Journal of Molecular Structure 980 (2010) 18–23

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Journal of Molecular Structure

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Structure of glycosylated Cu/Zn-superoxide dismutase from Kluyveromyces yeastNBIMCC 1984

Pavlina Dolashka-Angelova a,*, Vesela Moshtanska a, Anna Kujumdzieva b, Boris Atanasov a,Vencislava Petrova b, Wolfgang Voelter c, Jozef Van Beeumen d

a Institute of Organic Chemistry with Centrum of Phytochemistry, Bulgarian Academy of Sciences, G. Bonchev 9, 1113 Sofia, Bulgariab The Sofia University, Biological Faculty, Department of General and Industrial Microbiology, 8 ‘‘Dragan Tsankov” St., 1421 Sofia, Bulgariac Interfacultary Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Straße 4, D-72076 Tübingen, Germanyd Laboratory of Protein Biochemistry and Biomolecular Engineering, Ghent University, KL Ledeganckstraat 35, 9000 Ghent, Belgium

a r t i c l e i n f o

Article history:Received 2 May 2010Received in revised form 30 June 2010Accepted 30 June 2010Available online 6 July 2010

Keywords:GlycoproteinCu/Zn-superoxide dismutaseMALDI–TOF–TOFQ-TrapYeast Kluyveromyces marxianus

0022-2860/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.molstruc.2010.06.031

* Corresponding author. Address: Institute of OrganPhytochemistry, Bulgarian Academy of Sciences, G. BoTel.: +359 29606163; fax: +359 28700225.

E-mail addresses: pda54@abv.bg, dolashka@orAngelova).

a b s t r a c t

The primary structure of Cu/Zn-superoxide dismutase from Kluyveromyces marxianus NBIMCC 1984 waselucidated by N-terminal sequence analysis of the intact protein and by determination of the amino acidsequences of tryptic peptides by MALDI–TOF–TOF tandem mass spectrometry. The molecular mass of onesubunit of the homodimer SOD, containing 152 amino acid residues, was calculated to be 15858.3 Dawhile a value of 17096.63 Da was obtained by MALDI–TOF MS. This difference is explained by the pres-ence of N-glycosylation of one linkage site, -Asn-Ile/Leu-Thr-, and a glycan chain with the structure Hex5

GlcNAc2. Glycosylation of K. marxianus superoxide dismutase is a post-translational modification. Recentdevelopments in mass spectrometry have enabled detailed structural analyses of covalent modificationsof proteins. Therefore, in this paper, we introduce a covalent modification of Cu/Zn-SOD from K. marxi-anus NBIMCC 1984, by analysis of the enzymatic liberated N-glycan from the enzyme using MALDI–TOF and tandem mass spectrometry on a Q-Trap mass spectrometer.

This is the first report of the structure of the oligosaccharide of a naturally-glycosylated superoxide dis-mutase, determined by mass spectrometry.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

SODs are metalloenzymes that detoxify superoxide radicals bytheir conversion to hydrogen peroxide and oxygen [1–3]. They areclassified into four groups: Mn-SOD [4], Cu/Zn-SOD [5], Fe-SOD[6] and Ni-SOD [7]. Cu/Zn-SODs are generally found in the cytosolof eukaryotic cells, in chloroplasts, and in some prokaryotes [8–12].

However, also an extracellular Cu/Zn-SOD exists in all kingdomsof life. In higher organisms, including human, it is believed to be se-creted in the extracellular matrix to protect against ROS, but also topreserve NO bioactivity and, as such, it is widely studied for its rolein a wide range of tissues and disease [13]. Human EC-SOD (SOD3)is a tetramer bearing a single N-linked glycan of hitherto unknownstructure, except for the recombinant product in CHO cells [14]. Thecrystal structure, providing evidence for heparin and collagen bind-ing sites, has recently been resolved [15]. No biological activity isattributed to the glycan, as a non-glycated recombinant variant

ll rights reserved.

ic Chemistry with Centrum ofnchev 9, 1113 Sofia, Bulgaria.

gchm.bas.bg (P. Dolashka-

maintains all biological function [16]. Human EC-SOD is associatedwith a wide range of diseases [17–19]. There is strong evidence for atherapeutic value of extracellular Cu/Zn-SOD as an antioxidant totreat, amongst others, burn wounds [19], heart diseases [20] andphysiological decline caused by aging [21].

Currently, obtaining SOD for therapeutic applications still relieslargely on extractions from tissue. Efforts for large-scale produc-tion of EC-SOD in Escherichia coli failed, due to the formation ofinclusion bodies and despite severe efforts for refolding to regainactivity [22,23]. We postulate that the poor solubility is mainlydue to absence of the glycan chain in bacterial expression. Indeed,murine EC-SOD was successfully produced in Pichia pastoris, a fun-gal strain known as a good host for glycoprotein expression[24,25]. Recently, human EC-SOD was successfully produced at aconcentration of 0.44 mg/L [26]. An alternative host for expressionof EC-SOD is Kluyveromyces marxianus, an important industrialyeast used in classical applications (biomass, ethanol, enzymes)and also a host for heterologous protein production [27,28]. Re-cently, the K. marxianus L3 strain was shown to possess the highestnatural SOD production upon fermentative growth [29]. However,this work was focusing on the intracellular (SOD1) Cu/Zn andMn-SOD types. We recently detected and partially purified a natu-rally-glycosylated extracellular Cu/Zn SOD from this strain [30],

P. Dolashka-Angelova et al. / Journal of Molecular Structure 980 (2010) 18–23 19

which opens ways for the production of glycosylated human EC-SOD. Moreover, some of us discovered potential antiviral and anti-cancer properties of another naturally glycosylated fungal SOD,isolated from Humicola lutea 103 [31,32].

Therefore, the aim of this study was the purification and struc-tural characterization of the naturally-glycosylated superoxidedismutase from yeast strain K. marxianus NBIMCC 1984. Thelarge-scale biotechnological production of EC-SOD with filamen-tous fungi was so far never developed; K. marxianus thereforewould prove to be attractive organisms, for industrial-scale fer-mentative production of food- or pharmaceutical-grade SOD.

2. Materials and methods

2.1. Purification of Cu/Zn-SOD from K. marxianus NBIMCC 1984

Cu/Zn-SOD was purified from the yeast strain K. marxianusNBIMCC 1984 as described by Nedeva et al. [30]. Additionally,

Table 1Molecular masses and amino acid sequences of isolated peptides of Cu/Zn-SOD fromK. marxianus NBIMCC 1984 on a Nucleosil C18 column of a HPLC system. Assignmentsof either Leu or Ile (isobaric) were made on the basis of homology with the sequencesof other SODs (see Fig. 3).

Fraction elut.time (min)

Mass[M + H]+

Position Amino acidsequences

190 1773.51(1571.01)

29–42 EVWN(I/L)TGNSPNA(I/L)R

210 1588.8 136–152 TGNAGSRPACGV(I/L)G(I/L)TN220 2718.8 43–68 GFHSHEFGDNTNGCTSAGPHFDPSAK230 2139.7 115–135 TVVVHGGQDD(I/L)GKGGNEES(I/L)K

1002.2 96–104 GSKQD(I/L) (I/L) (I/L)K250 1681.9 79–95 HVGD(I/L)GN(I/L)STDAQGVAK290 1177.3 69–78 EHG(I/L)PPDQQR

1324.7 115–127 TVVVHGGQDD(I/L)GK833.9 128–135 GGNEES(I/L)K

420 976.1 8–17 GDSNVSGIVK490 1133.3 105–114 (I/L)IFQNSVVGR

1327.5 18–28 FEQESEDQSTK

Fig. 1. Reverse phase chromatography of tryptic peptides of Cu/Zn-SOD from K. marHyPURITY C18, Thermo Quest), and eluted as given in Section 2. (Insert) Orsinol/H2SO4 tes42: B1-49, B2-52, B3-53, B4-190 , B5-140 , B6-H2O.

the enzyme was purified on a Mono Q column 5/5 using a FPLC sys-tem. The pure SOD fraction was eluted with 50 mM Tris/HCl(pH 7.0) with a 0–1.0 M NaCl gradient and at a flow rate of 1 ml/min. The SOD activity was measured using the nitroblue tetrazo-lium (NBT) reduction method of Beauchamp and Fridovich [33].One unit of SOD activity was defined as the amount of SOD re-quired to inhibit the reduction of NBT by 50%, measured at560 nm, and was expressed as units per mg protein [U/mg protein].The protein content was estimated by the Lowry procedure [34],using crystalline bovine serum albumin as standard.

2.2. Pyridylethylation and enzymatic digestion of Cu/Zn-SOD from K.marxianus NBIMCC 1984

Three milligrams of Cu/Zn-SOD from K. marxianus NBIMCC 1984were dissolved in 1.0 ml of 0.25 M Tris/HCl, pH 8.5, 6 M guanidine–HCl, and 1 mM EDTA. An ethanolic solution of 30 ll DTT was addedand the mixture was incubated under nitrogen for 2 h at roomtemperature in the dark. Neat 4-vinylpyridine (100-fold molar ex-cess of the expected cysteinyl residues) was added and the mixturewas incubated under nitrogen for 2 h at room temperature in thedark. The pyridylethylated protein was desalted by reverse phaseHPLC on an Aquapore RP-300 column (2.1 � 30 mm; Applied Bio-systems, Weiterstadt, Germany).

Fifty microlitres of a trypsin solution (1 mg/ml) was added to0.50 ml of 25 mM Tris/HCl, pH 9.0, containing 1 mg pyridylethylat-ed SOD (E:S 1:50) and the reaction mixture was incubated over-night at room temperature. The digest was applied to an HPLCHypersil column (250 � 4.6 mm; 5 ll HyPURITY C18, ThermoQuest), eluted with eluent A (0.1% TFA in water) and eluent B(80% acetonitrile in A), using a gradient program of 0% B for5 min and then 0–100% B in 60 min; the flow rate was 0.7 ml/min. The UV absorbance of the elution was monitored at 214 nm.Peak fractions were dried and, after dissolving in 0.1% (v/v) TFA,were analysed by MALDI–TOF–TOF mass spectrometry on a 4700Proteomics Analyser (Applied Biosystems, Framingham, MA). Themass spectrometer uses a 200 Hz frequency-tripled Nd–YAG laseroperating at a wavelength of 355 nm. For analysis, about 50 pmol

xianus NBIMCC 1984, separated on a HPLC Hypersil column (250 � 4.6 mm; 5 llt on isolated fractions from HPLC system: A1-210 , A2-220 , A3-230; A4-250 , A5-29, A6-

20 P. Dolashka-Angelova et al. / Journal of Molecular Structure 980 (2010) 18–23

of the HPLC fractions were dissolved in 0.1% (v/v) TFA and appliedto the target. Analysis was carried out using a-cyano-4-hydroxy-cinnamic acid as a matrix. The instrument was calibrated using apeptide mixture solution provided by the manufacturer.

2.3. Carbohydrate analysis and sequencing of glycan

The lyophilized peptides isolated on a HPLC Hypersil columnwere dissolved in 10 ll water and 1 ll of this solution was appliedto a thin layer plate and air-dried, taking care to restrict the size ofthe spot to 2–3 mm in diameter. The plate was sprayed with orci-nol/H2SO4 and heated for 20 min at 100 �C.

For deglycosylation, approximately 2 mg of Cu/Zn-SOD from K.marxianus NBIMCC 1984 were treated with PNGase F (2 units)(Roche Diagnostics GmbH, Mannheim, Germany). The glycan wasisolated as described by Sandra et al. [35]. After incubation for24 h at 37 �C, the exoglycosidase digestion was diluted 10-fold

β1 β2

β4

β6

β8

1 10 20K. marxianus 1984 Q A V A V L K G D S N V S G I V K F E QK. marxianus L3 N A V A V L K G D S N V S G I V R F E QS. cerevisiae Q A V A V L K G D A G V S G V V K F E QC. famata Q A V A V L R G D S K V I G V V N F E QA.niger K A V A V I R G D S K V S G T V T F E QA.oryzae K A V A V L R G D S K I S G T V T F E QA. fumigatus K A V A V L R G D S K I T G T V T F E QH. lutea K A V A V L R G D S K I T G T V T F E QN.crassa K A V A V V R G D S N V K G T V I F E Q

50 60K. marxianus1984 H S H E F G D N T N G C T S A G P H F DK. marxianus L3 H I H E F G D N S N G C T S A G P H F NS. cerevisiae H I H E F G D A T N G C V S A G P H F NC. famata H V H T F G D N T N G C T S A G P H F NA.niger H V H Q F G D N T N G C T S A G P H F NA.oryzae H V H Q F G D N T N G C T S A G P H F NA. fumigatus H V H Q F G D N T N G C T S A G P H F NH. lutea H I H Q E G D N T N G C T S A G P H Y NN.crassa H I H T F G D N T N G C T S A G P H F N

110K. marxianus1984 D A Q G V A K G S K Q D L L I K L I F QK. marxianus L3 D A Q G V A K G S V T D K H V K L I G PS. cerevisiae D E N G V A K G S F K D S L I K L I G PC. famata D T S G V A K G S K Q D L F V K L I G QA.niger D A E G N A V G S K Q D K L V K L I G AA.oryzae D A E G N A V G S K Q D K L I K L I G AA. fumigatus D A E G N A V G S K Q D K L I K L I G AH. lutea D A E G N A V G S V Q D K L I K V I G AN.crassa D A Q G N A K G T V T D N L V K L I G P

140 150K. marxianus1984 K T G N A G S R P A C G V I G L T NK. marxianus L3 K T G N A G G R V A C G V I G I S NS. cerevisiae K T G N A G P R P A C G V I G L T NC. famata K T G N A G A R L A C G V I G L T N K PA.niger K T G N A G P R P A C G V I G I A AA.oryzae K T G N A G A R P A C G V I G I AA. fumigatus K T G N A G A R P A C G V I G I AH. lutea K T G N A G P R P A C G V I G I AN.crassa K T G N A G P R P A C G V I G I S Q

Fig. 2. Alignment of the amino acid sequence of Cu/Zn-SOD from K. marxianus NBIMCparentheses): Saccharomyces cerevisiae (AAT99430), K. marxianus L3 (29), Candida famAspergillus fumigatus (Q9Y8D9), Humicola lutea 103 (P83684), Neurospora crassa (P07509

and 1 ll of a 1:1 sugar–matrix mixture was applied onto the MAL-DI target. The matrix was a dihydroxybenzoic acid solution in 50%CH3CN (10 mg/ml). A total of 1500 shots were acquired in the MSmode. Spectra from m/z 900 to 3000 were recorded. The digestionproducts were typically observed as [M + Na]+ ions.

2.4. Q-Trap analyses (MS and MS/MS)

Off-line ESI-MS measurements of the glycan was performed onthe Q-Trap mass spectrometer, equipped with a nanospray ionsource (Proxeon, Odense, Denmark) using Proxeon medium nano-spray needles. Typically, 10 ll of sample in 50% MeOH was intro-duced. The needle voltage was set at 1000 V. In the product ionscanning mode, the scan speed was set to 1000 Da/s, with Q-trap-ping being activated. The trap fill-time was 200 ms in the MS/MS-scan modes. For operation in the MS/MS modes, the resolution ofQ1 was set to ‘low’. Excitation time was set at 100 ms.

β3 β4

β5

β7

30 40E S E D Q S T K E V W N I T G N S P N A L R G FE S E D Q S T K I S W E I T G N D A N A L R G FA S E S E P T T V S Y E I A G N S P N A E R G FS S E S D P T F I T W E I S G N D A N A L R G FA N E N T P T T I S W N I T G H D A N A E R G FA D A N A P T T V S W N I T G H D A N A E R A FA D E N S P T T V S W N I K G N D P N A K R G FA N E S A P T T V S W N I T G H D P A E R G ME S E S A P T T I T Y D I S G N D P N A K R G F

70 80 90P S A K E H G L P P D Q Q R H V G D L G N I S TP Y K K T H G A P G D E T R H V G D L G N I S TP F K K T H G A P T D E V R H V G D M G N V K TP F T K E H G A P E D D N R H V G D L G N V T TP Y G K T H G A P E D D E R H V G D L G N F K TP F G K E H G A P E D E N R H V G D L G N F K TP F G K T H G A P E D S E R H V G D L G N F E TP F K K T H G A P T D E V R H V G D L G N I K TP H G G T T H G D R T A E V R H V G D L G N I E T

120 130N S V V G R T V V V H G G Q D D L G K G G N E E S LL S V I G R T V V V H G G Q D D L G K G G N E E S LT S V V G R S V V I H A G Q D D L G K G D T E E S LN S I L G R T V V I H A G T D D L G K G G N A E S KE S V L G R T L V V H A G T D D L G R G G N E E S KE S V L G R T L V I H A G T D D L G R S E H P E S KE S V L G R T L V V H A G T D D L G R G G N E E S KE S I L G R T I V V H A G T D D L G R G G N E E S KE S V I G R T V V V H A G T D D L G K G G N E E S L

N S

C 1984 to SODs from other sources (SWISS-PROT/TrEMBL Accession numbers inata (AAK82335), Aspergillus niger 26 (AAU4413), Aspergillus oryzae (BAE58164.1),), Cu/Zn-SODs.

P. Dolashka-Angelova et al. / Journal of Molecular Structure 980 (2010) 18–23 21

2.5. Bioinformatics

Using programmes such as UCSFChimera, DeepView, MolIDEand RasMol, combining the most frequent modeling steps in asemi-automatic interactive way, a three-dimensional proteinstructure of SOD dimer was generated [36].

3. Results and discussion

Yeast cells produce two forms of superoxide dismutase: thecopper- and zinc-containing SOD (Cu/Zn-SOD), encoded by theSOD1 gene which is the predominant form and protects cytosolicconstituents from oxidation [37], and the manganese containingenzyme (Mn-SOD), encoded by the gene SOD2, which is locatedin the mitochondrial matrix [38]. The lactose-fermenting yeast K.marxianus, besides having a high SOD activity, presents a numberof gainful large-scale fermentation characteristics in comparisonto other yeast species: it has a fast and high biomass yield, it hasa high temperature of growth, and is safe in use (not methylo-trophic and food grade status) [39,40]. Besides these advantages,a new Cu/Zn-SOD could be isolated and characterized from thethermotolerant yeast strain K. marxianus NBIMCC 1984.

The enzyme was purified on a Mono Q column as described inExperiments. It is a homodimer and the molecular mass measured

Fig. 3. MALDI–MS/MS, spectrum of peptide at m/z 1681.89 of C

Fig. 4. MALDI–MS/MS, spectrum of peptide moiety still carrying one HexNAc (m/z 177voltage of 95 V and a collision energy of 80 eV.

by MALDI–MS is 17096.6 [M + Na]+. Under the acidic pH of 0.1% (v/v) TFA, the homodimer dissociates into monomers with a molecu-lar mass of 17074.6 Da, which is in agreement with the value of34 kDa deduced from 10% PAGE electrophoresis (data not shown)[30].

The primary structure of Cu/Zn-SOD from K. marxianus NBIMCC1984 was elucidated by N-terminal sequencing of the intactprotein combined with the determination of the amino acid se-quences of a set of overlapping peptides generated by proteolyticcleavage (Table 1). Fig. 1 presents the reverse phase HPLC isolationprofile of the tryptic fractions, which were collected and subse-quently sequenced by MALDI–MS/MS. The spectrum of the peptidewith mass 1681.89 is shown in Fig. 2. It allows to deduce the se-quence HVGD(L/I)GN(L/I)STDAQGVAK from the series of y- and b-ions. Based on the amino acid sequence of other peptides the pri-mary structure of Cu/Zn-SOD from K. marxianus NBIMCC 1984 isrepresent in Fig. 3. As for all the other peptides of the protein se-quence in which the isobaric residues Leu or Ile might occur, theassignment was made on the basis of sequence homology withseveral other SODs.

K. marxianus SOD contains two Cys residues, Cys57 and Cys147,only one tryptophan residue at position 32, and three putativelinkage sites at positions 11 (-Asn-Val-Ser-), 33 (-Asn-Leu/Ile-Thr-) and 87 (-Asn-Leu/Ile-Ser-). Very high homology was observed

u/Zn-SOD isolated from strain K. marxianus NBIMCC 1984.

3.51), of Cu/Zn-SOD isolated from strain K. marxianus NBIMCC 1984, using a cone

Man5 GlcNAc2

22 P. Dolashka-Angelova et al. / Journal of Molecular Structure 980 (2010) 18–23

in several regions in comparison with SODs from the species Sac-charomyces cerevisiae (AAT99430), K. marxianus L3 [29], Candida fa-mata (AAK82335), Aspergillus niger 26 (AAU4413), Aspergillusoryzae (BAE58164.1), Aspergillus fumigatus (Q9Y8D9), H. lutea 103(P83684), Neurospora crassa (P07509). Ser27, Asp65, and Phe109are replacing the conserved residues Pro27, Asn65, and Gly109,respectively. However, in the zinc-binding region (loop IV), Pro66is well-conserved as in the other Cu/Zn-SODs. Pro62, Pro66,Pro75 and Pro76 are important to preserve the conformation of ab-turn. In Cu/Zn-superoxide dismutase from deep-sea yeast Cryp-tococcus liquefaciens strain N6 was found that Arg75 substitutingprolines in other Cu/Zn-SODs increased loop flexibility [12].

Also the conserved region around the copper ion (composed ofHis46, His48 and His121) in the active site is present and is likelyassociated with the critical role it may play in the structural stabil-ity of Cu/Zn-SOD. It was found that the unfolding of human Cu/Zn-SOD loaded with copper ions is slower than that of the zinc-loadedprotein, indicating that the copper ion, with three of its four li-gands belonging to the b-barrel motif, contributes more than thezinc ion to the kinetic stability of SOD [41].

Based on the primary structure of Cu/Zn-SOD from K. marxianusNBIMCC 1984, a 3-D model of one subunit was built using the pro-gramme RasMol and the homologous S. cerevisiae Cu/Zn-SOD astemplate. The SOD molecule has a highly conserved typical struc-

Fig. 5. MALDI–TOF–MS spectra of the N-glycan isolated from Cu/Zn-SOD from K.marxianus NBIMCC 1984. One microlitre of a 1:1 sugar–matrix mixture was appliedonto the MALDI target. The matrix DHB (10 mg/ml dihydroxybenzoic acid solutionin 50% AcN) was used.

Fig. 6. MS/MS spectra on a Q-Trap and structure with fragmentation nomenclature of tfrom K. marxianus NBIMCC 1984.

tural scaffold common to all SODs, with mainly antiparallel b-sheets connected by long variable loops and very short a-helices(data not shown).

Post-translational modifications of proteins control manybiological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spec-trometry have enabled detailed structural analyses of covalentmodifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. We here showthe existence of covalent modification of superoxide dismutase, asglycosylation.

This paper extends our previous studies on the glycosylated Cu/Zn-SOD from K. marxianus NBIMCC 1984. Based on the identifiedamino acid sequence, the molecular mass of Cu/Zn-SOD from K.marxianus NBIMCC 1984 was calculated to be 15858.3 Da, whichdiffers from the value obtained from MALDI–MS analysis of the in-tact protein (17096.63 Da). The Cu/Zn-SOD sequence revealedthree potential N-linkage sites (Asn11, Asn33, and Asn87), suggest-ing that the enzyme might be glycosylated. The spectra of two pep-tides with masses 1681.89 and 1773.51 are shown in Figs. 2 and 4,respectively. The sequence of the peptide at m/z 1681.89 contains

he single charged [M + Na]+ of the glycan at m/z 1235.52, isolated from Cu/Zn-SOD

Man5 GlcNAc2

Cu Zn

Fig. 7. Ribbon presentation of the three-dimensional model structure of Cu/Zn-SODisolated from yeast K. marxianus NBIMCC 1984, established by the programMODELLER, and the position of carbohydrate structure attached to the polypeptidechain.

P. Dolashka-Angelova et al. / Journal of Molecular Structure 980 (2010) 18–23 23

one potential linkage site -Asn-(Leu/Ile)-Ser-, but the mass calcu-lated from the amino acid sequence fits the value obtained byMALDI, which means that no carbohydrate chain is attached to thislinkage site. The peptide gives also a negative orcinol/H2SO4 test(Fig. 1, position A4). Analysis of this peptide has proven thatAsn87 is not glycosylated.

One putative linkage site also was observed in the sequence ofthe peptide -EVWN(I/L)TGNSPNA(I/L)R- with mass of 1773.51. Evi-dently the linkage site (-Asn-Leu/Ile-Thr-) at position 33–35 is theglycosylated one. This linkage site is conserved in several SODs[31]. The orcinol/H2SO4 test was effectively positive, confirmingthat one glycan is present in the fraction eluted at 19 min. The ami-no acid sequence of the peptide chain EVWN(I/L)TGNSPNA(I/L)Rwas determined based on the singly-charged ions (Fig. 4). The dif-ference between two single ions at m/z 1571.01 and m/z 1773.51 iscorresponding to one GlcNAc connected to the peptide, which alsodemonstrates that the peptide of 1773.51 Da is glycosylated. Theion at 1359.23 is the same peptide as y11 [at m/z 1156.74, N(I/L)TGNSPNA(I/L)R], with one GlcNAc connected to linkage siteAsp-Ile/Leu-Thr.

Determination of the carbohydrate structure in the polypeptidechain of Cu/Zn-SOD from K. marxianus NBIMCC 1984 included iso-lation and MALDI–TOF characterization of the glycan from the in-tact enzyme, carried out as described under ExperimentalProcedures. A single peak at 1257.3 [M + Na]+, was detected whichsuggests a uniform oligosaccharide chain (Fig. 5). The structure ofthis carbohydrate chain was determined by ESI Q-Trap MS/MS ofthe singly-charged ion with a mass of 1235.52 Da (M + H)+ asshown in Fig. 6. The sequence can easily be read when consideringthe y-ions and the combination of b- and y-ions (m/z 222.1, 325.1,425.1, 811.2, 1014.3). The structure, given as inset in the figure, is aclassical high mannose type of sugar (GlcNAc2, Man5), with a cal-culated mass of 1234.4 Da.

Based on the primary structure we also made a 3-D model of thedimer. The position of the carbohydrate chain was identified to beon the surface of the molecule (Fig. 7). The glycosylation of SODs isof interest both from scientific and clinical point of view. So far,only one naturally-glycosylated enzyme was identified in fungalSODs: one molecule of N-acetylglucosamine is connected to thepolypeptide chain of H. lutea 103 SOD [31]. We have previouslystudied the protective effects of this naturally-glycosylated HLSODcompared to non-glycosylated SODs. After treatment the experi-mental influenza A/Aichi infection of mice was found that HlSODprotects mice from mortality, the survival having been prolongedby 5.2 days [31]. Apart from Hl SOD, glycosylated SOD is alsoknown in erythrocytes and rat tissues, and in Schistosoma mansoni[42]. The presence of mono-, di, tri-, and tetra-glycated SODs wasdetected in human erythrocytes [43]. The structure of the largestcarbohydrate chain has not yet been determined [14].

Acknowledgments

This work was supported by the Fund for Scientific Research-Flanders (FWO-Vlaanderen) through Project VS.011.06N and agrant from the National Science Fund, Ministry of Education andScience – Republic of Bulgaria, Project No. <201/06-2492.

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