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Copper‑Containing Amine Oxidase Purified from Lathyrus Sativus As A INTERNATIONAL JOURNAL OF MOleCular meDICine 45: 1583-1590, 2020 Copper‑containing amine oxidase purified fromLathyrus sativus as a modulator of human neutrophil functions PAOLA PIETRANGELI1*, Elisabetta CApuOzzO1*, MIRCEA AlExANDRu Mateescu2 and LUCIA MARCOCCI1 1Department of Biochemical Sciences ‘A. Rossi Fanelli’, Sapienza university of Rome, I‑00185 Rome, Italy; 2Department of Chemistry, Research Chair on Enteric Dysfunctions ‘Allerdys’ and CERMO‑FC Centre, university of Quebec at Montreal (uQAM), Montreal, (QC) H3C 3p8, Canada Received August 2, 2019; Accepted January 27, 2020 DOI: 10.3892/ijmm.2020.4535 Abstract. Over the last few decades, copper-containing amine the fMLP-dependent superoxide generation, elastase release oxidase (Cu-AO) from vegetal sources, and belonging to the and cell migration, and interferes with the process of calcium class of diamine oxidase, has been documented to exhibit flux, supporting the idea that plant Cu‑AO can interact with beneficial effects in both in vivo and ex vivo animal models human neutrophils to modulate their inflammatory function. of inflammatory or allergic conditions, including asthma‑like Therefore, the importance of these properties on the possible reaction and myocardial or intestinal ischemia‑reperfusion use of vegetal Cu‑AO to control inflammatory conditions, injuries. The aim of the present study was to assess the particularly intestinal inflammation, is discussed in the current potential of vegetal Cu‑AO as an anti‑inflammatory and study. an antiallergic agent and to clarify its antioxidant proper- ties. In cell‑free systems, the reactive oxygen species and Introduction reactive nitrogen species scavenging properties of Cu-AO that is purified from Lathyrus sativus were investigated. Its An inflammatory response is the result of a complex and effect on the formyl‑methionyl‑leucyl‑phenylalanine peptide interconnected series of events of the immune system aimed at (fMLP)-activated cellular functions of human neutrophils were destroying foreign agents or altered cells, but inevitably leading subsequently analyzed. The obtained results demonstrated that to healthy tissue damage at the site of injury (1). In particular, Cu-AO is not a scavenger of superoxide or nitric oxide, and activated neutrophils, which are key effectors of the innate does not decompose hydrogen peroxide. However, it inhibits immune responses, generate reactive oxygen species (ROS) and reactive nitrogen species (RNS), proteolytic enzymes and neutrophil extracellular traps to eliminate pathogens and dying cells (2). However, these molecules can also interact with nucleic acids, proteins and lipids of the surrounding Correspondence to: Professor Lucia Marcocci, Department of healthy tissues, causing changes in their structure and function Biochemical Sciences ‘A. Rossi Fanelli’, Sapienza university of with detrimental consequences for cellular homeostasis (3). Rome, piazzale Aldo Moro 5, I‑00185 Rome, Italy E-mail: [email protected] The neutrophil-mediated activation of the immune response, as a causative or secondary factor, occurs in a broad range *Contributed equally of pathologies, including post-ischemic reperfusion damage, neurodegeneration, obstructive pulmonary disease, obesity, Abbreviations: AAp, aminoantipyrine; BSA, bovine serum albumin; diabetes and cancer (4). The excessive activated neutrophil BSAO, bovine serum Cu‑AO; CRAC, calcium‑release activated recruitment also takes place in a variety of intestinal dysfunc- channels; Cu‑AO, copper‑containing amine oxidase; DAO, diamine tions and inflammatory conditions, including intestinal oxidase; DCHBS, 3,5‑dichloro‑2‑hydroxybenzensulfonic acid; ischemia and inflammatory bowel diseases (IBD), including EGTA, ethylene glycol‑bis(β‑aminoethyl ether)‑N,N,N',N'‑tetraacetic Crohn's disease and ulcerative colitis (5,6). Therefore, there acid; fMLP, formyl‑methionyl‑leucyl‑phenylalanine peptide; HRp, is a growing effort to understand the mechanisms associated horseradish peroxidase; IBD, inflammatory bowel disease; lSAO, with the action of neutrophils in inflammatory sites, and to Cu-AO purified from Lathyrus sativus; pBS, phosphate‑buffered saline; NO, nitric oxide; ROS, reactive oxygen species; RNS, identify novel factors that may modulate neutrophil behavior reactive nitrogen species; SOCE, store‑operated calcium entry; in order to develop therapeutic strategies to limit or neutralize TG, thapsigargin; VAp‑1, vascular adhesion protein 1 their destructive action. Serotonin, histamine, the putrescine diamine, the spermine Key words: copper-containing amine oxidase, diamine oxidase, and spermidine polyamines exert immunomodulatory, neuro- histaminase, neutrophils, oxidative stress, inflammation, active and proliferative functions (7). Consequently, during the inflammatory bowel disease last few decades copper-containing amine oxidases (Cu-AOs) have been proposed as efficient anti‑inflammatory and anti‑allergic agents (8), due to their ability to metabolize the 1584 PIETRANGELI et al: MODULATION OF HuMAN NEuTROpHIl FuNCTIONS BY VEGETAl Cu-AO primary amine groups of these biogenic amines as follows (9): (H2O2), N‑methoxysuccinyl‑ Ala‑Ala‑​Pro‑Val‑7‑amido‑4‑ R‑CH2‑NH2+O2+H2O → RCHO+NH3+H2O2. The beneficial methylcoumarin (MeO‑Suc‑Ala‑Ala‑​Pro‑Val‑AMC), porcine effects of Cu-AOs from vegetal sources have been documented pancreas elastase, sulphanilamide, sodium diethyldithiocarba- both in in vivo and ex vivo animal models of inflammatory or mate (DDC), sodium nitroprusside, sodium nitrite, thapsigargin allergic conditions, including asthma‑like reaction (10) and (TG), trypan blue and xanthine were purchased from Merck myocardial (11) or intestinal (12) ischemia‑reperfusion injuries, KGaA. Fura‑2‑acetoxymethyl ester (Fura‑2AM) was purchased in which the treatment with vegetal Cu-AO was demonstrated to from Thermo Fisher Scientific, Inc., polymorphprep™ solu- promote the alleviation of these dysfunctions and the decrease tion from Sentinel Diagnostics and Superdex D200 FPLC of tissue histamine level, neutrophil infiltration and cellular column from GE Healthcare. ultrafiltration devices with oxidative damage. Also, the removal of histamine, which is a cut‑off of 30 kDa were obtained from Sartorious AG. an efficient substrate of vegetal Cu‑AO (13) (for this reason QCM™ Chemotaxis 3 µm 96‑well cell migration assay system the enzyme is also called histaminase, as well as diamine was from Merck KGaA. Other chemicals were analytical grade oxidase (DAO) for its ability to metabolize diamines), was and were used without further purification. associated with the anti‑inflammatory function of this enzyme and its anti‑allergic properties. Histamine, which is a major LSAO purification and characterization. lSAO was prepared mast cell‑derived mediator, is not only an efficient modulator from Lathyrus sativus seedlings, according to a large-scale of endothelial cell contraction and vascular permeability, but preparation method (19). Following purification, samples also a pro‑inflammatory amine able to induce the expression were dialyzed at 4˚C overnight against 50 mM phosphate of endothelial cell adhesion molecules, thus promoting the buffer (1:1,000; pH 7.4), concentrated on ultrafiltration devices rolling and extravasation of leukocytes (14). and further purified by size exclusion chromatography on a Cu‑AO purified from bovine serum (BSAO) has been Superdex D200 FPLC column, equilibrated and eluted with reported to exert a cardioprotective effect in isolated rat hearts 50 mM phosphate buffer (pH 7.4) at a flow rate of 0.5 ml/min exposed to electrolysis‑generated ROS, and to have a scav- using a FPLC System (GE Healthcare). Aliquots with the enging capacity against ROS and their by‑products generated highest AO‑specific activity were pulled together, concen- by electrolysis in Krebs‑Henseleit buffer (superoxide, hydroxyl trated using ultrafiltration and stored at ‑20˚C until use. radical, singlet oxygen, hydrogen peroxide and hypochlorous The purified protein (10 µg/lane) moved as a single band on acid) (15), despite the fact that Cu‑AOs generate hydrogen 7.5% SDS‑pAGE. lSAO preparations were characterized for peroxide. It has been hypothesized that the beneficial effects protein content and enzymatic activity was measured in terms of plant Cu‑AO are associated with its scavenging capacity of H2O2 generation from the oxidation of 3 mM putrescine, as of ROS and RNS that are generated by activated neutrophils previously reported (20). lSAO preparations with a specific and the consequent reduction of tissue oxidative damage (8). activity of 40±5 U/mg protein were used. 1 U enzyme oxidizes However, direct evidence of the scavenging activity of vegetal 1 µmol substrate/min. Cu‑AO is lacking. Catalytically inactive lSAO samples were prepared With the aim to clarify the antioxidant and anti‑inflamma- upon the depletion of copper from the active site by DDC tory properties of vegetal Cu‑AO and further define its potential treatment (21). The residual copper content was measured as use as a novel antiallergic and anti‑inflammatory agent, the previously described (22). present study analyzed the scavenging properties of Cu‑AO purified fromLathyrus sativus (lSAO) seedlings against ROS Neutrophil isolation. Neutrophils were purified from heparin- and RNS generated chemically in cell‑free systems. Moreover, ized human blood, as previously reported (23), freshly drawn vascular adhesion protein 1 (VAp‑1) is a peculiar Cu‑AO of the from repeat healthy blood donors at the
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