Biochem. J. (2015) 469, 211–221

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doi:10.1042/BJ20140768

Oxygen reactivity of mammalian sulfite oxidase provides a concept for the treatment of sulfite oxidase deficiency Abdel A. Belaidi*1,2 , Juliane R¨oper*, Sita Arjune*, Sabina Krizowski*, Aleksandra Trifunovic† and Guenter Schwarz*2,3 *Institute of Biochemistry, Department of Chemistry, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, Zuelpicher Straße 47, 50674 Cologne, Germany †CECAD Research Center, University of Cologne, Joseph-Stelzmann-Straße 26, 50931 Cologne, Germany

Mammalian sulfite oxidase (SO) is a dimeric enzyme consisting of a molybdenum cofactor- (Moco) and haem-containing domain and catalyses the oxidation of toxic sulfite to sulfate. Following sulfite oxidation, electrons are passed from Moco via the haem cofactor to cytochrome c, the terminal electron acceptor. In contrast, plant SO (PSO) lacks the haem domain and electrons shuttle from Moco to molecular oxygen. Given the high similarity between plant and mammalian SO Moco domains, factors that determine the reactivity of PSO towards oxygen, remained unknown. In the present study, we generated mammalian haemdeficient and truncated SO variants and demonstrated their oxygen reactivity by hydrogen peroxide formation and oxygenconsumption studies. We found that intramolecular electron transfer between Moco and haem showed an inverse correlation to SO oxygen reactivity. Haem-deficient SO variants exhibited oxygen-dependent sulfite oxidation similar to PSO, which was confirmed further using haem-deficient human SO in a cell-

based assay. This finding suggests the possibility to use oxygenreactive SO variants in sulfite detoxification, as the loss of SO activity is causing severe neurodegeneration. Therefore we evaluated the potential use of PEG attachment (PEGylation) as a modification method for future enzyme substitution therapies using oxygen-reactive SO variants, which might use blooddissolved oxygen as the electron acceptor. PEGylation has been shown to increase the half-life of other therapeutic proteins. PEGylation resulted in the modification of up to eight surfaceexposed lysine residues of SO, an increased conformational stability and similar kinetic properties compared with wild-type SO.

INTRODUCTION

eukaryotic molybdenum enzyme [7]. As a result, electrons derived from sulfite oxidation are passed directly to molecular oxygen, a process that generates hydrogen peroxide (H2 O2 ), which, in the presence of the enzyme catalase, is converted further into water and oxygen [8]. The latter explains the peroxisomal localization of PSO [9], whereas animal SO is localized in the mitochondrial inter-membrane space, where it uses cytochrome c as the terminal electron acceptor [10]. SO from chicken was the first reported crystal structure of a eukaryotic molybdenum enzyme and has been fundamental in understanding the relationship between the molybdenum and haem domains in animal SO [11]. Within the crystal structure of chicken SO, a large distance of 32 Å (1 Å = 0.1 nm) was found between the molybdenum and haem domain [11], which would not support the high electron transfer rates measured between both domains using laser flash photolysis methods [12]. Thus it has been suggested that animal SO may undergo conformational change that brings the molybdenum and haem domains in close proximity in order to allow fast electron transfer. On the basis of the decrease in IET as a function of viscosity [13] and residue deletions within the tether connecting the molybdenum

Molybdenum enzymes catalyse key redox reactions in the global carbon, sulfur and nitrogen cycles [1]. Their overall reaction is characterized by the transfer of an oxygen atom to or from a substrate in a two-electron transfer reaction [2]. Molybdenum is bound to the pterin-based molybdenum cofactor (Moco) of those enzymes, which in most cases harbour additional prosthetic groups for intramolecular electron transfer (IET). Most molybdenum enzymes are found in bacteria; in eukaryotes only five molybdenum enzymes are known so far [3]. In mammals, the most important molybdenum enzyme is sulfite oxidase (SO), which is mainly found in liver, where it catalyses the oxidation of sulfite, which is generated throughout the catabolism of cysteine [4]. Animal SO is a dimeric enzyme, which harbours a cytochrome b5 -type haem domain in addition to a Moco domain [5]. The catalytic cycle of SO involves electron transfer from sulfite to Moco, followed by two electron-transfer steps via the cytochrome b5 domain to the terminal electron acceptor cytochrome c [6]. The orthologous plant SO (PSO) lacks the haem domain and thereby constitutes the simplest

Key words: hydrogen peroxide, intramolecular electron transfer, molybdenum cofactor deficiency, oxygen reactivity, PEGylation, sulfite oxidase.

Abbreviations: cPMP, cyclic pyranopterin monophosphate; FDA, Food and Drug Administration; HEK, human embryonic kidney; HSO, human SO; HSOMo , human SO molybdenum domain; IET, intramolecular electron transfer; MoCD, Moco deficiency; Moco, molybdenum cofactor; MOCS , molybdenum cofactor synthesis; MSO, murine SO; MSOMo , murine SO molybdenum domain; NHS, N -hydroxysuccinimide; PSO, plant SO; SO, sulfite oxidase; SOD, SO deficiency; wt, wild-type. 1 Present address: The Florey Institute for Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Parkville, Victoria 3052, Australia. 2 Abdel A. Belaidi and Guenter Schwarz are co-inventors on a patent application related to the use of PEGylated human sulfite oxidase for enzyme substitution therapy in molybdenum cofactor deficiency. 3 To whom correspondence should be addressed (email [email protected]).  c 2015 Authors; published by Portland Press Limited

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and haem domains [14], it was confirmed that animal SO undergoes dynamic conformational changes, which guide the haem domain in docking through electrostatic interactions to the molybdenum domain thus allowing efficient electron transfer. The reaction mechanism of SO can be divided into a reductive and oxidative half-reaction [15]. In the reductive half-reaction, sulfite binds at the MoVI centre and is oxidized to sulfate by the transfer of two electrons to the molybdenum centre yielding the reduced MoIV species. According to the respective reduction potentials, one electron is transferred via IET to the haem domain creating a paramagnetic MoV intermediate state, which can be detected using EPR spectroscopy [16]. The oxidative half-reaction is initiated with the transfer of one electron from haem to the final electron acceptor cytochrome c. Then the second electron can leave the MoV centre by a second IET step via haem to a second cytochrome c yielding the fully oxidized form of the enzyme [15]. The crystal structure of PSO from Arabidopsis thaliana depicted the close resemblance of the molybdenum domain between animal and plant SOs [7] suggesting a similar reductive half-reaction during the catalytic cycle of sulfite oxidation for animal and plant SOs [15]. However, the absence of the haem domain in PSO implicates a different oxidative halfreaction from that in animal SO. In fact, it has been shown that the molybdenum domain of PSO reacts directly with oxygen leading to the formation of superoxide ions as the immediate product of the oxidative half-reaction, which is spontaneously dismutated to H2 O2 [17]. The PSO reaction with oxygen is very unusual as none of the molybdenum enzymes characterized so far are able to react at significant rates with oxygen [17]. Furthermore, plant and animal SO share a structurally very similar molybdenum centre and residues important for substrate binding are strictly conserved [15]. Thus the factors responsible for PSO reactivity towards oxygen remain unknown. The impact of mutations on IET between the molybdenum and haem domains have been investigated using the human SO enzyme [18,19]. Interestingly, the authors showed also a reactivity of human SO with ferricyanide as the electron acceptor, which, unlike cytochrome c, can directly be reduced by the molybdenum centre. However, oxygen reactivity studies were not performed with human SO [19]. Defects in any step of the biosynthesis of Moco lead to Moco deficiency (MoCD), a rare inherited metabolic disorder resulting in the loss of activity of all molybdenum enzymes, and affected patients usually die in early childhood [20]. Mutations in the SUOX gene result in SO deficiency (SOD); approximately 30 cases have been reported so far [21]. MoCD and SOD are clinically very similar, which qualifies SO as the most important molybdenum enzyme in humans [22]. Both deficiencies are characterized by a severe neurodegenerative phenotype [20] resulting from the accumulation of sulfite and other toxic metabolites such as S-sulfocysteine [24]. MoCD can be grouped into three types according to the underlying genetic defect [1]. Type A deficiency affects two-thirds of all patients and is caused by mutations in the MOCS1 (molybdenum cofactor synthesis 1) gene [25]. Type B patients accumulate the first Moco intermediate cPMP (cyclic pyranopterin monophosphate) [26] due to defects in the MOCS2 gene [27]. Type C deficiency affects the GPHN (gephyrin) gene [28] with only two cases reported to date [29,30]. Until the last few years, no effective therapy was available for MoCD, and death in early childhood has been the usual outcome [1]. The ability to purify the first Moco intermediate cPMP [26] was the starting point for the establishment of the first treatment approach towards MoCD type A and, since 2010, several replacement therapies with cPMP have been reported for  c 2015 Authors; published by Portland Press Limited

MoCD type A patients [31,32]. Treated patients were exposed to repetitive intravenous injections of cPMP, which resulted in the restoration of molybdenum enzyme activities and the normalization of all disease biomarkers [31,32]. However, cPMP is the only reported stable Moco intermediate and similar therapies for MoCD type B and C are not feasible [21]. Knowing that the clinical symptoms observed in MoCD are mainly caused by the loss of SO activity, a possible replacement therapy with purified SO may be considered. However, such a therapy is limited by the fact that SO catalytic activity requires mitochondrial translocation, which will result in unfolding of the protein and loss of Moco. In the present study, we investigated mammalian SO from two different aspects. Given the high similarity of PSO and animal SO, we first aimed to identify factors that restrict the reactivity of animal SO towards oxygen. We investigated the production of H2 O2 by different mammalian SO variants with altered or defective IET between the molybdenum and haem domains and found a significant reactivity towards oxygen for IET-restricted SO variants with nearly comparable rates between PSO and haem domain-deficient mammalian SO. Secondly, we investigated the potential use of oxygen-reactive SO proteins in a replacement therapy towards MoCD as such an enzyme may use blood-dissolved oxygen as an electron acceptor, thus overcoming mitochondrial translocation. As high immunogenicity and a reduced protein half-life are common for protein-based therapeutics, we explored the possibility to use PEG-based protein modification (PEGylation) as a tool to increase the half-life of a protein in a biological environment without loss of activity. PEGylation of proteins has been shown in previous studies to effectively increase the serum half-life of proteins and decrease their immunogenicity [33,34]. Furthermore, PEGylated proteins have been approved by the U.S. FDA (Food and Drug Administration) for human administration and are successfully used in many diseases such as hepatitis and cancer [35,36]. We used the PEGylation of surface-exposed lysine residues of SO by the N-hydroxysuccinimide (NHS) ester method and found a significant impact on the oligomerization state of the protein, whereas kinetic properties and oxygen reactivity were only moderately altered. Finally, cell-based assays showed that PEGylated oxygen-reactive mammalian SO in combination with catalase were effective in preventing sulfite-mediated toxicity, suggesting a dual replacement therapy as a possible novel therapeutic route towards the treatment of MoCD and SOD. EXPERIMENTAL Molecular biology

Expression constructs for human and murine SO molybdenum domain (HSOMo and MSOMo ) were generated by cloning the coding sequence for the dimerization and Moco domains [human SO (HSO), GenBank® accession number AY056018.1, residues 110–488; murine SO (MSO), GenBank® accession number BC027197.1, residues 168–546] into pQE80L (Qiagen) using SalI and HindIII restriction sites. The same restriction sites were used for cloning of HSO wild-type (wt). HSO deletion variants HSOKVATV and HSOKVAPTV were generated by fusion PCR and cloned into the pQE80L vector using SacI and SalI restriction sites. MSO (wt) and MSOhaem were generated as described previously [10]. For catalase expression, the coding sequence of human catalase (GenBank® accession number BC110398.1) was PCR-cloned into pQE80L using SalI and HindIII restriction sites. For recombinant expression of PSO, the previously described rAt-SO construct was used [37].

Oxygen reactivity of mammalian sulfite oxidase Protein expression and purification

All SO proteins were expressed in Escherichia coli TP1000 [38] as described previously [28]. Human catalase was expressed in E. coli BL21(DE3) cells. Expression was induced with 0.1 mM IPTG at an A600 of 0.1 and continued for 15 h at 30 ◦ C. All Histagged proteins were purified by Ni2 + -nitrilotriacetic acid (NiNTA) affinity as recommended by the manufacturer (Qiagen). For PSO, a second purification step consisting of an anion-exchange chromatography was performed as described previously [7]. All purified proteins were exchanged into the same buffer (20 mM Tris/HCl, pH 8.0, and 50 mM NaCl) and stored at − 80 ◦ C. Determination of Moco saturation

Moco saturation was determined by denaturing 500 pmol of protein using acid iodine oxidation and alkaline phosphatase treatment resulting in the formation of the stable Moco oxidation product FormA-dephospho, which was quantified further using reverse-phase HPLC as described in [39]. Determination of SO activity

For PSO, HSOMo and MSOMo , activities were either measured using the sulfite:ferricyande or sulfite:cytochrome c assay. Sulfite:ferricyanide activity was measured by monitoring the reduction of ferricyanide [Fe(CN)6 ] at 420 nm (ε420 = 1020 M − 1 ·cm − 1 ) [40]. The assay included the following components: 140 μl of 100 mM Tris/acetate (pH 8), 20 μl of protein solution and 20 μl of 4 mM Fe(CN)6 , and the reaction was started by the addition of 20 μl of sodium sulfite (various concentrations). Activities were measured at an enzyme concentration of 50 nM and 500 nM for the plant and mammalian SO proteins respectively. Sulfite:cytochrome c activity was determined for HSO and deletion variants HSOKVATV and HSOKVAPTV by monitoring the absorption change of cytochrome c at 550 nm (ε550 = 19630 M − 1 ·cm − 1 ) [18]. Briefly, equal proteins concentrations (10 nM) were incubated in a 200 μl final volume with a mixture containing 50 mM Tris/acetate (pH 8) and sodium sulfite (various concentrations), and the reaction was started by adding 12 μl of cytochrome c (10 mg/ml). SO activity in crude protein extracts was determined in a similar way with the following modifications: 50 μg of protein crude extract was used and the assay buffer mixture contained 50 mM Tris/acetate (pH 8), 0.2 mM deoxycholic acid, 0.1 mM potassium cyanide and 0.5 mM sodium sulfite. All activities were measured at room temperature (25 ◦ C) using a 96-well plate reader (BioTeK). H2 O2 quantification

Quantification of H2 O2 is based on the formation of a complex between Xylenol Orange and ferric ions (Fe3 + ), which is produced by the peroxide-dependent oxidation of ferrous iron (Fe2 + ). The method was performed using a commercial kit (National Diagnostics) and detection was carried out colorimetrically following the protocol of the manufacturer. Quantification was carried out after an incubation time of 30 min at room temperature (25 ◦ C) by measuring the absorption at 560 nm using a 96-well plate reader. Oxygraph measurement

Oxygen consumption was measured using an Oroboros Oxygraph 2k Instrument. First, a 2 ml solution containing 100 mM Tris/HCl

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(pH 8.0) and 250 nM purified enzyme was introduced into the Oxygraph chamber and maintained at 37 ◦ C for approximately 15 min to equilibrate oxygen concentration in the chamber. The reaction was started by addition of 60 μM sulfite and the slopes corresponding to the linear change in oxygen concentration of the plots were used to calculate the oxygenconsumption rates. Data were analysed using DatLab4 software (version 4.3). Western blotting

Western blots were performed on mouse crude extracts of liver, kidney and brain derived from three different animals. The protein concentration was determined using the Bradford method and 50 μg of each crude extract was separated by SDS/PAGE (10 % gel). Primary antibodies used were anti-sulfite oxidase (Eurogentec) and anti-actin (Santa Cruz Biotechnology). Secondary antibodies coupled to horseradish peroxidase (Abcam) were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific), and analysis of the protein bands was performed with the Chemoluminescence DeVision HQ2 camera system and the Gel-Pro Analyzer software (Decon Science Tec). PEGylation reaction

For PEGylation of plant and mammalian SO proteins, the NHS method was used [41]. Three different PEG reagents (mPEG NHS ester; Celares) different in size and chemistry were applied (for details, see Supplementary Figure S3). Briefly, the SO proteins were exchanged into PBS and concentrated to 5– 10 mg/ml. PEG reagent was added to the protein solution at a 20-fold molar excess and the solution was incubated for 30 min at room temperature (25 ◦ C). Finally, excess of non-reacted PEG was removed by size-exclusion chromatography using a HR16/30 Superdex 200 column (GE Healthcare). Cell viability

Cell viability studies were conducted in human embryonic kidney (HEK)-293 cells using the MTT assay (Promega). Briefly, 80 μl of HEK-293 cell suspension (containing 2×104 cells) were dispensed into each well of a 96-well tissue culture plate and incubated overnight at 37 ◦ C in a humidified 5 % CO2 atmosphere. Next, 10 μl of SO and/or catalase proteins were added to each well at a final concentration of 0.5 μM and allowed to incubate for 30 min at 37 ◦ C. Then, 10 μl of sulfite (various concentrations) was added to each well and incubated for 15 h at 37 ◦ C in a humidified 5 % CO2 atmosphere. H2 O2 toxicity was also investigated in the absence and presence of catalase using a concentration range of 0– 0.5 mM enzyme. Finally, cell viability was evaluated using the MTT dye according to the supplier’s protocol, and absorption at 570 nm (reference 650 nm) was recorded using a well plate reader (Tecan). RESULTS Electron transfer between Moco and haem determines SO reactivity towards oxygen

SO from chicken (Gallus gallus) and plant (Arabidopsis thaliana) share 47 % sequence identity despite the lack of the haem domain in PSO. However, only PSO has been reported to react  c 2015 Authors; published by Portland Press Limited

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Figure 1

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The haem domain affects H2 O2 formation in mammalian SO

Sulfite-dependent formation of H2 O2 by MSO, HSO, PSO and their variants in the presence and absence of 0.5 μM catalase. H2 O2 formation is shown after 30 min of incubation of 1 μM wt MSO (A), wt HSO (B), PSO (C), MSOhaem (D), HSOKVATV (E) and HSOKVAPTV (F). Linear regression curves were determined for the activity without catalase (slopes: C, y = 0.96x − 1.30; D, y = 0.72x + 0.92; E, y = 0.41x + 0.72; F, y = 0.54x + 0.11). All experiments were repeated at least three times (n = 3) and results are means + − S.D.

with oxygen, a process involving the formation of superoxide ions, which are further dismutated to H2 O2 [17]. Given the similar kinetic properties in sulfite oxidation between mammalian and plant SOs and their different domain structure, we asked to what extent the haem domain affects oxygen reactivity in mammalian SO. Therefore we first determined H2 O2 production as a function of sulfite oxidation in both animal and plant SOs. For this purpose, a colorimetric method that quantifies all organic peroxides including superoxide ions and H2 O2 was used, and the exclusive production of H2 O2 was probed by the addition of recombinantly expressed and purified human catalase. We first determined sulfite-dependent H2 O2 production (for 30 min) of wt MSO and HSO, and compared this with that of PSO. H2 O2 formation was low in the presence of MSO showing 15 μM H2 O2 formation with 75 μM sulfite (Figure 1A), whereas HSO did not lead to any significant H2 O2 production (