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Effects of glycosylation and pH conditions in thedynamics of human arylsulfatase AMadza Yasodara Farias Virgens a b , Laercio Pol-Fachin c , Hugo Verli c & Maria Luiza Saraiva-Pereira a b c da Laboratório de Identificação Genética, Centro de Pesquisas, Hospital de Clínicas de PortoAlegre, Rua Ramiro Barcelos, 2350, Porto Alegre, 90035-903, RS, Brazilb Departamento de Genética, Universidade Federal do Rio Grande do Sul, Av. BentoGonçalves, 9500, Porto Alegre, 91500-970, RS, Brazilc Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves9500, Porto Alegre 15005, 91500-970, RS, Brazild Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Rua RamiroBarcelos, 2600, Porto Alegre, 90035-003, RS, BrazilVersion of record first published: 13 Apr 2013.

To cite this article: Madza Yasodara Farias Virgens , Laercio Pol-Fachin , Hugo Verli & Maria Luiza Saraiva-Pereira (2013):Effects of glycosylation and pH conditions in the dynamics of human arylsulfatase A, Journal of Biomolecular Structure andDynamics, DOI:10.1080/07391102.2013.780982

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Effects of glycosylation and pH conditions in the dynamics of human arylsulfatase A

Madza Yasodara Farias Virgensa,b, Laercio Pol-Fachinc, Hugo Verlic* and Maria Luiza Saraiva-Pereiraa,b,c,d

aLaboratório de Identificação Genética, Centro de Pesquisas, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350,Porto Alegre, 90035-903, RS, Brazil; bDepartamento de Genética, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves,9500, Porto Alegre, 91500-970, RS, Brazil; cCentro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. BentoGonçalves 9500, Porto Alegre 15005, 91500-970, RS, Brazil; dDepartamento de Bioquímica, Universidade Federal do Rio Grandedo Sul, Rua Ramiro Barcelos, 2600, Porto Alegre, 90035-003, RS, Brazil

Communicated by Ramaswamy H. Sarma

(Received 12 December 2012; final version received 23 February 2013)

Arylsulfatase A (ARSA) is a lysosomal sulfatase that catalyzes the hydrolysis of cerebroside sulfate. Its deficiency resultsin Metachromatic Leukodystrophy, whereas a minor condition called ARSA pseudodeficiency occurs in healthy individu-als, which has been associated with the substitution of the glycosylated Asn350 by a Ser and with the loss of the polyad-enylation signal. In this work, we have investigated ARSA dynamics employing molecular dynamics simulations inresponse to (1) different pH’s, as, beyond its natural lysossomal environment, it has been recently identified incytoplasmatic medium and (2) glycan occupancies, including its normal glycosylation state, presenting three high man-nose-type oligosaccharides. Accordingly, four systems were studied considering ARSA under different conditions: (1)nonglycosylated at pH� 7 (ARSApH7); (2) non-glycosylated at pH� 5 (ARSApH5); (3) triple glycosylated at pH� 5(ARSAglyc,pH5); and (4) ARSA-N350S mutant at pH� 5 (ARSAN350S,pH5). Lowering pH and increasing glycosylationwas found to reduce the flexibility of the enzyme. In addition, at acidic pH, the glycosylated enzyme presented a highersecondary conformational stability when compared to its nonglycosylated counterpart, supporting experimental findingson triple glycosylation as the essential state of ARSA. The N350S mutant exhibited a consistent degree of unfolding,which may be related to its in vitro reduced stability. Finally, the obtained data are discussed in the search for structuralevidences able to contribute to the understanding of biological activity of ARSA and molecular etiology of ARSApseudodeficiency, as determined by ARSA-N350S in the absence of polyadenylation defect.

Keywords: ARSA; glycan; magnesium; mutation; sulfatase

Introduction

Sulfatases are an evolutionary highly conserved familyof proteins essential for degradation and remodeling ofsulfate esters (Galperin & Jedrzejas, 2001; Ghosh, 2007;Lukatela et al., 1998). The physiological importance ofthese enzymes in humans is illustrated by seven distinctsevere lysosomal storage disorders, each one caused bythe deficiency of one specific sulfatase (Neufeld &Muenzer, 2001; von Figura, Gieselmann, & Jaeken,2001). Among such diseases, metachromatic leukodys-trophy (MLD) is the one caused by arylsulfatase A(ARSA) deficiency, comprising an autosomal recessiveinborn error of metabolism (von Figura et al., 2001).Such insufficiency results in a lysosomal accumulationof cerebroside sulfate, the ARSA major physiologicalsubstrate, in the white matter of the central nervoussystem and peripheral nerves, with severe neurological

consequences (von Figura et al., 2001). ARSA issynthesized as a 507 amino acid polypeptide that, aftertranslocation into the endoplasmic reticulum (ER), iscleaved at its N-terminal region, yielding an α/β 489amino acid mature enzyme. Structurally, while the coreof the enzyme is conserved between sulfatases, at theC-terminus, a loop including the long helix, known asαI, is formed by nearly 80 amino acid residues, stretch-ing over the whole ARSA monomer. As well, a clusterof six Cys residues knots 20 C-terminal amino acid resi-dues by forming disulfides bonds, which is unique toARSA (Stein et al., 1989; von Figura et al., 2001)(Figure 1(A)).

In the ER, ARSA is N-glycosylated at three sites(Asn158, Asn184 and Asn350) with core fucosylatedhigh mannose-type oligosaccharides (Hoja-Lukowicz,Ciolczyk, Bergquist, Litynska, & Laidler, 2000; Laidler

*Corresponding author. Email: hverli@cbiot.ufrgs.br

Journal of Biomolecular Structure and Dynamics, 2013http://dx.doi.org/10.1080/07391102.2013.780982

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& Litynska, 1997) (Figure 1(B)). As well, formylglycine(FGly), a key catalytic residue in its active site, isgenerated by the FGly-generating enzyme through oxida-tion of the CH2SH group of a conserved Cys to an alde-hyde (Dierks et al., 2005). Subsequently, dimers areformed and transported to the Golgi apparatus, wherethey receive mannose-6-phosphate (M6P) recognitionmarkers at the Asn158 and Asn350 linked oligosaccha-rides and bind to M6P receptors (Sommerlade et al.,1994). Such complexes are transported to endosomes fordissociation, allowing ARSA to be subsequently deliv-ered to lysosomes, where the enzyme octamerizes, in apH-dependent manner (Vagedes, Saenger, & Knapp,2002) (Figure 1(C)). Within the lysosomal compartment,lysosomal proteins usually undergo limited proteolysisand partial hydrolysis of their oligosaccharides, includingdephosphorylation of M6P residues. For this reason, asfor the majority of lysosomal proteins isolated from tis-sues, lysosomal resident ARSA is devoid of M6P resi-dues in its glycan chains (Bresciani & von Figura,1996).

An important function of high mannose-type oligo-saccharides in ARSA, as above stated, is its delivery tolysosomes, a common pathway for many lysosomal

hydrolases (Gieselmann, Schmidt, & von Figura, 1992;Harvey, Carey, & Morris, 1998). In nature, the carbohy-drate moiety of glycoproteins has been associated withseveral structural and functional roles, as stabilizing largeregions of the backbone structure, protein folding orintracellular targeting (Drickamer & Taylor, 1998; Impe-riali, 1997; Imperiali & O’Connor, 1999; Wormald &Dwek, 1999). In this context, additional implicationsconcerning ARSA glycosylation in its catalytic activityhave been suggested by in vitro studies, pointing toreduced stability of ARSA when a substitution of a gly-cosylated Asn350 to Ser (N350S) occurs, abolishing theglycosylation at this site (Fluharty, Meek, & Kihara,1983; Waheed, Steckel, Hasilik, & von Figura, 1983).Moreover, ARSA carbohydrate component modifications,as sialylation, phosphorylation or sulfation, are oftenassociated with the appearance of tumors (Nakamura,Gasa, & Makita, 1984). Despite these mentioned in vitroand in vivo findings, the molecular basis of glycosylationon ARSA activity and conformation remains mostlyunclear.

Besides its lysosomal pathway, ARSA has beenrecently proposed to be involved as an extracellular com-ponent, existing on the cell surface beyond lysosomes

Figure 1. ARSA general structure and topology. In (A), ARSA topology is shown, where α-helices are shown as columns andβ-strands as arrows, numbered from N- to C-termini. Cys residues are circled, with disulfide bridges presented as thick black lines.The active site region is represented by the shaded ellipse. The glycosylation sites and amino acid residues forming the salt-bridgeAsp335–Arg370 are boxed, and the salt-bridge residues are shown within dashed lines. ARSA region involved in oligomerizarion(β-sheets 15–18 and helix I) is colored in gray. Adapted with permission from Lukatela et al. (1998). In (B), a scheme of ARSA corefucosylated high mannose-type oligosaccharides is presented. In (C), a model for triple glycosylated ARSA octameric association ispresented, where the protein structure is presented as cartoon representation, and ARSA glycans are shown in the oligomeric structureas surface.

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(Mitsunaga-Nakatsubo, Kusunoki, Kawakami, Akasaka,& Akimoto, 2009). However, while the cell-surfaceARSA might be able to bind sulfates in the extracellularmatrix, it would not hydrolyze them in such conditions,as its optimal pH is 4.5 (Mitsunaga-Nakatsubo et al.,2009). So far, only the pH dependence of the ARSAoctamerization has been explained on the molecularlevel. The importance of pH over ARSA activity or spe-cific structural differences between lysosomal and cell-surface ARSA need further explanation.

In this context, this work intends to investigate ARSAdynamics in response to changes in pH and glycosylationthrough a series of molecular dynamics (MD) simula-tions. Accordingly, the retrieved data were compared withprevious in vitro studies, including direct-site mutagene-sis, pulse-chase, and X-ray crystallography, offeringinsights into the impact of pH and glycosylation onARSA activity and stability.

Materials and methods

Nomenclature and software

The nomenclature recommendations and symbols areused as proposed by IUPAC (IUPAC-IUBMB Commis-sion on Biochemical Nomenclature, 1996). The relativeorientation of a pair of contiguous carbohydrate residuesis described, for different types of linkages, by two orthree torsional angles at the glycosidic linkage. For a(1→X) linkage, where “X” is 3, 4, or 6 for the (1→3),(1→4) or (1→6) linkages, respectively, ϕ and ψ aredefined as shown below:

/ ¼ O5–C1–OX0–CX0 ð1Þ

w ¼ C1–OX0–CX0–CðX� 1Þ0 ð2Þ

For a (1→6) linkage, ω is defined as shown below:

x ¼ O60–C60–C50–C40 ð3Þ

The manipulation of structures was performed withMOLDEN (Schaftenaar & Noordik, 2000), VMD (Hum-phrey, Dalke, & Schulten, 1996), SPDBV (Guex &Peitch, 1997), and PYMOL (DeLano, 2002). The sec-ondary structure content analyses were performed withDSSP (Kabsch & Sander, 1983) and PROCHECK(Laskowski, McArthur, Moss, & Thornton, 1993).Dimeric and octameric ARSA structures were obtainedwith PISA (Krissinel & Henrick, 2007), and electrostaticsurface analyses were performed with APBS tools exten-sion of PYMOL (Baker, Sept, Joseph, Holst, & McCam-mon, 2001). All the MD calculations and remaininganalysis were performed using GROMACS 3.3.3 simula-

tion suite (van der Spoel et al., 2005) and GROMOS9643a1 force field (Scott et al., 1999).

Topology construction

Human ARSA in its nonglycosylated monomeric formwas retrieved from PDB under code 1AUK (Lukatelaet al., 1998). The residues in the gap between Gly443and Ala448 and the missing C-termini, from Asp504 toAla507, were added based on ARSA protein sequenceretrieved from NCBI under code P15289 (Stein et al.,1989) using Swiss-PDB Viewer program. As well, theFGly69 residue was constructed using MOLDEN in itsaldehydic nonhydrated form, being further submitted tothe PRODRG server (Schuttelkopf & van Aalten, 2004),from which its topology was retrieved and added by HF/6-31G⁄⁄-derived Löwdin charges, as reported (Becker,Guimarães, & Verli, 2005; Pol-Fachin, Fernandes, &Verli, 2009; Verli & Guimarães, 2004). The glycosylatedARSA structures were filled with core fucosylated highmannose-type oligosaccharides, as proposed based on aconsensus between previous mass spectrometry studies(Hoja-Lukowicz et al., 2000; Laidler & Litynska, 1997),using glycosciences modeling tools (Lütteke et al.,2006). These obtained models for glycosylated ARSAhad their glycosidic linkage geometries adjusted to themain conformations for each linkage, based on their rela-tive abundances in the isolated disaccharides in water, asdescribed previously (Fernandes, Sachett, Pol-Fachin, &Verli, 2010). The glycan parameters were obtained fromthe PRODRG server and added by HF/6-31⁄⁄-derivedLöwdin charges, as reported (Becker et al., 2005; Pol-Fachin et al., 2009; Verli & Guimarães, 2004). Improperdihedrals were included to preserve the hexopyranoseconformations in accordance with their expected formsin aqueous solution: 4C1 for D-GlcpNAc, 1C4 forL-Fucp, 4C1 for D-Manp, and 4C1 for D-Galp. Addition-ally, N-glycosidic linkages between the carbohydrate andamino acid residues were treated as previously described(Pol-Fachin et al., 2009). This MD protocol has beenproved to be accurate in conformational analysis of car-bohydrates, being able to explain dihedral angle distribu-tions for glycosidic linkages found in NMR experiments(Becker et al., 2005; Fernandes et al., 2010; Pol-Fachinet al., 2009; Verli & Guimarães, 2004).

MD simulations

Four ARSA MD simulations were performed: (1) non-glycosylated ARSA at pH� 7 (ARSApH7); (2) nonglyco-sylated at pH� 5 (ARSApH5); (3) triple glycosylated atpH� 5 (ARSAglyc,pH5); and (4) ARSA-N350S mutant atpH� 5 (ARSAN350S,pH5). Although it is known thatARSA at pH� 7 exists as a dimer and at pH� 5 as anoctamer (Sommerlade et al., 1994; Vagedes et al., 2002),only ARSA monomeric structures were considered in

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order to limit the systems size and reduce computationalcosts, as reported (Fowler & Coveney, 2006; Lv, Jiang,Yu, & Lu, 2011). Each structure was solvated in atriclinic box using periodic boundary conditions and sim-ple point charge (SPC) water model (Berendsen, Grigera,& Straatsma, 1987), employing a 10Å distance from theoutside of each structure and the box edge. The proton-ation states were set by GROMACS software forARSApH7 and adjusted according to the pKa values ofthe amino acid residues side chains for pH� 5 systems(supplementary material, Table S1) as verified usingPDB2PQR (Dolinsky et al., 2007). Counter ions (sodiumor chloride) were added to neutralize the systemscharges, whenever needed. The employed MD protocolwas based on previous studies, as described (Beckeret al., 2005; Pol-Fachin et al., 2009; Verli & Guimarães,2004). The Lincs method (Hess, Bekker, Berendsen, &Fraaije, 1997) was applied to constrain covalent bondlengths, allowing an integration step of 2 fs after an ini-tial energy minimization using Steepest Descent algo-rithm. Electrostatic interactions were calculated withParticle Mesh Ewald Method (Darden, York, & Peder-sen, 1993). Temperature and pressure were kept constantby coupling (glyco)proteins, ions, and solvent to externaltemperature and pressure baths with Berendsen thermo-stat and barostat, employing coupling constants of τ= .1and .5 ps, respectively (Berendsen, Postma, DiNola, &Haak, 1984). The dielectric constant was treated as ɛ= 1,and the reference temperature was adjusted to 310K.The systems were slowly heated from 50 to 310K, insteps of 5 ps, each one increasing the reference tempera-ture by 50K and extended to 50 ns.

Results and discussion

Structural effects of pH and triple glycosylation

In order to search for structural evidences of acid pHimportance for the optimal lysosomal activity of ARSA,we performed 50 ns MD simulations of monomeric non-glycosylated ARSA as ARSApH7 and ARSApH5, in con-ditions resembling the cytoplasmic and lysosomal pH,respectively. Based on these data, lower values of overallroot mean square deviation (RMSD) are observed forARSApH5 when compared to ARSApH7 (Figure 2(A)).Regarding such analysis, while the overall RMSD ofARSApH7 gradually increased to approximately 5Å, theoverall RMSD of ARSApH5 remained stable around3.5Å during most of simulation time, except by asudden increase to � 4Å around 37 ns of simulationtime. This increase was observed to be associated withalterations in ARSA helix secondary structure content,especially the helix D folding and helix E and I unfold-ing (supplementary data, Figure S1). Despite a reductionin global flexibility of ARSApH5 when compared to

ARSApH7, specific regions presented an increase inflexibility (Figure 3(A) and (B)), while the β-twistedhydrophobic core proved to be very rigid in ARSApH5

(Figure 3(B)), which is compatible with the optimal lyso-somal activity of ARSA, the regions close to the glyco-sylation site at Asn350, which also includes a regionadjacent to the structurally important salt-bridgeAsp335–Arg370, became more flexible (Figure 3(A) and(B)). This pattern of high flexibility in regions three-dimensionally close to glycosylation sites in ARSApH5

system led us to postulate a possible stabilizing role ofARSA glycans, which were missing in the systems dis-cussed so far, as reported for others glycoproteins (Impe-riali & O’Connor, 1999; Petrescu, Wormald, & Dwek,2006; Wormald & Dwek, 1999).

In order to test this hypothesis and obtain insightsinto the structural role of ARSA glycosylation, a 50 nsMD simulation of monomeric triple glycosylated ARSAat pH� 5 (ARSAglyc,pH5) was performed, which pre-sented lower overall RMSD when compared toARSApH5 or ARSApH7 (Figure 2(A)), that is, around3.4Å during the entire simulation time. As previouslyreported for a set of other glycoproteins (Pol-Fachin &Verli, 2011), a stabilizing effect of the ARSA N-linkedglycans could be observed, in which the regions three-dimensionally close to glycosylation sites became lessflexible when compared to ARSApH5, especially that oneclose to the glycosylation at Asn350 site (in Figure 3(B)and (C)). Moreover, ARSAglyc,pH5 secondary structurecontent was very constant during simulations, in contrastto the behavior observed in the absence of glycosylation(that is, when comparing the maintenance of secondarycontent of helices I, E and D between ARSApH5/ARSApH7 and ARSAglyc,pH5).

The glycans at Asn158 and Asn184 composed anarrangement with the protein region where the ARSA-unique cluster of six Cys residues knots its C-terminalthrough three disulfide bonds (Figure 4(A) and (C)). Asreported for other glycoproteins (van Veen, Geerts, vanBerkel, & Nuijens, 2004), this glycan arrangement maywork as a steric protective factor against proteolyticattacks in this region, considering the intense proteolyticactivity in lysosomes (Winchester, 2005). Likewise, theglycosylation at Asn350 site bends over and interactswith the salt-bridge Asp335–Arg370 loop and itsadjacent regions during all simulation time. Thus,Asn350-linked glycan may not only be important to thepronounced decrease of flexibility of this entire region(Figure 3(B) and (C)), but it also can afford a stericprotection against proteolysis of this critical portion(supplementary data, Figure S2).

ARSA-N350S mutant

About .2–.5% of healthy population presents ARSAactivity levels similar to those of MLD patients, in a

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nonpathogenic condition, known as ARSA-PD, causedby the homozygozity for ARSA-PD allele. The normaland ARSA-PD differ by two adenine to guanine tran-sitions, being one related to the substitution of a gly-cosylated Asn350 to Ser (N350S), abolishing theglycosylation at this site, and the other concerned withthe alteration of the ARSA major polyadenylation sig-nal (Gieselmann, Polten, Kreysing, & von Figura,1989). Historically, the reduction in ARSA activitycaused by ARSA-PD has been attributed to the lastmentioned cause (Gieselmann et al., 1989; Gieselmannet al., 1992; Harvey et al., 1998; Sommerlade et al.,1994). Moreover, the enzymatic activity of the wild

type and N350S polymorphic enzymes have beenobserved to be both similar (Gieselmann et al., 1989)and different (Harvey et al., 1998), with a �43% inARSA-N350S in vitro activity loss. However, consider-ing that MLD heterozygous patients have been identi-fied carrying the N350S allele in the absence of thepolyadenylation defect, with �50% of ARSA activity(Barth, Ward, Harris, Saad, & Fensom, 1994; Franciset al., 1993; Shen, Li, Waye, Francis, & Chang,1993), and that the N350S allele have been recentlyassociated with alcoholism (Chung et al., 2002; Park,Poretz, Stein, Nora, & Manowitz, 1996), we performeda MD simulation of monomeric ARSA-N350S enzyme

Figure 2. ARSA dynamic profile during MD simulations. A superimposition of the final frames of each simulated system forARSApH7 (black), ARSApH5 (red), ARSAglyc,pH5 (purple), and ARSAN350S,pH5 (blue) is shown in the top of the figure. The glycansfor ARSAglyc,pH5 and ARSAN350S,pH5 are labeled using colors corresponding to their respective systems. In (A), all-atom RMSD forARSApH7 (black), ARSApH5 (red), ARSAglyc,pH5 (purple), and ARSAN350S,pH5 (blue) is presented. In (B), Asp335-Cγ to Arg370-Cζdistance for ARSApH7 (black), ARSApH5 (red), ARSAglyc,pH5 (purple), and ARSAN350S,pH5 (blue) is shown. Below eachcorresponding plot, tables present average and standard deviation values, as well as maximum values achieved during eachsimulation. Asp335-Cγ to Arg370-Cζ distance in 1AUK PDB structure is also presented for comparison effects.

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Figure 3. Secondary structure modifications and global flexibility. A dictionary of protein secondary structure (DSSP) and root meansquare fluctuation (RMSF) analysis per residue are presented as a function of time for: (A) ARSApH7, (B) ARSApH5, (C) ARSAglyc,

pH5, and (D) ARSAN350S,pH5. Helices D, E and I are highlighted in DSSP plots by small letters, while glycosylation sites areindicated by arrows.

Figure 4. N-glycosidic linkage conformational analisys and glycan reorientation. The ϕN-Gly dihedral angle measure is presented as afunction of time for the (A) glycans at Asn158 (pink) and at Asn184 (purple) for the ARSAglyc,pH5 system and for (B) glycans atAsn158 (dark blue) and at Asn184 (light blue) for the ARSAN350S,pH5 system, together to a frame superimposition of structuresobtained at every 10 ns of simulation evolution. In (C), the approximation between each of the glycans at Asn158 (pink) and Asn184(purple) to the ARSA 18 C-termini amino acid residues is shown, together to the final of ARSAglyc,pH5 system. In (D), thedetachment between each of the glycans at Asn158 (light blue) and Asn184 (dark blue) to the ARSA 18 C-termini amino acidresidues is presented, together to the final frame of ARSAN350S,pH5 system. In the structures, the protein moiety is represented ascartoon, while glycan chains and disulfide bonds are presented as sticks.

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at pH� 5 (ARSAN350S,pH5) in order to reveal the sig-nificance of the loss of the glycosylation at Asn350for ARSA function.

Our results show that the overall RMSD ofARSAN350S,pH5 gradually increased to �4.5Å duringsimulation, presenting a progressive trajectory in asimilar behavior to the overall RMSD of ARSApH7

(Figure 2(A)). Moreover, an increased global flexibilityof ARSAN350S,pH5 is observed when compared toARSAglyc,pH5. Specifically, modifications occur in thearrangement between the glycans at Asn158, Asn184,and the C-termini (Figure 4(A) and (D)), the glycan atAsn184 departed from the C-termini and started interact-ing with neighbor helices D and E which, consequently,destabilized and unfolded (Figure 3(C) and (D)). Thehelix E (the longest helix of ARSA) presented a loss of2.5 turns in its C-terminal portion (component of thehat-shaped monomer base) in ARSAN350S,pH5 (Figure 3(D)), which represents the most significant secondarystructure change among all tested systems. In view ofthat the reported loss of �43% in ARSA-N350S in vitroactivity (Harvey et al., 1998) may be related to theabove-mentioned ARSAN350S,pH5 secondary structurecontent changes. Such hypothesis is reinforced by circu-lar dichroism studies, which demonstrated ARSA inacti-vation due to secondary structure loss during its turnoverprocess (Waheed & van Etten, 1979a).

In addition, as pulse chase experiments have previ-ously demonstrated (Ameen & Chang, 1987), the appar-ent rates of degradation of ARSA in lysosomes ofnormal ARSA and ARSA-PD cells are markedly differ-ent. In this context, while the apparent half-life of theradioactive ARSA has been estimated in 33 h in theARSA-PD cells, in normal cells only 12% of the labeledARSA are degraded in the same period (Ameen &Chang, 1987). Our results suggest that the reduced sta-bility and high proteolysis of ARSA N350S may occurdue to the structural effects of the loss of the glycan atAsn350 on the optimal arrangement between the remain-ing glycans and the structure of the C-terminal residues.

Asp335–Arg370 salt-bridge

Missense mutations may be able to disturb ARSA struc-ture and cause partial or complete loss of activity (vonFigura et al., 2001). This statement is illustrated by twomutations: related to the substitution of an Asp to a Val(D335V) and the other of an Arg to a Trp (R370W),which disturb the functionally important ARSA salt-bridge Asp335–Arg370 (Hess et al., 1996; Schestaget al., 2002). These two mutations have been described inpatients with severe MLD, causing the complete loss ofARSA enzymatic activity (Hess et al., 1996; Lugowska,Ploski, Wlodarski, & Tylki-Szymanska, 2010).

In this work, the maintenance of the salt-bridgeAsp335–Arg370 was observed in both ARSApH5 and

ARSAglyc,pH5 (Figure 2(B)). While such systems pre-sented the smallest average and standard deviation valuesfor the distance between Asp335-Cγ and Arg370-Cζ,both ARSApH7 and ARSAN350S,pH5 presented higheraverage and standard deviation values for such distance.In this context, it has been speculated that the formationof salt-bridges in the interior of a protein could be lessfavorable and even disfavor protein folding (Hendsch &Tidor, 1994; Honig & Nicholls, 1995). However, empiri-cal studies of direct-site mutagenesis had demonstratedthat the removal of ion pair members usually destabilizesthe native protein structure by 3–5 kcal.mol�1, so themaintenance of Coulomb interactions in the protein inte-rior may be related to an increase in folding stability andspecificity (Honig & Yang, 1995). Accordingly, theARSA salt-bridge Asp335–Arg370 is located in the inte-rior of the enzyme, being only partially accessible to sol-vent. Moreover, equivalent salt-bridges are conservedwithin sulfatase family (Galperin & Jedrzejas, 2001;Ghosh, 2007; Schestag et al., 2002), with the singleexception of iduronate sulfatase. The salt-bridgeAsp335–Arg370 importance for ARSA correct foldinghas been evidenced the previously described data, inwhich the retention of ARSA-D335V mutant in the ERoccurs as a result of its inappropriate fold (Schestaget al., 2002).

The three-dimensional disposition of ARSA salt-bridge residues suggests that it may work as a hook,helping to sustain, within each protein monomer, thestructures responsible for oligomerization close to theenzyme core, thus favoring a proper hat-shaped mono-mer. This hypothesis could be evidenced by our findings,as instability in ARSA Asp335–Arg370 salt-bridge isrelated to bigger distances between the centers of massof ARSA structures responsible for oligomerization andthe enzyme core (supplementary data, Figure S3). Inother words, the helix I consistently moves away fromprotein core in ARSAN350S,pH5 and in ARSApH7, that is,the systems in which the ARSA Asp335–Arg370 salt-bridge is very instable, and it remains close to proteincore in ARSAglyc,pH5 and ARSApH5, the systems inwhich the Asp335–Arg370 salt-bridge is stable. There-fore, these findings point to the participation of Asp335–Arg370 salt-bridge in the optimal association betweenportions of ARSA (supplementary data, Figure S3).

Electrostatic potential surface

The majority of ARSA ionizable residues are located onenzyme surface, especially at ARSA association inter-faces and catalytic pocket. Accordingly, our results pointthat the three major positive ARSA charged sites, at bothpH� 7 and pH� 5, are the following: (1) the octamericinterface, (2) the loop components of dimeric interface,and (3) the catalytic pocket itself (supplementary data,Figure S4). The role of amino acids at protein–protein

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interfaces is twofold; they are supposed to promote theprotein–protein association, but also allow proteins to besoluble. In this context, while hydrophobic residues atthe protein surface may favor association unspecifically,allowing many different configurations, hydrophilicresidues can both disfavor association or act specifically,allowing for interfacial recognition of protein surfacesand lead to a definite configuration of the associatedmolecules (Vagedes et al., 2002). In this sense, the pat-tern of distribution of ARSA positive-charged residues,with most of them spread on association interfaces, canbe related to a more specific manner of its association.In addition, this hypothesis is reinforced by the findingthat ARSA dissociation is strongly favored by anincrease in the ionic strength, thus suggesting that ionicand/or hydrogen bond formation are the driving forcesfor ARSA association and that hydrophobic interactionsplay only a minor role in stabilizing the ARSA quater-nary structure (Waheed & van Etten, 1979b).

Glycans analysis

The N-glycosidic linkage conformation has been exten-sively studied by both NMR and X-ray methods (Davis,Hirani, Bartlett, & Reid, 1994; Imberty & Pérez, 1995;Petrescu, Petrescu, Dwek, & Wormald, 1999). Thesestudies show that the GlcNAc-(1→N)-Asn linkage is rel-atively rigid and planar, with a tendency to extend theglycan residue away from the peptide backbone and intothe solvent. Moreover, previous X-ray data indicate thatthe ϕN-Gly dihedral angle presents higher flexibility com-pared to ψN-Gly dihedral angle, with 80° and 40° ampli-tude, respectively (Imberty & Pérez, 1995). This largeamplitude of the ϕN-Gly also suggests the co-existence ofmultiple conformer populations in solution (Pol-Fachinet al., 2009). In agreement with these findings, all ARSAglycans at ARSAglyc,pH5 system presented similar confor-mational patterns around the GlcNAc-(1→N)-Asn duringthe performed simulations, in a behavior mostly indepen-dent on the surrounding protein scaffold (Figure 4(A)).However, the ϕN-Gly of both glycans in ARSA-N350Ssystem presented a distinct behavior (Figure 4(B)). Whilethe distance between the glycan at Asn184 and theARSA C-termini progressively increased in ARSA-N350S enzyme system (Figure 4(D)), such departure isreflected by changes in the ϕN-Gly conformational profile.In the same way, the distinct behavior of the ϕN-Gly atAsn158 glycan of ARSAN350S,pH5 system also followschanges in the glycan orientation.

Additionally, in order to search for possible confor-mational influences of the ARSA core on the glycosidiclinkages composing the studied high-mannose glycans,their conformational profile was analyzed and comparedwith the pattern observed for the proper disaccharideunits in solution (supplementary data, Figures S5 andS6). Accordingly, disaccharidic linkages in different car-

bohydrate trees from ARSA did not present major newconformer populations independently on the site ofattachment to ARSA but showed distinct preferencesover the conformations already observed in solution forisolated disaccharides units (Fernandes et al., 2010; Pol-Fachin et al., 2009). Such behavior denotes a minorinfluence of the protein scaffold on glycan conformation,as previously demonstrated (Fernandes et al., 2010; Pol-Fachin et al., 2009). The only exception to this patternwas the distinct behavior of the ϕ angle from β-D-Glcp-NAc-(1→4)-β-D-GlcpNAc of the glycan at Asn184 inthe ARSAN350S,pH5 system that follows the glycan globalreorientation discussed above (supplementary data,Figure S5).

Mg+2 coordination

The ARSA active site residues and architecture arehighly conserved among eukaryotic and prokaryotic sul-fatases, indicating a common catalytic mechanism sharedby members of the family (Galperin & Jedrzejas, 2001;Ghosh, 2007). In addition, the active center residues ofARSA and Arylsulfatase B show remarkable structuralhomology (Galperin & Jedrzejas, 2001; Ghosh, 2007).These two sulfatases contain divalent metal cations thatare coordinated by three Asp side chains, an Asn residueand the key residue FGly. In the case of ARSA, the resi-dues Asp29, Asp30, Asp281, Asn282, and FGly69 areresponsible for the Mg+2 octahedral coordination (Wal-dow, Schmidt, Dierks, von Bulow, & von Figura, 1999).

In all systems simulated in this work, the Mg+2 ionremained correctly positioned in the active site pocket,except in the ARSAN350S,pH5 system. The metal ionapparently escapes as a result of the global destabiliza-tion of this system (Figure 5). The metal escape alsoleads to a loss of electrostatic potential at the catalyticpocket, which became strongly negative (supplementarydata, Figure S7). This behavior agrees with previouslysuggested hypothesis about the large importance of Mg+2

for ARSA binding to sulfate groups by originating afavorable positive microenvironment (Schenk et al.,2009). It should be also considered that the specificactivities of wild type and N350S enzymes were consid-ered equivalent (Fluharty et al., 1983) and that N350SARSA was detected with reduced activity in transfectionexperiments (Harvey et al., 1998) and in vivo (Barthet al., 1994; Francis et al., 1993; Shen et al., 1993).Altogether, and considering the results obtained inARSAN350S,pH5 MD simulation, we suggest that thereduced stability of the N350S polymorphic enzyme mayfacilitate Mg+2 escape, and the absence of such ion inthe catalytic pocket of a fraction of physiologicallyoccurring enzymes may contribute for its measuredreduced activity.

Accordingly, overall distances between oxygen atoms(Asp29-Oδ1, Asp30-Oδ1, Asp281-Oδ1 and Oδ2,

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Asn282-Oδ1, and FGly-Oγ) and Mg+2 were measured(Table 1), in which the smaller distances between theoxygen donors and the Mg+2 were noted for ARSAglyc,

pH5. On the other hand, consistent changes in oxygendonors coordination were not found to occur when com-

paring different tested pH, that is, the overall distancesbetween oxygen atoms and Mg+2 for ARSApH7 toARSApH5 did not deviate from each other consistently.This indicates that varying pH in this magnitude is notdirectly related to changes in ARSA active site coordina-

Figure 5. Mg+2 coodination analysis. The amino acid residues composing ARSA catalytic site, as well as those responsible forMg+2 octahedral coordination (Asp29, Asp30, Asp281, Asn282, and FGly69) are presented in the trajectory final frame of (A)ARSApH7, (B) ARSApH5, (C) ARSAglyc,pH5, and (D) ARSAN350S,pH5 systems. At ARSAN350S,pH5 system, the Mg+2 escape isillustrated within the simulation box.

Table 1. Octahedral Mg+2 coordination.

Oxygen donors

Distance from Mg+2 (Å)

1AUK

ARSA MD simulations

ARSApH7 ARSApH5 ARSAglyc,pH5 ARSAN350S,pH5

Asp29 Oδ1 2.30 2.07 ± .01 2.60 ± .09 2.79 ± .09 28.54 ± 12.94Asp30 Oδ1 2.23 2.11 ± .01 2.07 ± .01 2.08 ± .01 25.52 ± 16.31Asp281 Oδ1 2.66 2.08 ± .01 2.07 ± .01 2.06 ± .01 29.30 ± 14.31

Oδ2 2.19 2.09 ± .01 2.11 ± .01 2.06 ± .01 29.37 ± 14.12Asn282 Oδ1 2.59 2.19 ± .02 2.18 ± .02 2.02 ± .01 26.58 ± 16.20FGly69 Oγ 3.02 4.21 ± .05 4.10 ± .06 2.01 ± .01 28.06 ± 16.02

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tion architecture, and, consequently, it may not be relatedto the partial loss of ARSA catalytic activity in neutralenvironment. However, it is not possible to discard amore direct role of pH in the proper global architectureof ARSA active site during catalysis at all, once anincrease in positive charges are observed to occur frompH 7 to pH 5 around the active site, as previously dem-onstrated (Schenk et al., 2009), and this factor may pro-portionate a more suitable environment for binding thesulfate group.

Concluding remarks

In the current work, we were able to provide newinsights into how ARSA dynamics can be affected byenvironmental characteristics (as different pH conditions)and intrinsic components (as different glycan occupan-cies). In addition, several general and specific hypothe-ses, early formulated by in vitro findings, could beconfirmed by the employed approach. Based on thecomparison between ARSApH7 and ARSApH5 systems,nonglycosylated ARSA may have its catalytic functionabolished by structural changes associated with flexibilityincrease at cytoplasmatic pH. Despite ARSApH5 has beenobserved to be less flexible than ARSApH7, the second-ary structure conformational stability of ARSA onlybecomes apparent in ARSAglyc,pH5 state. In this context,the structural role of ARSA glycans appears to betwofold: (1) ARSA glycans specific disposition suggeststhat they may afford a steric protection against proteo-lytic attacks in lysosomes and (2) the absence of ARSAthird glycan in ARSAN350S,pH5 system results in a degreeof unfolding which involves glycosylation in the mainte-nance of ARSA secondary structure content.

The discussed hypothesis that ARSA-N350S poly-morphism may also significantly contribute for thepseudodeficiency condition, in the absence of ARSApolyadenylation defect, is evidenced by the existingpseudodeficient individuals presenting this allelic pro-file, with about 50% of normal ARSA activity, andby the additional finding of the 43% in vitro activityloss in ARSA-N350S. Our results provide, as a struc-tural explanation for this loss in ARSA activity, acombined effect of the protein unfolding and highersusceptibility to proteolytic attacks in lysosomal envi-ronment. While the ARSA protein moiety suffersimportant modifications derived from the interactionswith its attached glycans, as becoming more rigid inspecific regions, the glycan moieties vary their orien-tation in relation to the enzyme, without new con-former populations on their composing glycosidiclinkages. This observation reinforces the conception ofa minor influence of the protein scaffold on glycanconformation. With these findings, we hope to con-tribute to the comprehension of ARSA biological

activity, as well as to the effects of N-linked glycansin glycoproteins.

List of abbreviationsARSA arylsulfatase AARSAN350S,pH5 ARSA-N350S mutant MD simulation at

pH� 5DSSP dictionary of protein secondary structureFGly formylglycineM6P mannose 6-phosphateMLD metachromatic leukodystrophyARSApH5 nonglycosylated MD simulation at pH� 5ARSApH7 non-glycosylated MD simulation at

pH� 7RMSD root mean square deviationRMSF root mean square fluctuationARSAglyc,pH5 triple glycosylated MD simulation at

pH� 5

Acknowledgments

This study was supported by the Brazilian Funding Agen-cies Conselho Nacional de Desenvolvimento Científicoe Tecnológico (CNPq), MCT; by Coordenação deAperfeiçoamento de Pessoal de Nível Superior (CAPES),MEC, Brasília, DF, Brazil; by Fundação de Amparo à Pesquisado Estado do Rio Grande do Sul (FAPERGS); and by theFundo de Incentivo à Pesquisa e Eventos do Hospital deClínicas de Porto Alegre (FIPE-HCPA).

Supplementary material

The supplementary material for this paper is availableonline at http://dx.doi.10.1080/07391102.2013.780982.

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