Implication for Functions of the Ectopic Adipocyte Copper Amine Oxidase (AOC3) from Purified Enzyme and Cell-Based Kinetic Studies

Abstract

AOC3 is highly expressed in adipocytes and smooth muscle cells, but its function in these cells is currently unknown. The in vivo substrate(s) of AOC3 is/are also unknown, but could provide an invaluable clue to the enzyme's function. Expression of untagged, soluble human AOC3 in insect cells provides a relatively simple means of obtaining pure enzyme. Characterization of enzyme indicates a 6% titer for the active site 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor and corrected k cat values as high as 7 s−1. Substrate kinetic profiling shows that the enzyme accepts a variety of primary amines with different chemical features, including nonphysiological branched-chain and aliphatic amines, with measured k cat/Km values between 102 and 104 M−1 s−1. Km(O2) approximates the partial pressure of oxygen found in the interstitial space. Comparison of the properties of purified murine to human enzyme indicates k cat/Km values that are within 3 to 4-fold, with the exception of methylamine and aminoacetone that are ca. 10-fold more active with human AOC3. With drug development efforts investigating AOC3 as an anti-inflammatory target, these studies suggest that caution is called for when screening the efficacy of inhibitors designed against human enzymes in non-transgenic mouse models. Differentiated murine 3T3-L1 adipocytes show a uniform distribution of AOC3 on the cell surface and whole cell Km values that are reasonably close to values measured using purified enzymes. The latter studies support a relevance of the kinetic parameters measured with isolated AOC3 variants to adipocyte function. From our studies, a number of possible substrates with relatively high k cat/Km have been discovered, including dopamine and cysteamine, which may implicate a role for adipocyte AOC3 in insulin-signaling and fatty acid metabolism, respectively. Finally, the demonstrated AOC3 turnover of primary amines that are non-native to human tissue suggests possible roles for the adipocyte enzyme in subcutaneous bacterial infiltration and obesity.

Intrigues and Intricacies of the Biosynthetic Pathways for the Enzymatic Quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ

“…148,149 Even in the case of the mammalian TPQ-dependent enzymes (where several intracellular enzymes are documented), 150,151 important ectopic enzymatic variants have been identified on the outer plasma membrane of the endothelium of the vasculature and of adipocytes. 152-154 Soluble forms of TPQ-containing CAOs have also been identified in serum. 155,156 These considerations raise a very intriguing possibility: that the elusive evolutionary link among the structurally divergent quinone cofactors lies with the subcellular location in which their respective enzymes function.…”

Structural Analysis of Aliphatic versus Aromatic Substrate Specificity in a Copper Amine Oxidase from Hansenula polymorpha

“…5-7 The presence of the extracellular CAO in adipocytes is also well documented, and while initially ascribed to a role in moderating glucose uptake, 8 may also present a first line of defense against invading pathogens. 9 The products generated during CAO catalysis can be highly cytotoxic, in particular formaldehyde, which has been implicated in protein crosslinking correlated with cellular damage in late-diabetic vascular complications and atherosclerosis. 10 …”

“…14 In contrast, VAP-1 that is localized to the outer plasma membrane of adipocytes recognizes a wide range of aliphatic and aromatic amines of variable size. 9 …”

Systemic Manifestations of Mucosal Diseases

“…Thus, in the presence of an inflamed gut, increased portal vein levels of certain amines (e.g., methylamine) could activate VAP-1-dependent hepatic MAdCAM-1 expression, thereby promoting the recruitment of mucosal effector cells to the liver (Liaskou et al, 2011). Substrates for VAP-1 include not only dietary amines but those produced by gut commensals and enteric pathogens such as Bacteroides fragilis, Salmonella typhimurium, Yersinia entercolitica, Escherichia coli, and Clostridium perfringens, providing a potential link among the microbiota, mucosal immunity, and pathogenesis of PSC (Shen et al, 2012).…”