n u s c By modeling histamine as the off-copper TPQ-Schiff base intermediate, it was discovered r ip that Asp186 might be within hydrogen bonding distance (3.2 Å) of the imidazole nitrogen of t histamine (43). Therefore, it was postulated that Asp186 might play an important role in binding diamine substrates in the active site of AOC1, though further studies are necessary to evaluate this hypothesis. The crystal structure also confirmed that AOC1 is N- glycosylated at Asn110, Asn538 and Asn745 (three of the four predicted N-glycosylation sites), and the electron density suggests that Asn168 (the remaining predicted site) is not N- A glycosylated (43). The importance of the N-linked glycans for the biochemical and u t physiological functions of AOC1 has not been examined. h o r M Crystal structures of AOC1 complexed with berenil (K = 13 ± 1 nM) or pentamidine (K = i i a n 290 ± 19 nM) have also been solved (43). Berenil and pentamidine are two antiprotozoal u s aromatic diamidine pharmaceutical compounds that noncovalently inhibit AOC1 in mixed c r fashions (43). In the active sites of the two inhibited forms of AOC1, the TPQ cofactor was ip t detected in the inactive, on-copper conformation. Whether the binding of these inhibitors induces the conformational change of TPQ from off-copper (active) to on-copper (inactive) was not discussed. Amine oxidase, copper-containing 2, AOC2 (EC 1.4.3.21) A AOC2 was originally cloned from the retina in 1997 (44). The mRNA of AOC2 has also u t been detected in adipose tissue and was found to be upregulated during in vitro adipocyte h o differentiation (57,58). Additionally, AOC2 has also been detected at the mRNA level in r M many tissues (lung, brain, kidney, cartilage, tonsil, and heart); however, AOC2 amine a n oxidase activity (using tyramine as a substrate) has only been detected in the retina (59). u s Therefore, AOC2 is alternatively known as retina-specific amine oxidase (RAO). c r ip t Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 6 A recombinant form of AOC2 (rAOC2) produced in human embryonic kidney (HEK293) A cells was detected at the cellular surface (59). In that study, crude cell lysates were used to u th conduct kinetic studies. The preferred in vitro substrates for AOC2 were 2- o r phenylethylamine, tryptamine and p-tyramine, instead of methylamine and benzylamine (the M preferred substrates of AOC3, see below). AOC2 does not oxidize histamine (the preferred a n substrate for AOC1) or spermidine (Table 1). u s c r Since AOC2 shares 65% sequence homology with AOC3, homology modeling based on the ip t structure of AOC3 (60) was performed to create a structure model for AOC2 (depicted in Figure 4 of (59)). The monomers of AOC2 and AOC3 superimpose with a root mean square deviation (RMSD) of 0.91 Å. The active site of AOC2 appears to be much larger than AOC3, most likely because Val205 and Asn388 in the active site of AOC2 are smaller than the corresponding residues, Met211 and Tyr394, in that of AOC3 (Figure 4B and 4C in (59)). Not surprisingly, the differences in active site size and structure of AOC2 and AOC3 A u help explain their substrate preferences. For example, docking experiments revealed that the t h o aromatic ring of benzylamine is sandwiched between Tyr384 and Leu469 in AOC3 (Figure r M 4D in (59)). However, in the AOC2 model, benzylamine is stabilized only by Tyr378, due to a the replacement of Leu469 by Gly463 (Figure 4E in (59)). Additionally, 2- n u s phenylethylamine, a good in vitro substrate for AOC2 (but not AOC3), fits generously into c r the modeled active site cavity of AOC2, due to the extra space generated from the Leu469 to ip t Gly463 substitution (Figure 4G in (59)). However, 2-phenylethylamine (Figure 4F in (59)), p-tyramine, and tryptamine cannot be docked in the same position in the AOC3 active site; the additional -CH - groups makes their hydrocarbon chains longer, so that the aromatic ring 2 collides with the surroundings. Amine oxidase, copper-containing 3, AOC3 (EC 1.4.3.21) A u AOC3 is the most studied of the three human CAOs, and has been reviewed previously t h o (5,11,61-63). Alternative names for AOC3 are semicarbazide-sensitive amine oxidase r M (SSAO), vascular adhesion protein-1 (VAP-1), plasma amine oxidase (PAO) and primary a n amine oxidase. AOC3 is found in adipocytes, smooth muscle cells and endothelial cells, and u s is highly expressed in the lung, aorta, liver and ileum. AOC3 is a type II membrane-bound c r protein; soluble AOC3 is released upon proteolytic cleavage of the C-terminus by a ip t metalloprotease (64). Healthy humans have a low level of soluble AOC3 activity in their sera, while an elevated level of AOC3 activity has been observed in the sera of patients suffering from diabetes, congestive heart failure, and liver disorders. The affected organs are thought to be the source of the soluble AOC3 (65,66). Recombinant forms of AOC3 (rAOC3) have been produced in Chinese hamster ovary A u (CHO) cells (46,60), Ax endothelial cells (67), HEK293 cells (68,69), and Drosophila S2 t h cells (70). Recombinant AOC3s produced in HEK and S2 cells were expressed without the o r transmembrane domain (i.e. residues 29-763 were expressed). In addition to the currently M a known endogenous substrates for AOC3 (i.e. methylamine and aminoacetone (71,72)), n u rAOC3 oxidizes benzylamine in vitro, but does not oxidize the diamines histamine or s c putrescine, unlike rAOC1 (Table 1)(49,70). r ip t Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 7 The AOC3 monomer has six predicted N-linked and three putative O-linked glycosylation A sites. A series of rAOC3s with single mutations at each of the 9 glycosylation sites were u th transiently-expressed in Ax cells (a rat high endothelial venule-derived cell line)(67). rAOC3 o r was shown to be glycosylated at all six putative N-linked glycosylation sites, while no O- M linked glycosylation was detected. Among the six N-linked glycans, three N-linked a n carbohydrates are located on the top of the “cap” of AOC3, and could modulate AOC3- u s mediated lymphocyte adherence to the endothelium: when two or all three apical N-linked c r ip glycans were omitted from AOC3, the consequent lymphocyte adhesion to the endothelium t was reduced by 25–35% under non-static assay conditions. Further, the glycosylation was shown to affect the catalytic activity of rAOC3. It was hypothesized that removal of the apical highly sialylated carbohydrates would effect changes in the charge of the rAOC3 molecule, thereby affecting the structural flexibility of rAOC3 and altering its enzymatic activity. A u Recently, two independent detailed biochemical studies were conducted on a rAOC3 t h o produced in HEK293-EBNA1 cells and insect cells (68,70). These rAOC3s were purified to r M >95% homogeneity from serum-free media and were detected as a single band at ~ 100 kDa a (68,70). The stoichiometric amount of titratable TPQ cofactor was ~19% and ~6%, n u s respectively, for the rAOC3s produced in HEK cells and insect cells. Incubation of the c rip partially biogenized rAOC3 from HEK cells in buffer containing excess Cu2+ or O2 did not t change the amount of titratable TPQ. It was concluded that either 1) Cu2+ was replaced by another metal (most likely Zn2+, which does not support TPQ biogenesis (73)) in a large fraction of the purified rAOC3, or that 2) the TPQ cofactor was somehow not able to react fully with phenylhydrazine. Recombinant AOC3 produced in insect cells accepted a variety of primary amines with A different chemical properties (i.e. nonphysiological branched-chain and aliphatic amines), u th with apparent (k /K ) values on the order of 102 to 104 M−1s−1 (70). The K (O ) o cat m m 2 r approximated the partial pressure of oxygen found in the interstitial space. The apparent M a (kcat/Km) values for most of the screened amines only differed 3- to 4-fold between purified n u murine and human rAOC3; however, human rAOC3 was ~10-fold more active towards s c methylamine and aminoacetone (70). r ip t The pH-dependency curve of the steady-state kinetic parameters of rAOC3 produced in HEK cells was fit using nonlinear regression (68). A bell-shaped curve was fit to the apparent k versus pH plot, with two macroscopic pK values (7.0 ± 0.2 and 10.0 ± 0.4) cat a representing ionizable groups in the rAOC3-substrate complex. The pH-dependency of the apparent (k /K ) revealed a single pK value (9.0 ± 0.1) that was assigned to the primary cat m a A amino group of benzylamine. u t h o A kinetic isotope effect (KIE) of 6 to 7.6 was obtained on apparent (kcat/Km) over the pH r M range of 6 to 10 using d2-benzylamine. The KIE on apparent kcat was found to be close to a unity over the same pH range. The unusual KIE values on (k /K ) were explained by a n cat m u s mechanistic scheme including multiple isotopically sensitive steps (typical of CAOs). c r Analysis of quantitative structure-activity relationships (QSAR) using para-substituted ip t protiated and deuterated phenylethylamines was also conducted. With phenylethylamines, a Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 8 large KIE on apparent k (8.01 ± 0.28 with phenylethylamine) was observed, indicating cat A that C–H bond breakage is limiting for TPQ reduction. Poor correlations were observed u th between steady-state rate constants and QSAR parameters. o r M The X-ray crystal structure of rAOC3 expressed in CHO cells was solved and refined to 2.9 a n Å (60), and the structures of two forms of rAOC3 expressed in HEK293 cells (i.e. the wild- u s type (WT) and 2-hydrazinopyridine (2-HP)-inhibited forms) were solved and refined to 2.5 c r Å and 2.9 Å, respectively (55). The major difference between the WT-rAOC3s produced in ip t CHO and HEK cells is the conformation of the TPQ cofactor, i.e. on-copper (inactive) versus off-copper (active) TPQ. TPQ cofactor is known to have some mobility in the active site, and depending on the crystallization conditions, these two forms have been detected routinely (7,12,21). An additional disparity is that the structure of WT-rAOC3 produced in HEK293 cells contains an intermolecular disulfide bridge between Cys41 and Cys748; however, the authors acknowledged the possibility that this was an artifact from the A u crystallization procedure (55). t h o r Overall, the crystal structures of the rAOC3s expressed in CHO and HEK cells are very M a similar to each other while differing from other CAOs in some important ways. As n u mentioned above, rAOC3 possesses an active site cavity that is markedly smaller than that of s c AOC2, owing to the presence of three active site amino acids with much bulkier side chains r ip than those found in AOC2 (59). Additionally, the much narrower substrate entry channel of t rAOC3 distinguishes it from human rAOC1 and CAOs from lower organisms. In both rAOC3 structures, Leu469 is proposed to function as a gate, controlling substrate access to the active site cavity (55,60). Leu468 and Leu469 are located at the bottom part of the substrate entry channel ‘funnel,’ which might sterically hinder larger substrates from entering the active site cavity. This is likely to contribute to the preference of AOC3 for A small amine substrates (e.g. methylamine and aminoacetone) over larger amines (e.g. u t h benzylamine and phenylethylamine) (Table1). o r M In addition to Leu469, Met211 and Tyr394 reside at the bottleneck of the substrate entry a n channel (Figure 7A in (74)), and a triple mutant form (M211V/Y394N/L469G) of rAOC3 u s exhibited substrate specificity similar to that reported for rAOC2 (59). In order to understand c r ip which of the three residues is critical for defining the substrate specificity of AOC3, single t mutants (M211V, Y394N, or L469G) were transiently expressed in CHO cells, and crude cell lysates were used to obtain kinetic parameters (74). Leu469 and Met211 (but not Tyr394) were found to be critical for substrate recognition, and mutation of either of Leu469 or Met211 to the corresponding amino acids in AOC2 (i.e. L469G or M211V) changed the substrate specificity of AOC3. It was proposed that the larger active site of the M211V and A L469G mutants and the absence of large hydrophobic side chains make the correct u t positioning of methylamine (a small substrate) difficult. h o r M Despite these important differences between AOC3 and other CAOs, the active site a configuration of the 2-HP-inhibited form of rAOC3 is very similar to a previously n u s characterized 2-HP-inhibited form of a CAO from E. coli (31,32), and confirmed that c r Asp386 is the active site base for AOC3 and that the pyridine ring of the 2-HP is involved in ip t Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 9 π-stacking interactions with Tyr (Tyr384). This Tyr residue is also conserved in AOC1 A (Tyr371) and AOC2 (Tyr378) (43,59). u t h o More recently, X-ray crystal structures of two imidazole-bound forms (on-copper and off- r M copper TPQ) of the soluble, proteolytically cleaved form of native AOC3 isolated from a n human serum were solved to 2.6-2.95 Å resolution (74). The overall structures of the u s imidazole-bound forms are largely similar to those of rAOC3s, except that Cys748 is c r reduced in the structures of the native AOC3, whereas Cys748 is involved in either an ip t intermolecular or intramolecular disulfide bridge in the rAOC3 structures (55,60,75). It was found that at high concentration (100 mM), imidazole could covalently bind to the active carbonyl group of TPQ at C5. The saturation state of the bond between the N1 nitrogen of imidazole and TPQ was not clear at 2.95 Å resolution; however, imidazole most likely forms a substrate Schiff base-like adduct. The N3 of imidazole was within the necessary distance to hydrogen bond with Asp386, the active site base. The imidazole-bound (TPQ off-copper) A u form of AOC3 could not be derivatized with p-nitrophenylhydrazine and was inactive toward t h o oxidation of substrate amines. Subsequently, it was determined that imidazole inhibits r M competitively, with an IC50 of 1.28 – 8.6 mM. A second molecule of imidazole was also a seen in the AOC3 active site, away from TPQ and was involved in hydrogen bonding n u s interactions with Tyr394 and the main chain nitrogen of Thr212 (through a water molecule), c r and hydrophobic interactions with Leu469 and Tyr176. Based on these observations, the ip t authors noted the potential for inhibitor design based on secondary amine inhibition and/or the selectivity of inhibitors bridging the active site and the secondary imidazole binding site, which appears to be unique to AOC3. In addition to the CAO catalytic domain, AOC3 has an adhesion domain that targets leukocytes for transmigration (76). Both sites and the amine oxidase activity of AOC3 are A critical for AOC3-mediated induction of leukocyte rolling, adhesion and transmigration in u t h response to inflammatory stimuli (77). Inhibition of AOC3 has been shown to be effective in o r mice models of inflammation (in the eyes, carrageenan-injected air pouch, and lungs), M a rheumatoid arthritis, liver fibrosis, and stroke (78,79). These results indicate that AOC3 has n u potential as a therapeutic target for inflammation and fibrosis. Consequently, several s c pharmaceutical companies have developed alkylhydrazino-, guanidine-, and imidazole- r ip derivatives as AOC3 inhibitors with therapeutic potential (reviewed in (63)). An alternative t strategy is to use monoclonal antibodies against AOC3 to disrupt its role in leukocyte trafficking (reviewed in (61)). In vitro and in vivo experiments show that genetically engineered chimeric monoclonal mouse-human antibodies can block sites used by AOC3 to promote leukocyte transmigration in humans without leading to side effects caused by immunogenecity or activation of effector functions (80). BTT-1023 (81), a fully human A monoclonal antibody that specifically binds to AOC3, has been developed and has shown u t h promising efficacy and safety in early clinical studies in rheumatoid arthritis and psoriasis o r patients, and in a range of preclinical models of inflammatory diseases, including chronic M a obstructive pulmonary disease (COPD), certain neurological conditions, and certain niche n u liver inflammatory fibrotic diseases. Currently, it is undergoing phase 2 clinical trials. s c r ip t Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11. Finney et al. Page 10 LYSYL OXIDASE FAMILY A u Protein-lysine 6-Oxidase, LOX (EC.1.4.3.13) t h o r Protein-lysine 6-oxidase, which is more commonly referred to as lysyl oxidase, is expressed M highly in the heart, placenta, skeletal muscle, kidney, lung and pancreas (82). LOX is a n initially translated as pre-pro-LOX containing an N-terminal secretion signal (pre), a highly u s acidic propeptide (pro) and the C-terminal catalytic domain (LOX) (Figure 8). LOX is c r ip proposed to be N-glycosylated at the predicted N-glycosylation sites (Asn81, Asn97, and t Asn144) in the propeptide domain (83). There are no N- or O-glycosylation sites predicted in the LOX catalytic domain. After being secreted from cells, the propeptide is proteolytically cleaved by bone morphogenetic protein-1 (BMP-1), releasing mature LOX (47,84). Recombinant forms of secreted LOX have been prepared from CHO and RFL cell growth A u media (83,85), and biochemical characterization of LOX has been conducted using crude t h o cell lysate and/or crude medium, with the cell lysate or medium from mock-transfected cells r M serving as negative controls (83,85). For LOX activity assays conducted using crude lysate/ a media, the activity is generally expressed in terms of BAPN-inhibitable amine oxidase n u s activity, since BAPN is specific for the LOX-family of proteins, and does not inhibit CAOs c r or maoA or maoB (34). Studies have implicated that pro-LOX is catalytically latent, so ip t processing by BMP-1 has been proposed to be essential for the LOX amine oxidase activity (47). However, no biochemical study using purified proteins has yet compared the relative activities of pro-LOX and mature LOX. In order to assess the importance of N-glycosylation of the propeptide for secretion and protein maturation, a triple mutant form (N81Q/N97Q/N144Q) of pro-LOX was expressed A in CHO cells (83). The triple mutant was secreted into the medium and underwent BMP-1 u t h cleavage, suggesting that N-glycosylation of the propeptide is not essential for secretion or o r proteolytic activation. Intriguingly, the catalytic activity of the triple mutant in the crude M a medium was ~ 40% of that of WT-LOX. Because the propeptide and the associated N-linked n u glycans are not retained by mature LOX, these results suggest that the N-linked glycans in s c the propeptide may play an important role in LTQ biogenesis prior to secretion. r ip t When the propeptide domain was omitted altogether by fusing the catalytic domain of LOX to the signal peptide, LOX was not secreted; instead, it was rapidly degraded in the cells via endoplasmic reticulum-associated protein degradation (ERAD)(83). These data indicate that the propeptide is essential for proper folding and secretion of LOX. Interestingly, the propeptide may also play a role in the recognition of LOX substrates in the extracellular A milieu. To support this, the propeptide domain of pro-LOX was shown to be essential for u t deposition of pro-LOX onto elastic fibers produced in cultures of rat lung fibroblast cells h o (RFL-6) (85). Additionally, when the pre-propeptide without the C-terminal LOX catalytic r M domain was expressed in RFL-6 cells, it was secreted into the medium and still co-localized a n with elastic fibers. u s c r Aberrant expression of LOX has been linked to many diseases. Downregulation or decreased ip t activity of LOX is associated with connective tissue disorders, such as cutis laxa (86) or Arch Biochem Biophys. Author manuscript; available in PMC 2018 June 11.