Expression, characterization and homology modeling of a novel eukaryotic GH84 β-N-acetylglucosaminidase from Penicillium chrysogenum

Protein Expression and Purification 95 (2014) 204–210

Contents lists available at ScienceDirect

Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep

Expression, characterization and homology modeling of a novel eukaryotic GH84 b-N-acetylglucosaminidase from Penicillium chrysogenum Kristy´na Slámová a,⇑, Natallia Kulik b, Martin Fiala a, Jana Krejzová-Hofmeisterová a,c, Rüdiger Ettrich b, Vladimír Krˇen a a

Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenˇská 1083, 14220 Praha 4, Czech Republic Department of Structure and Function of Proteins, Institute of Nanobiology and Structural Biology of GCRC, Zámek 136, 37333 Nové Hrady, Czech Republic c Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Technická 5, 16628 Praha 6, Czech Republic b

a r t i c l e

i n f o

Article history: Received 21 November 2013 and in revised form 3 January 2014 Available online 14 January 2014 Keywords: b-N-acetylglucosaminidase Homology modeling O-GlcNAcase Pichia pastoris Yeast expression system

a b s t r a c t b-N-acetylglucosaminidases from the family 84 of glycoside hydrolases form a small group of glycosidases in eukaryotes responsible for the modification of nuclear and cytosolic proteins with O-GlcNAc, thus they are involved in a number of important cell processes. Here, the first fungal b-N-acetylglucosaminidase from Penicillium chrysogenum was expressed in Pichia pastoris and secreted into the media, purified and characterized. Moreover, homology modeling and substrate and inhibitor docking were performed to obtain structural information on this new member of the GH84 family. Surprisingly, we found that this fungal b-N-acetylglucosaminidase with its sequence and structure perfectly fitting to the GH84 family displays biochemical properties rather resembling the b-N-acetylhexosaminidases from the family 20 of glycoside hydrolases. This work helped to increase the knowledge on the scarcely studied glycosidase family and revealed a new type of eukaryotic b-N-acetylglucosaminidase. Ó 2014 Elsevier Inc. All rights reserved.

Introduction b-N-acetylglucosaminidases1 from eukaryotic organisms (O-GlcNAcases; OGA; EC 3.2.1.169; GH84) form a small and structurally not much explored group of glycosidases strictly specific for the hydrolysis of a single N-acetylglucosamine moiety bound to proteins [1,2]. The modification of nuclear and cytosolic proteins with O -GlcNAc is widespread in higher eukaryotes and it is involved in a number of important cell processes, such as transcription, ubiquitination, cell cycle and stress response [3,4]. The studies employing kinase and phosphatase inhibitors have proved the reciprocal relationship between protein phosphorylation and O-GlcNAcylation, suggesting that protein O-GlcNAc modification forms an alternative signalling mechanism [5]. Due to its universal occurrence and ⇑ Corresponding author. Tel.: +420 296442766; fax: +420 296442509. E-mail addresses: [email protected], [email protected] (K. Slámová). 1 Abbreviations used: aa, amino acid; BtOGA, b-N-acetylglucosaminidase from Bacteriodes thetaiotaomicron; CpOGA, b-N-acetylglucosaminidase from Clostridium perfringens; GH84, glycoside hydrolase family 84; NAG-thiazoline, 1,2-dideoxy -2 0 -methyl- a - D -glucopyrano-[2,1-d]- D 2 0 -thiazoline; OGA, b-N-acetylglucosa minidase; O-GlcNAc, N-acetylglucosamine O-linked to protein backbone via Ser/Thr residues; O-GlcNAcase, b-N-acetylglucosaminidase; OgrOGA, hyaluronoglucosaminidase from Oceanicola granulosus; pdb, protein data bank; pNP-b-GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy-b-D-glucopyranoside. 1046-5928/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2014.01.002

importance, protein O-GlcNAcylation is involved in the etiology of severe afflictions such as diabetes, cancer and neurodegenerative disorders like Alzheimer’s disease [6,7]. Together with peptide N-acetylglucosaminyltransferase, b-N-acetylglucosaminidase is responsible for protein O-GlcNAc cycling, which makes this enzyme crucial for the regulation of a large number of physiological events. Up to date, only a very limited number of GH84 O-GlcNAcases have been isolated and characterized and only three crystal structures of the bacterial GH84 enzymes have been resolved: the multimodular b-N-acetylglucosaminidase from the pathogen Clostridium perfringens [8,9], the b-N-acetylglucosaminidase from the human gut symbiont Bacteroides thetaiotaomicron [10,11] and the enzyme from Oceanicola granulosus sharing high homology with human O-GlcNAcase [12]. Due to the availability of their structures, these enzymes have been widely used in model studies of O-GlcNAcase inhibition and many enzyme-inhibitor crystal structures have been reported [13–15]. Among the eukaryotic O-GlcNAcases, only the representatives from rat [16], mouse [17] and humans [2,18,19] have been characterized, identifying the mammalian enzyme as a multimodular protein comprising C-terminal histone acetyltransferase domain and N-terminal GH84 domain. The coordination of these activities is required for chromatin acetylation and the removal of O-GlcNAc repressor in the process of gene transcription initiation [17]. In

K. Slámová et al. / Protein Expression and Purification 95 (2014) 204–210

humans, the enzyme providing protein O-GlcNAc cleavage was originally identified as the MGEA5 hyaluronidase (meningioma expressed antigen 5; mgea5 gene); moreover, it was found to occur in two splice variants separately located in cell compartments: full-length (130 kDa) in the cytoplasm and spliced variant (75 kDa) in the nucleus [18]. As no crystal structure of eukaryotic GH84 b-N-acetylglucosaminidase has been resolved so far, recently the molecular model of human O-GlcNAcase based on structures of the homologous bacterial enzymes has been reported [20]. The detailed structural and kinetic studies have provided evidence that GH84 b-N-acetylglucosaminidases employ the substrate-assisted catalytic mechanism, which proceeds via the oxazoline reaction intermediate instead of the classical enzyme– substrate covalent intermediate typical for most glycosidases [21], with the key catalytic residues identified as Asp174-Asp175 in the human enzyme [22]. Due to the importance of O-GlcNAcase for human health, the research of specific inhibitors based on the knowledge of the structure and catalytic mechanism is quickly progressing (reviewed in [23]). Among all compounds designed and tested as O-GlcNAcase inhibitors, thiamet-G is the lead molecule, as it has been shown to slow down neurodegeneration and stabilize protein tau against aggregation by hindering the action of O-GlcNAcase in a mouse model, which makes thiamet-G a potential Alzheimer’s therapeutic [24,25]. Here we report on the first non-mammalian eukaryotic b-N-acetylglucosaminidase from Penicillium chrysogenum, which was expressed in a yeast expression system, purified and characterized. Moreover, the molecular model of this enzyme was prepared using homology modeling. Overall, this work provides complex biochemical and computational information on the first fungal GH84 b-N-acetylglucosaminidase, increasing the knowledge base of this scarcely studied family of glycosidases.

Materials and methods Expression of b-N-acetylglucosaminidase in Pichia pastoris The gene of b-N-acetylglucosaminidase from P. chrysogenum Wisconsin 54–1255 (GenBank ID: CAP80500; NCBI: XP_002557703.1) [26] was prepared synthetically (Generay, China) and cloned into the yeast expression vector pPICZaA via the EcoRI and KpnI restriction sites, omitting the predicted signal propeptide (aa 1–24). The expression vector pPICZaA-GH84Pc encoding also for the yeast a-factor for extracellular protein targeting and zeocin resistance was used for the b-N-acetylglucosaminidase expression in P. pastoris KM71H. The vector (10 lg) was linearized by restriction endonuclease SacI (New England Biolabs, USA) and phenol/chloroform extraction and ethanol precipitation were carried out before transformation. The yeast strain P. pastoris KM71H (Invitrogen, USA) was cryopreserved at 80 °C in 15% (v/v) glycerol (OD600 = 50  100), added into YPD (Yeast Extract Peptone Dextrose Medium: 1% yeast extract; 2% peptone; 2% glucose) medium, or maintained on YPD plates (YPD medium with 2% agar). For the P. pastoris cultivation, YPD, BMGY (Buffered Glycerol–complex Medium: 1% yeast extract; 2% peptone; 100 mM potassium phosphate, pH 6.0; 1.34% YNB (Yeast Nitrogen Base, Invitrogen, USA); 4  105% biotin; 1% glycerol), and BMMY (Buffered Methanol–complex Medium: the same as BMGY, 0.5% methanol is added instead of 1% glycerol) media were used. All the cultivation media used are described in the manufacturer’s instructions (EasySelect Pichia Expression Kit, Invitrogen, USA). For the preparation of P. pastoris competent cells 5 mL of YPD medium was inoculated with 50 lL of cryopreserved culture and cultivated overnight at 30 °C. 250 mL of YPD medium in 1 l conical flask was inoculated with 50 lL of the night culture and cultivated

205

at 30 °C until OD600 reached 1.3–1.5 (OD600 = 1 corresponded approximately to 5.107 cells/mL). The culture was centrifuged (4000g, 10 min, 4 °C) and the pellet was resuspended in 500 mL of ice-cold sterile water. The cells were centrifuged as described above. The pellet was resuspended in 250 mL of ice-cold sterile water and centrifuged as described above. Then the pellet was resuspended in 20 mL of ice-cold 1 M sorbitol and centrifuged. Finally the pellet was resuspended in 1 mL of ice-cold 1 M sorbitol. Such prepared competent P. pastoris cells were transformed with the expression vector by electroporation: 80 lL of freshly prepared P. pastoris cells were incubated with 10 lg of the linearized expression vector containing b-N-acetylglucosaminidase gene in an electroporation cuvette for 5 min on ice and then electroporated (Pichia program, Electroporator, Bio-Rad, DE). 1 mL of ice-cold 1 M sorbitol was added to the transformed cells and the transformed cells were incubated for 2 h at 30 °C in a 15 ml tube. The electroporated cells were maintained in various concentrations (spread 10, 25, 50 and 100 lL culture each on separate YPD plates) under the selection pressure of zeocin (100 lg/mL) on YPD agar plates for 2–4 days at 28 °C. For the production of the recombinant enzyme, the colonies were inoculated into 100 mL of BMGY medium in 1 l flask and incubated overnight at 28 °C on a rotary shaker (220 rpm) until OD600 reached 2–6. Then the cells were collected by centrifugation (3000g, 10 min, 20 °C) and the pellet was resuspended in 50 mL of BMMY medium in 300 ml baffled flask. The recombinant b-N-acetylglucosaminidase production was induced by methanol (0.5% v/v) every 24 h and flasks were stirred on a rotary shaker at 28 °C and 220 rpm for 3 days. Purification of b-N-acetylglucosaminidase Recombinant b-N-acetylglucosaminidase was purified from the culture medium of P. pastoris after 3 days of cultivation as described above. The cells were removed by filtration and centrifugation (3000g, 10 min, 20 °C), and the medium was diluted 3 with water and its pH was adjusted to 6.5. The diluted culture medium containing b-N-acetylglucosaminidase was loaded onto the anion exchange chromatography column (Q-Sepharose (Merck, DE) equilibrated by 10 mM sodium citrate–phosphate buffer pH 6.5 using Äkta Purifier protein chromatography system (Amersham Biosciences, SE). Under these conditions, b-N-acetylglucosaminidase was not retained in the column, while most of the other proteins were bound to the matrix, thus, the flow-through was collected and concentrated by ultrafiltration (10 kDa cut off membrane, Millipore, US). The concentrate was further purified by gel permeation chromatography (Superdex 200, Amersham Biosciences, SE) employing the 50 mM sodium citrate–phosphate buffer pH 7.0, 150 mM NaCl, as a mobile phase. The active fractions were collected and utilized in further experiments. Protein concentration was estimated using the Bradford method [27]. Enzyme assays b-N-acetylglucosaminidase activity was assayed in end-point experiments using p-nitrophenyl 2-acetamido-2-deoxy-b-D-glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-b-D-galactopyranoside as substrates; starting concentration 2 mM. One unit of enzymatic activity was defined as the amount of enzyme releasing 1 lmol of p-nitrophenol per minute in 50 mM citrate–phosphate buffer at pH 7.0 and 37 °C. After incubation of the reaction mixture at 37 °C for 10 min, the liberated p-nitrophenol was determined spectrophotometrically at 420 nm under alkaline conditions (50 lL of the reaction mixture was added to 1 mL of 0.1 M Na2CO3). The effect of pH on b-N-acetylglucosaminidase activity (pNP-bGlcNAc) was measured at 37 °C as described above; the series of

206

K. Slámová et al. / Protein Expression and Purification 95 (2014) 204–210

buffers composed of 50 mM citric acid, NaH2PO4, Na2HPO4 and H3PO4 in the range of pH 1.0–11.0 were used. The temperature activity profile was determined as described above in citrate–phosphate buffer, pH 3.5, in the range of 25–80 °C. Michaelis–Menten kinetics of b-N-acetylglucosaminidase reactions was measured in a kinetic assay at 348 nm (isosbestic point of p-nitrophenol), using pNP-b-GlcNAc as substrate with its concentration in the range of 0.025–0.5 mM (citrate–phosphate buffer pH 7.0, 22 °C); in the inhibition assays the concentration of the inhibitor NAG-thiazoline ranged from 0.01 to 1 mM. All data were measured in triplicates, the non-linear regression was calculated using Enzfitter (Biosoft, UK). Analytical transglycosylation reaction The reaction mixtures (total volume 50 lL) contained 25–200 mM pNP-b-GlcNAc used both as glycosyl donor and acceptor in 50 mM citrate–phosphate buffer at pH 7.0. The reaction was started by the addition of b-N-acetylglucosaminidase from P. chrysogenum (0.1 U) and was incubated at 35 °C with shaking for 2 h. The reaction progress was monitored by TLC (propane-2ol:water:ammonia = 7:2:1) on aluminium sheets precoated with Silica Gel 60 (F254 Merck, DE), and the plates were visualized by UV light (254 nm) and charring with a mixture of 5% sulfuric acid in ethanol. Homology modeling and molecular docking The search for homologues was performed employing BLAST [28], secondary structure prediction for sequence was done by consensus secondary structure prediction [29]. The multiple sequence structure based alignment was constructed with T-coffee [30] and manually corrected based on the templates alignment done with MUSTANG algorithm implemented in YASARA [31]. The set of homology models was prepared with Modeller [32] and the best one was selected based on the quality parameters of models estimated with ProSA [33] and Procheck [34]. The highest ranked models of fungal and human b-N-acetylglucosaminidases were further refined by molecular dynamics in TIP3 water model with YASARA (periodic boundary condition, PME used for long-range interaction [35], YASARA 2 force field [36], NPT ensemble). The angles between a-helices of fungal and BtOGA enzymes were calculated as angles between Ca atoms of terminal and overlaid residues from structures previously overlaid by MUSTANG algorithm [31] in YASARA. Local docking of compounds into the active site of BtOGA, human and fungal b-N-acetylglucosaminidases as well as binding energy calculations were done by AutoDock 4.2.3 (grid space 0.275 Å, Lamarckian genetic algorithm, number of runs 100) [37,38]. Active site amino acids were identified based on the active site in the crystal structures used as templates. Substrate– enzyme simulation was performed and analyzed with YASARA (periodic boundary condition, PME used for long-range interaction [35], YASARA 2 force field ([36], NPT ensemble). Interactions in the active site were shown with LigPlot+ [39], analysis of substrate/ inhibitor–enzyme complexes were done with YASARA. Results Expression and purification of b-N-acetylglucosaminidase The gene of b-N-acetylglucosaminidase from the filamentous fungus P. chrysogenum was prepared synthetically on a commercial basis, the codon usage in the sequence was optimized for expression in P. pastoris and the predicted signal sequence of the gene was omitted (aa 1–24); the optimized nucleotide sequence is presented

in the Supplementary material (Fig. S1). The synthetic gene was cloned into the pPICZaA yeast expression vector containing the a-factor for the extracellular secretion of the produced protein and the gene for zeocin resistance. The b-N-acetylglucosaminidase was expressed and secreted into the culture media of P. pastoris KM71H, the highest activity was obtained after 3 days upon induction by methanol, reaching 0.05–0.1 U/mL. As the production of the b-N-acetylglucosaminidase was very sensitive to aeration level, the baffled flasks with 50 mL of culture media were used; in larger media volumes and in standard flasks the production of the enzyme dropped significantly. The b-N-acetylglucosaminidase was purified from the P. pastoris culture media, after separation of the yeast cells by centrifugation the medium was diluted and its pH was adjusted to 6.5. In the first step, the anion-exchange chromatography was used in a reverse mode, which means that the protein of interest was not retained in the column while most of the ballast proteins and organic media components remain bound to the matrix. Unfortunately, we were not successful in finding a suitable method for complete retention of b-N-acetylglucosaminidase on a cation or anion-exchange column, as the activity was always distributed both in the flowthrough and in the gradient-eluted fractions, thus, we decided on this unusual mode to clear most contaminants from the medium. After this step the volume of the pre-purified medium was minimized employing ultrafiltration in order to obtain suitable samples for gel filtration, which yielded pure b-N-acetylglucosaminidase fractions (Fig. S2). The molecular weight of the enzyme was estimated to be 65 kDa (10% SDS–PAGE, Fig. S2), which corresponds to the theoretical molecular weight calculated for the protein sequence (68 kDa). The final yield of pure b-N-acetylglucosaminidase reached 34% with the specific activity 1.2 U/ mg; approximately 2.5 mg of pure enzyme were obtained starting from 300 mL of culture media. Characterization of b-N-acetylglucosaminidase from P. chrysogenum The purified b-N-acetylglucosaminidase was subjected to standard biochemical characterization, providing broad pH optimum in the range of pH 3–6 with the peak at pH 3.5 (37 °C; Fig. S3) and the temperature optimum of 10 min reaction at 60 °C (pH 3.5; Fig. S4). The Michaelis–Menten kinetics determined with the artificial chromogenic substrate pNP-b-GlcNAc yielded the KM value 0.188 ± 0.040 mM and Vmax value 0.212 ± 0.012 mmol/mg/min. In the inhibition assays with the mechanism-based b-N-acetylglucosaminidase inhibitor NAG-thiazoline and pNP-b-GlcNAc as substrate the inhibition constant of 0.103 ± 0.016 mM was obtained. Surprisingly, the galacto-analogue of the substrate (pNP-b-GalNAc, 2 mM) was hydrolyzed by the b-N-acetylglucosaminidase on a considerable level as the GalNAcase activity was found to be ca 20% of the activity with the standard substrate pNP-b-GlcNAc, which is absolutely unique among the GH84 b-N-acetylglucosaminidases. Moreover, the fungal b-N-acetylglucosaminidase catalyzed the transglycosylation reaction using various concentrations of pNP-bGlcNAc both as glycosyl donor and acceptor on analytical scale. Homology modeling and docking The complete sequence of b-N-acetylglucosaminidase from P. chrysogenum Wisconsin 54-1255 (NCBI: XP_002557703.1) was used for the BLAST search [28] for potential homology modeling templates. The closest homology sequences with resolved threedimensional structures deposited in the Protein Data Base [40] are listed here: crystal structure of b-N-acetylglucosaminidase from C. perfringens (PDB: 2xpk; sequence identity to query sequence is 29%, coverage 83%; further CpOGA) [41]; crystal structure of b-N-acetylglucosaminidase from B. thetaiotaomicron (PDB: 2chn; sequence

K. Slámová et al. / Protein Expression and Purification 95 (2014) 204–210

identity is 29%, coverage 83%; further BtOGA) [10]; and hyaluronoglucosaminidase from O. granulosus (PDB: 2xsb; sequence identity is 33% over 42% of query sequence; further OgrOGA) [12]. The sequence of the b-N-acetylglucosaminidase domain of the human bifunctional protein NCOAT isoform b (NCBI: NP_001135906.1) was also accepted as a reference for homology modeling, based on the templates used for fungal b-N-acetylglucosaminidase, as sequences of the catalytic domain of fungal and human enzymes share 35% identity. BLAST search and domain identification revealed that residues 180-500 of the studied fungal enzyme display high identity to the b-N-acetylglucosaminidase catalytic domain. Moreover, the predicted secondary structure [29] for the N- and C-terminal domains of the fungal b-N-acetylglucosaminidase is similar to BtOGA N-terminal and a-helical domains; the multiple sequence alignment of the catalytic domains used for model generation is presented in Fig. 1; the more elaborate MSA in color is shown in Figure S5 in the Supplementary material. The catalytic domain of GH84 b-N-acetylglucosaminidases is highly conserved, some differences were observed in loops close to the active site. Loop 1 between sheet b10 and helix a8 (numbering of secondary structure elements is based on BtOGA structure [42]) was found to be the longest in the fungal enzyme. Human b-N-acetylglucosaminidase and OgrOGA feature the corresponding loop of comparable size

207

to the fungal enzyme, while in other templates Loop 1 appears significantly shorter; on the other hand, Loop 2 between a10 and a11 in the fungal b-N-acetylglucosaminidase is shorter than in other enzymes. Loop 3 connecting catalytic and a-helical domain is located close to the active site. The overall structure of b-N-acetylglucosaminidase from P. chrysogenum comprises three domains: a small a + b zincin-like domain at the N-terminus, followed by a catalytic (b/a)6 TIM-barrel domain and a C-terminal a-helical bundle domain, which is slightly shifted in orientation compared to the templates (Fig. S6). The corresponding differences in angles between a-helices of fungal and BtOGA enzymes are as follows: for a21 18.630°, for a22 27.707° and for a23 21.217°. The terminal a-helical domain is not well conserved among b-N-acetylglucosaminidases, helices 21-23 are also twisted in CpOGA and OrgOGA (Fig. S7). Amino acid residues in the active site of b-N-acetylglucosaminidases are mostly conserved. In the fungal enzyme these residues have been identified as Gly191, Tyr193, Lys222, Asp298, Asp299, Tyr353, Thr378, Trp407, Asn409, Asp414 and Asn442; aspartates 298 and 299 being the key catalytic amino acids providing the substrate-assisted catalytic mechanism. The aromatic residues of Tyr193 and Tyr353 are supposed to participate in displaced p–p stacking with the reducing end of the artificial substrate pNP -b-GlcNAc. The substrate and the inhibitor NAG-thiazoline were

Fig. 1. Multiple sequence alignment of catalytic domain of b-N-acetylglucosaminidases: 2chn is BtOGA, 2xsb is OgrOGA, 2xpk is CpOGA, fungi stands for the sequence of Penicillium chrysogenum OGA, human stands for human OGA. ClustalW symbols for similarity are shown under aligned sequences (‘’ is used for positions which have a fully conserved residue; ‘:’ means conservation between groups of strongly similar properties; ‘.’ means conservation with weakly similar properties). Secondary structure assignment for BtOGA and secondary structure prediction for the fungal enzyme is shown by different font style: bold font is used for b-sheet, underlined for a-helix. Active site amino acid residues are marked by small italic style letters with labels according to fungal b-N-acetylglucosaminidase.

208

K. Slámová et al. / Protein Expression and Purification 95 (2014) 204–210

docked in the active site of human, fungal and BtOGA b-N-acetylglucosaminidases and the resulting complexes were used for molecular dynamics (MD) simulation in YASARA [43]. Schematic figures of the substrate pNP-b-GlcNAc docked into the active site of P. chrysogenum b-N-acetylglucosaminidase including hydrogen bonds between the enzyme and the ligand is presented in Fig. 2; more figures (in color) are shown in the Supplementary material (S8–S10) (Table 1). Averaged binding energies calculated by AutoDock [38] for a representative set of complexes from the stable period of molecular dynamics simulation show that the substrate binds to the active site of all enzymes with comparable energies (Table 2) and the inhibitor NAG-thiazoline displays slightly higher binding energy than the substrate. Discussion b-N-acetylglucosaminidases from the family 84 of glycoside hydrolases (www.cazy.org) form a small group of glycosidases, whose eukaryotic members have not been explored much so far, even though their physiological impact in higher eukaryotes is significant. In order to broaden the knowledge on this enzyme family, we expressed the gene of b-N-acetylglucosaminidase from the filamentous fungus P. chrysogenum Wisconsin 54-1255 in the yeast expression system of P. pastoris, purified it from the yeast culture medium, characterized the obtained enzyme and computed its homology model. Thus, we obtained complex biochemical and structural information on the first member of the GH84 family originated from a fungus. The b-N-acetylglucosaminidase from P. chrysogenum was produced by P. pastoris and secreted, reaching up to 0.1 U/mL after 3 days upon induction by methanol under vigorous shaking in baffled flasks. Unfortunately, the enzyme production decreased significantly when standard cultivation flasks and larger volumes than 50 mL per flask were used, which strongly limited the yield of the enzyme. Moreover, the obtained b-N-acetylglucosaminidase was not easy to purify, as it did not interact with the ion exchange columns as would be expected. Finally, the reversed mode of the anion exchange chromatography was found suitable for the removal of most contaminating low and high molecular weight components of the culture media followed by ultrafiltration and gel

Fig. 2. Artificial substrate pNP-b-GlcNAc in the active site of fungal b-N-acetylglucosaminidase after 10 ns of MD simulation. Hydrogen bonds are shown by dashed lines. Other residues able to participate in hydrophobic interaction are shown schematically.

chromatography to gain the homogeneous enzyme with the average yield of 34%. The purified b-N-acetylglucosaminidase was subjected to biochemical characterization, showing a broad pH optimum in the slightly acidic range and a temperature optimum at 60 °C. The kinetic experiments determined the KM value for the standard substrate pNP-b-GlcNAc as 0.188 mM, while the inhibition constant for NAG-thiazoline, the generally recognized strong competitive inhibitor of b-N-acetylglucosaminidases and b-N-acetylhexosaminidases, was as high as 0.103 mM. These data show that the activity of the studied enzyme is influenced by this inhibitor only weakly, while with other related enzymes its strong inhibition potential has been proven, e.g. KI of 70 nM was determined for the human b-N-acetylglucosaminidase, albeit under different experimental conditions [21]. Interestingly, we have documented for the first time the hydrolysis of the substrate in galacto-configuration by a GH84 b-N-acetylglucosaminidase, which was not negligible, reaching the up to 20% of the original activity towards pNP-b-GlcNAc. We have also tested the enzyme for transglycosylation activity, which was also successful; using various concentrations of pNP-b-GlcNAc both as glycosyl donor and acceptor the fungal b-N-acetylglucosaminidase catalyzed the formation of oligosaccharides, as observed by TLC. Together, the GalNAc-cleaving and transglycosylation activities of the b-N-acetylglucosaminidase from P. chrysogenum and the values of pH and temperature optima resemble rather fungal b-N-acetylhexosaminidases from the GH20 family [44,45] than the GH84 O-GlcNAcases described so far. This suggests that we have identified a mixed-type enzyme with the sequence and structure of GH84 b-N-acetylglucosaminidase and qualities related to GH20 b-N-acetylhexosaminidases with unclear physiological function in the fungal organism. We were also interested in the structure of the b-N-acetylglucosaminidase from P. chrysogenum, unfortunately, due to the low production of the enzyme we were not able to obtain sufficient amount for its crystallization. So we opted for the construction of a homology model based on known crystal structures of three bacterial GH84 b-N-acetylglucosaminidases (PDB: 2xpk; 2chn; 2xsb). The multiple sequence alignments showed high homology mainly in the catalytic domains of the compared enzymes, and also the amino acid composition of the active site is highly conserved, while the structure analysis revealed significant differences in C- and Nterminal domains, such as mutual orientation of catalytic and ahelical domains. Moreover, the substrate pNP-b-GlcNAc and the mechanism-based inhibitor NAG-thiazoline were docked into the active site of the fungal, human and bacterial b-N-acetylglucosaminidases; the respective binding energies were calculated by AutoDock (Table 2) and the dynamics in positions and hydrogen bonds of the ligands were observed in the course of molecular dynamics simulations. The values of binding energies of the substrate with different enzymes are on a practically identical level and the inhibitor generally displays moderately higher binding energies than the substrate. Additionally, we have identified some non-conserved amino acid residues that could influence the binding of the substrate and the inhibitor, as can be illustrated by the following examples. Val349 in the fungal enzyme corresponds to Cys residue in BtOGA, OgrOGA and human OGA. The methyl group of the valine residue is able to provide additional hydrophobic interaction with the methyl group of the substrate and inhibitor, being separated just 0.4 nm, improving its binding to NAG-thiazoline. Fungal Val412, corresponding to the smaller alanine in human and OgrOGA (Fig. 1), keeps hydrophobic interaction with the C-6 atom of the substrate, while reducing the free space surrounding the C-6 of the substrate. In this work, we have expressed and characterized the first fungal b-N-acetylglucosaminidase from the family 84 of glycoside hydrolases. The sequence and modeled structure of the enzyme

209

K. Slámová et al. / Protein Expression and Purification 95 (2014) 204–210 Table 1 Purification of b-N-acetylglucosaminidase from P. chysogenum from the culture medium of P. pastoris.

Medium Q-Sepharose Ultrafiltration Superdex 200

Proteins (mg)

Activity (U)

Spec. activity (U/mg)

Purification factor

Yield (%)

55.6 25.3 10.5 2.5

8.8 6.8 4.4 3.0

0.16 0.27 0.42 1.20

0 1.7 2.6 7.5

100 77 50 34

Table 2 Binding energies (kcal/mol) of substrate (pNP-b-GlcNAc) and inhibitor (NAG-thiazoline) docked in the active site of b-N-acetylglucosaminidases calculated by AutoDock. Ligand

pNP-b-GlcNAc NAG-thiazoline

Source of b-N-acetylglucosaminidase Human

Fungal

BtOGA

7.45 6.42

7.62 7.36

7.75 7.14

from the fungus P. chrysogenum classifies it to the GH84 family while its biochemical properties such as the ability to hydrolyze the substrate in galacto-configuration and the formation of oligosaccharides under the catalysis by this enzyme resemble rather the fungal b-N-acetylhexosaminidases from the family GH20. Thus, we have found a new mixed-type b-N-acetylglucosaminidase, whose physiological function in its original organism remains unrecognized. Acknowledgments The access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum, provided under the program ‘‘Projects of Large Infrastructure for Research, Development, and Innovations’’ (LM2010005) is acknowledged. This work was supported by the Czech Science Foundation Grant P207/11/0629 and by the EU project NOVOSIDES FP7-KBBE-2010-4-265854.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pep.2014.01.002. References [1] C.R. Torres, G.W. Hart, Topography and polypeptide distribution of terminal Nacetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc, J. Biol. Chem. 259 (1984) 3308–3317. [2] Y. Gao, L. Wells, F.I. Comer, G.J. Parker, G.W. Hart, Dynamic O-glycosylation of nuclear and cytosolic proteins, cloning and characterization of a neutral, cytosolic b-N-acetylglucosaminidase from human brain, J. Biol. Chem. 276 (2001) 9838–9845. [3] G.W. Hart, M.P. Housley, C. Slawson, Cycling of O-linked b-Nacetylglucosamine on nucleocytoplasmic proteins, Nature 446 (2007) 1017– 1022. [4] C. Butkinaree, K. Park, G.W. Hart, O-Linked b-N-acetylglucosamine (O-GlcNAc): extensive crosstalk with phosphorylation to regulate signaling and transcription in response to nutrients and stress, Biochim. Biophys. Acta 2010 (1800) 96–106. [5] L.S. Griffith, B. Schmitz, O-linked N-acetylglucosamine levels in cerebellar neurons respond reciprocally to pertubations of phosphorylation, Eur. J. Biochem. 262 (1999) 824–831. [6] W.B. Dias, G.W. Hart, O-GlcNAc modification in diabetes and Alzheimer’s disease, Mol. BioSyst. 3 (2007) 766–772. [7] J.A. Hanover, M.W. Krause, D.C. Love, The hexosamine signaling pathway: OGlcNAc cycling in feast or famine, Biochim. Biophys. Acta 2009 (1800) 80–95. [8] H.C. Dorfmueller, V.S. Borodkin, M. Schimpl, S.M. Shepherd, N.A. Shapiro, D.M.F. van Aalten, GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-GlcNAcylation levels, J. Am. Chem. Soc. 128 (2006) 16484–16485.

[9] F.V. Rao, H.C. Dorfmueller, F. Villa, M. Allwood, I.M. Eggleston, D.M.F. van Aalten, Structural insights into the mechanism and inhibition of eukaryotic OGlcNAc hydrolysis, EMBO J. 25 (2006) 1569–1578. [10] R.J. Dennis, E.J. Taylor, M.S. Macauley, K.A. Stubbs, J.P. Turkenburg, S.J. Hart, G.N. Black, D.J. Vocadlo, G.J. Davies, Structure and mechanism of a bacterial bglucosaminidase having O-GlcNAcase activity, Nat. Struct. Mol. Biol. 13 (2006) 365–371. [11] B. Shanmugasundaram, A.W. Debowski, R.J. Dennis, G.J. Davies, D.J. Vocadlo, A. Vasella, Inhibition of O-GlcNAcase by a gluco-configured nagstatin and a PUGNAc-imidazole hybrid inhibitor, Chem. Commun. (2006) 4372–4374. [12] M. Schimpl, A.W. Schüttelkopf, V.S. Borodkin, D.M.F. van Aalten, Human OGA binds substrates in a conserved peptide recognition groove, Biochem. J. 432 (2010) 1–7. [13] G.E. Whitworth, M.S. Macauley, K.A. Stubbs, R.J. Dennis, E.J. Taylor, G.J. Davies, I.R. Greig, D.J. Vocadlo, Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise, J. Am. Chem. Soc. 129 (2007) 635–644. [14] M.S. Macauley, A.K. Bubb, C. Martinez-Fleites, G.J. Davies, D.J. Vocadlo, Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance, J. Biol. Chem. 283 (2008) 34687–34695. [15] Y. He, A.K. Bubb, K.A. Stubbs, T.M. Gloster, G.J. Davies, Inhibition of a bacterial O-GlcNAcase homologue by lactone and lactam derivatives: structural, kinetic and thermodynamic analyses, Amino Acids 40 (2011) 829–839. [16] D.L. Dong, G.W. Hart, Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol, J. Biol. Chem. 269 (1994) 19321–19330. [17] C. Toleman, A.J. Paterson, T.R. Whisenhunt, J.E. Kudlow, Characterization of the histone acetyltransferase (HAT) domain of a bifunctional protein with activable O-GlcNAcase and HAT activities, J. Biol. Chem. 279 (2004) 53665– 53673. [18] N. Comtesse, E. Maldener, E. Meese, Identification of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a b-N-acetylglucosaminidase, Biochem. Biophys. Res. Commun. 283 (2001) 634–640. [19] J. Li, C. Huang, L. Zhang, L. Lin, Z. Li, F. Zhang, P. Wang, Isoforms of human OGlcNAcase show distinct catalytic efficiencies, Biochemistry (Moscow) 75 (2010) 938–943. [20] N.A.N. de Alencar, P.R.M. Sousa, J.R.A. Silva, J. Lameira, C.N. Alves, S. Martí, V. Moliner, Computational analysis of human OGA structure in complex with PUGNAc and NAG-thiazoline derivatives, J. Chem. Inf. Model. 52 (2012) 2775– 2783. [21] M.S. Macauley, G.E. Whitworth, A.W. Debowski, D. Chin, D.J. Vocadlo, OGlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors, J. Biol. Chem. 280 (2005) 25313–25322. [22] N. Cetinbas, M.S. Macauley, K.A. Stubbs, R. Drapala, D.J. Vocadlo, Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants, Biochemistry 45 (2006) 3835– 3844. [23] M.S. Macauley, D.J. Vocadlo, Increasing O-GlcNAc levels: an overview of smallmolecule inhibitors of O-GlcNAcase, Biochim. Biophys. Acta 2010 (1800) 107– 121. [24] S.A. Yuzwa, M.S. Macauley, J.E. Heinonen, X. Shan, R.J. Dennis, Y. He, G.E. Whitworth, K.A. Stubbs, E.J. McEachern, G.J. Davies, D.J. Vocadlo, A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo, Nat. Chem. Biol. 4 (2008) 483–490. [25] S.A. Yuzwa, X. Shan, M.S. Maculey, T. Clark, Y. Skorobogatko, K. Vosseller, D.J. Vocadlo, Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation, Nat. Chem. Biol. 8 (2012) 393–399. [26] M.A. van den Berg, R. Albang, K. Albermann, J.H. Badger, J.-M. Daran, A.J.M. Driessen, C. Garcia-Estrada, N.D. Fedorova, D.M. Harris, W.H.M. Heijne, V. Joardar, J.A.K.W. Kiel, A. Kovalchuk, J.F. Martín, W.C. Nierman, J.G. Nijland, J.T. Pronk, J.A. Roubos, I.J. van der Klei, N.N.M.E. van Peij, M. Veenhuis, H. von Döhren, C. Wagner, J. Wortman, R.A.L. Bovenberg, Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum, Nat. Biotechnol. 26 (2008) 1161–1168. [27] M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [28] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410. [29] C. Combet, C. Blanchet, C. Geourjon, G. Deléage, NPS@: network protein sequence analysis, Trends Biochem. Sci. 25 (2000) 147–150.

210

K. Slámová et al. / Protein Expression and Purification 95 (2014) 204–210

[30] C. Notredame, D.G. Higgins, J. Heringa, T-Coffee: a novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. 302 (2000) 205–217. [31] A.S. Konagurthu, J.C. Whisstock, P.J. Stuckey, A.M. Lesk, MUSTANG: a multiple structural alignment algorithm, Proteins 64 (2006) 559–574. [32] A. Sali, T.L. Blundell, Comparative protein modelling by satisfaction of spatial restraints, J. Mol. Biol. 234 (1993) 779–815. [33] M. Wiederstein, M.J. Sippl, ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins, Nucleic Acids Res. 35 (2007) 407–410. [34] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: a program to check the stereochemical quality of protein structures, J. Appl. Crystallogr. 26 (1993) 283–291. [35] U. Essman, L. Perera, M.L. Berkowitz, T. Darden, H. Lee, L.G. Pedersen, A smooth particle mesh Ewald method, J. Chem. Phys. 103 (1995) 8577–8593. [36] E. Krieger, K. Joo, J. Lee, S. Raman, J. Thompson, M. Tyka, D. Baker, K. Karplus, Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: four approaches that performed well in CASP8, Proteins 77 (2009) 114–122. [37] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function, J. Comput. Chem. 19 (1998) 1639–1662. [38] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791.

[39] R.A. Laskowski, M.B. Swindells, LigPlot+: multiple ligand–protein interaction diagrams for drug discovery, J. Chem. Inf. Model. 51 (2011) 2778–2786. [40] F.C. Bernstein, T.F. Koetzle, G.J. Williams, E.E. Meyer, M.D. Brice, J.R. Rodgers, O. Kennard, T. Shimanouchi, M. Tasumi, The protein data bank: a computerbased archival file for macromolecular structures, J. Mol. Biol. 112 (1977) 535–542. [41] H.C. Dorfmueller, V.S. Borodkin, M. Schimpl, X. Zheng, R. Kime, K.D. Read, D.M.F. van Aalten, Cell-penetrant, nanomolar O-GlcNAcase inhibitors selective against lysosomal hexosaminidases, Chem. Biol. 17 (2010) 1250–1255. [42] W. Kabsch, C. Sander, Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features, Biopolymers 22 (1983) 2577–2637. [43] E. Krieger, G. Koraimann, G. Vriend, Increasing the precision of comparative models with YASARA NOVA – a self-parameterizing force field, Proteins 47 (2002) 393–402. [44] M. Scigelova, D.H.G. Crout, Microbial b-N-acetylhexosaminidases and their biotechnological applications, Enzyme Microb. Technol. 25 (1999) 3–14. [45] K. Slámová, P. Bojarová, D. Gerstorferová, B. Fliedrová, J. Hofmeisterová, M. Fiala, P. Pompach, V. Krˇen, Sequencing, cloning and high-yield expression of a fungal b-N-acetylhexosaminidase in Pichia pastoris, Protein Expr. Purif. 82 (2012) 212–217.