View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Universidade de Lisboa: Repositório.UL molecules Review Human Erythrocyte Acetylcholinesterase in Health and Disease Carlota Saldanha Instituto de Bioquímica, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Professor Egas Moniz, 1649-028 Lisboa, Portugal; [email protected] Received: 10 August 2017; Accepted: 4 September 2017; Published: 8 September 2017 Abstract: The biochemical properties of erythrocyte or human red blood cell (RBC) membrane acetylcholinesterase (AChE) and its applications on laboratory class and on research are reviewed. Evidence of the biochemical and the pathophysiological properties like the association between the RBC AChE enzyme activity and the clinical and biophysical parameters implicated in several diseases are overviewed, and the achievement of RBC AChE as a biomarker and as a prognostic factor are presented. Beyond its function as an enzyme, a special focus is highlighted in this review for a new function of the RBC AChE, namely a component of the signal transduction pathway of nitric oxide. Keywords: acetylcholinesterase; red blood cells; nitric oxide 1. Introduction Erythrocytes or red blood cells (RBC) are more than sacks of oxyhemoglobin or deoxyhemoglobin during the semi-life of 120 days in blood circulation [1]. Erythrocytes comport different signaling pathways which includes the final stage of apoptosis, also called eryptosis [2,3]. Exovesicules enriched with acetylcholinesterase (AChE) originated from membranes of aged erythrocytes appear in plasma [4]. Kinetic changes of the AChE enzyme have been observed in old erythrocytes [5]. Previously, AChE in erythrocytes was evidenced as a biomarker of membrane integrity [6]. Later on, increased impairment values of AChE enzyme activities were observed in several diseases as will be described below. The blood physiological functions at macro- and microcirculatory vessel networks are dependent on RBCs’ membrane integrity and the normal interaction with endothelium and other blood components [7]. Luminal vascular endothelial cells can rest in a stationary phase or be activated during an inflammatory response. The degree of resolution of that response creates a solved acute inflammation or unsolved chronic inflammation. In all situations, erythrocytes are a player [8]. Depending on the degree of endothelium integrity, the plasma acetylcholine (ACh) induces vasodilation or vasoconstriction through the amount of nitric oxide (NO) synthesized by endothelial cells and released to the vessels smooth muscle [9,10]. The NO released from endothelial cells to the lumen is scavenged by the erythrocytes through the band 3 protein, providing a route for an NO influx to, and an efflux from, erythrocytes [11–13]. NO is rescued by the hemoglobin molecule forming S-nitrosohemoglobin (SNOHb) inside the erythrocyte [12,13]. In blood circulation, where the erythrocyte senses tissues with low partial oxygen pressure, NO is transferred from β3 SNOHb to the thiol group of band 3 with an NO efflux to the lumen vessel [13]. Using in vitro inhibitors of protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP), phosphorylation and dephosphorylating of band 3 at tyrosine residues have been evidenced and the two forms exist in a dynamic equilibrium [14]. Dephosphorylate erythrocyte membrane band 3 is associated with oxyhemoglobin and with the glycolytic enzymes, glyceraldehyde dehydrogenase, aldolase, and phosphofructokinase, which disclose to the cytosol under phosphorylation band 3 state [15]. Molecules 2017, 22, 1499; doi:10.3390/molecules22091499 www.mdpi.com/journal/molecules Molecules 2017, 22, 1499 2 of 10 A higher erythrocyte aggregation tendency and increased membrane AChE enzyme activity is also evidenced when band 3 is phosphorylated, but not when it is dephosphorylated [16,17]. In addition, glutathione is an abundant molecule inside erythrocytes, which has a thiol group that can react with NO, forming nitrosothiols such as S-nitrosoglutathione (GSNO) [18]. The NO reservoir property Molecules 2017, 22, 1499 2 of 10 attributed to glutathione might be influenced by the inactivation of glutathione reductase induced by the oxidativeA stresshigher installederythrocyte in aggregation erythrocytes tendency [19]. and increased membrane AChE enzyme activity is Attemptingalso evidenced to pursuewhen band the 3 is challenge phosphorylated, of finding but not awhen physiological it is dephosphorylated function [16,17]. for erythrocyte In addition, glutathione is an abundant molecule inside erythrocytes, which has a thiol group that can membrane AChE, the nitric oxide discovery triggered by plasma ACh gave us a clue about the react with NO, forming nitrosothiols such as S-nitrosoglutathione (GSNO) [18]. The NO reservoir action ofproperty AChE. attributed Erythrocyte to glutathione membrane might AChE be influenc is involveded by the in inactivation the nitric of oxide glutathione (NO) reductase signal pathway as evidenced,induced for by the the firstoxidative time stress at the in startstalled of in this erythrocytes century, in[19]. several in vitro studies using blood samples from healthyAttempting donors as to described pursue the below. challenge of finding a physiological function for erythrocyte membrane AChE, the nitric oxide discovery triggered by plasma ACh gave us a clue about the action 2. Biochemicalof AChE. Properties Erythrocyte ofmembrane Human AChE Erythrocyte is involved Membrane in the nitric Acetylcholinesterase oxide (NO) signal pathway as evidenced, for the first time at the start of this century, in several in vitro studies using blood samples Humanfrom healthy erythrocyte donors acetylcholinesterase as described below. (AChE) discovered by Alles and Haves in 1940 was later, in 1961, classified as EC.3.1-1.7 by the Enzyme Commission [20,21]. Only in 1975 the appropriate process of extraction2. Biochemical and purification Properties ofof theHuman erythrocyte Erythrocyte membrane Membrane AChE Acetylcholinesterase confirmed it as a glycoprotein [22]. Later in 1985Human it was erythrocyte shown that acetylcholinesterase this enzyme, located (AChE) in thediscovered external by leaflet Alles and of the Haves erythrocyte in 1940 was membrane, is a dimericlater, protein in 1961, [classified23]. The as catalytic EC.3.1-1.7 efficiency by the Enzyme of the Commission dimeric form [20,21]. of AChE Only in depends 1975 the onappropriate the amphipathic process of extraction and purification of the erythrocyte membrane AChE confirmed it as a medium of extraction and purification [24,25]. AChE belongs to the glycosylphosphatidylinositol glycoprotein [22]. Later in 1985 it was shown that this enzyme, located in the external leaflet of the (GPI)-anchorederythrocyte protein membrane, family is a and dimeric bears protein the Yta [23]. blood The catalytic group efficiency antigen of [26 the,27 dimeric]. form of AChE Thedepends kinetic on profile the amphipathic of AChE shows medium a bell-shape of extraction curve, and purification (Figure1) [ [24,25].24], meaning AChE belongs that the to enzyme-free, the enzymeglycosylphosphatidylinositol substrate complex and the(GPI)-anchored acyl enzyme protein intermediate family and bears form the Yta all blood exist group in the antigen reaction [26,27]. medium; AChE is uncompetitive,The kinetic profile inhibited of AChE by show the excesss a bell-shape of substrate curve, (Figure concentration 1) [24], meaning which hasthat beenthe enzyme- shown also by others authorsfree, enzyme [28]. substrate This mechanism complex and showed the acyl a secondenzyme substrateintermediate molecule form all bindingexist in the to thereaction free anionic medium; AChE is uncompetitive, inhibited by the excess of substrate concentration which has been group ofshown the active also by center others of authors the acyl [28]. enzyme This mechanism complex [showed24]. The a second optimum substrate substrate molecule concentrations binding to values, which resultthe free from anionic the velocitygroup of the curve active profile, center areof th dependente acyl enzyme on complex the native, [24]. solubilized,The optimum or substrate purified forms of the enzyme,concentrations of the values, ionic strength which result of the from medium the velo andcity pHcurve values profile, and are of dependent the type on and the concentration native, of inhibitorsolubilized, or activator or purified compounds forms of [24 the]. Anotherenzyme, of parameter the ionic strength influent of the on medium optimum and substrate pH values values and is the methodof applied the type for and the concentration kinetic evaluation of inhibitor according or activator to thecompounds product, [24]. thiocholine Another parameter or acetate, influent that needs to on optimum substrate values is the method applied for the kinetic evaluation according to the be measuredproduct, when thiocholine acetylthiocholine or acetate, that is used needs as to the be syntheticmeasured analogwhen acetylthiocholine of acetylcholine is used (ACh), as the natural substratesynthetic of AChE analog [24, 29of ].acetylcholine A lower affinity (ACh), constantthe natural for substrate the substrate of AChE and [24,29]. a higher A lower optimal affinity substrate concentrationconstant value for the are substrate obtained and at a high higher ionic optimal strength substrate in relation concentration to the lowervalue are ionic obtained strength at high of