Elsevier

Cell Calcium

Volume 55, Issue 5, May 2014, Pages 261-268
Cell Calcium

Parvalbumin as a metal-dependent antioxidant

https://doi.org/10.1016/j.ceca.2014.03.001Get rights and content

Abstract

Parvalbumin (PA) is a Ca2+-binding protein of vertebrates massively expressed in tissues with high oxygen uptake and respectively elevated level of reactive oxygen species (ROS). To characterize antioxidant properties of PA, antioxidant capacity (AOC) of intact rat α-PA has been explored. ORAC, TEAC and hydrogen peroxide AOC assays evidence conformation-dependent oxidation of the PA. AOC value for the apo-PA 4-11-fold exceeds that for the Ca2+-loaded protein. Despite folded conformation of apo-PA, it has AOC equivalent to that of the proteolized protein. The most populated under resting conditions PA form, Mg2+-bound PA, has AOC similar to that of apo-PA. ROS-induced changes in absorption spectrum of PA evidence an oxidation of PA's phenylalanines in the ORAC assay. Sensitivity of PA oxidation to its conformation enabled characterization of its metal affinity and pH-dependent behavior: a transition with pKa of 7.6 has been revealed for the Ca2+-loaded PA. Since total AOC of PA under in vivo conditions may reach the level of reduced glutathione, we propose that PA might modulate intracellular redox equilibria and/or signaling in a calcium-dependent manner. We speculate that the oxidation-mediated damage of some of PA-GABAergic interneurons observed in schizophrenia is due to a decline in total AOC of the reduced glutathione–PA pair.

Introduction

Parvalbumin (PA) is a small acidic (106–109 residues, pI 4.1–5.5 (Swiss-Prot release 2013_04)) mainly cytosolic Ca2+-binding protein of vertebrates, which is found in fast-twitch muscles, specific neurons, certain cells of kidney and endocrine glands, Corti's cells, epidermal mucous cells and mucus of some frog and fish genera, etc. (reviewed in [1], [2], [3]). Since no protein targets have been found for PA to date, it is considered as a pure Ca2+-buffering protein. In vivo experiments demonstrated that PA promotes acceleration of muscle relaxation [4], [5], [6] presumably via facilitation of Ca2+ transport from myofibrils to sarcoplasmic reticulum. Besides, in vivo experiments showed that PA affects GABAergic synaptic transmission (reviewed in [7]), while PA expressed in the early part of the distal convoluted tubule in kidneys is engaged into the distal handling of electrolytes (reviewed in [8]). PAs found in the cutaneous mucus of amphibians and fishes are shown to participate in innate bacterial defense mechanisms by means of Ca2+ chelation and to serve as chemoattractants for different thamnophiine snakes [3]. PA is a major fish allergen [9]. Genetic approaches aimed at heart-specific expression of PA are suggested for therapy of relaxation abnormalities in different heart diseases and failures [10], [11]. PA is frequently expressed in mammalian tumors and considered as a marker of chromophobe carcinoma and oncocytoma (reviewed in [8]). PA level in PA-GABAergic interneurons is down-regulated in schizophrenia [12], [13], [14], [15].

Some experimental facts favor the view that PA is engaged into intracellular oxidative processes. For example, PA is massively expressed in the tissues characterized by highly efficient oxygen uptake: PA concentration in brain and skeletal muscles (oxygen consumption of 52 and 57 ml/min, respectively [16]) reaches 1.8 mM [7] and 4.9 g/kg of wet muscle tissue (ca 0.6 mM) [17], respectively. The consumed molecular oxygen is utilized in mitochondria during oxidative phosphorylation, ultimately leading to production of ATP molecules. Up to 0.5% of the oxygen consumed by mitochondria escape from the mitochondrial electron-transfer chain (complexes I and III) and experience reduction to superoxide anion (O2·), which is subsequently converted to hydrogen peroxide (H2O2) (reviewed in [18]). The latter is one of the most abundant (0.1 μM) and long living (half-life time of 10 μs) intracellular reactive oxygen species (ROS), which serves both as an inducer of oxidative damage and signaling molecule [18]. Though several enzymatic systems generate H2O2 in different cellular compartments, mitochondrial respiration is considered as the major intracellular source of ROS production. Hence, higher oxygen consumption in the PA-rich cells is associated with more intense production of ATP and ROS, including O2·and H2O2. Similarly, muscular exercise promotes formation of ROS (reviewed in [19]), affecting PA expression level [20]. Furthermore, PA expression positively correlates with relaxation speed in skeletal muscles [17], [21], [22] and, respectively, with their energy and oxygen requirements. PA immunoreactivity was found in (or near) mitochondria [23]. Finally, oxidative stress observed in schizophrenia [12], [24] is accompanied by a decrease in the PA level in PA-GABAergic interneurons [12], [13], [14], [15]. These facts suggest that PA might be involved into redox signaling and/or free radical scavenging. To explore the latter possibility, antioxidant capacities (AOC) of various metal bound and metal free forms of rat PA have been studied here using several techniques. The regularities found here are useful for an insight into an oxidation-mediated pathway of schizophrenia progression via suppression of the PA-GABAergic interneurons.

Section snippets

Materials

Tris, H3BO3, EDTA, NaCl, Fe(II)(NH4)2(SO4)2, KSCN, potassium persulfate, PMS, NADH, AAPH, ABTS, Trolox, and premium grade α-chymotrypsin from bovine pancreas were bought from Sigma–Aldrich Co. EGTA, standard solutions of CaCl2 and MgCl2 (Ca2+ content of 0.0005%), H2O2 and fluorescein were from Fluka. GSH was from AppliChem GmbH. NBT was from Roth. Phosphoric acid was from Merck. Phe was from Diam (Moscow, Russia). Sephadex G-25 was a product of Pharmacia LKB. Tris, NaCl, H2O2, potassium

Metal binding-induced changes in AOC of PA

Four in vitro AOC assays, differing by ROS/radicals and reaction involved (hydrogen atom or electron transfer), were applied to various states of intact rat α-PA at 37 °C/25 °C and pH 7.5/(8.1–8.3), using Trolox as a reference substance (Table 1). Two of them, hydrogen peroxide antioxidant capacity (H2O2 AOC) assay and superoxide radical (O2radical dot) scavenging assay, are of special interest due to the use of the biologically relevant ROS [18].

H2O2 is the most abundant (0.1 μM) and long living (half-life

Discussion

Three of the four in vitro AOC assays, differing by the ROS/radicals and reaction involved, manifest conformation-dependent oxidation of PA by both biologically relevant and model ROS/radicals (Table 1). This result reflects differences in solvent accessibility of oxidatively labile residues of different PA forms [36]. Only O2radical dot scavenging assay was insensitive to PA conformation, likely due to much higher reactivity of O2radical dot [18].

The apo-PA, the form being least populated under in vivo conditions

Conflict of interest statement

We confirm that there is no conflict of interest in this manuscript.

Acknowledgements

This work was supported by grants from the Program of the Russian Academy of Sciences «Molecular and Cellular Biology» (E.A.P.) and Russian Foundation for Basic Research14-04-31708 (N.V.A.). We are indebted to Victoria A. Rastrygina (IBI RAS, Pushchino) for the help with preparation of Fig. 3.

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