Mammalian glutaminyl cyclases and their isoenzymes have identical enzymatic characteristics Anett Stephan 1 , Michael Wermann 1 , Alex von Bohlen 2 , Birgit Koch 1 , Holger Cynis 1 , Hans-Ulrich Demuth 1 and Stephan Schilling 1 1 Probiodrug AG, Halle ⁄ Saale, Germany 2 Institute for Analytical Sciences, Dortmund, Germany Introduction In addition to proteolytic cleavage, glycosylation and amidation, N-terminal formation of 5-oxoproline (pyro- glutamate, pGlu) is a common post-translational event during the biosynthesis of secretory peptides and proteins, such as thyrotropin-releasing hormone (TRH), gastrin, fibronectin and neurotensin [1–3]. Glu- taminyl cyclases (QCs) have been identified in mammals, invertebrates and plants, catalyzing pGlu formation from glutaminyl precursors [4–8]. Moreover, compel- ling evidence suggests an involvement of QCs in diseases such as osteoporosis and Alzheimer’s disease (AD) [9,10]. QCs have been shown to catalyze pGlu formation at the N-terminus of amyloid peptides from glutamyl precursors, rendering them hydrophobic and Keywords Alzheimer’s disease; glutaminyl cyclase isoenzyme; glycosylation; Golgi apparatus; Pichia pastoris Correspondence S. Schilling, Probiodrug AG, Weinbergweg 22, 06120 Halle ⁄ Saale, Germany Fax: +49 345 5559901 Tel: +49 345 5559911 E-mail: stephan.schilling@probiodrug.de (Received 30 June 2009, revised 17 August 2009, accepted 28 August 2009) doi:10.1111/j.1742-4658.2009.07337.x Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate resi- dues at the N-terminus of several peptides and proteins from plants and animals. Recently, isoenzymes of mammalian QCs have been identified. In order to gain further insight into the biochemical characteristics of iso- QCs, the human and murine enzymes were expressed in the secretory pathway of Pichia pastoris. Replacement of the N-terminal signal anchor by an a-factor prepropeptide from Saccharomyces cerevisiae resulted in poor secretion of the protein. Insertion of an N-terminal glycosylation site and shortening of the N-terminus improved isoQC secretion 100-fold. A comparison of different recombinant isoQC proteins did not reveal an influence of mutagenic changes on catalytic activity. An initial character- ization showed identical modes of substrate conversion of human isoQC and murine isoQC. Both proteins displayed a broad substrate specificity and preference for hydrophobic substrates, similar to the related QC. Likewise, a determination of the zinc content and reactivation of the apo- isoQC revealed equimolar zinc present in QC and isoQC. Far-UV CD spectroscopic analysis of murine QC and isoQC indicated virtually identi- cal structural components. The present investigation provides the first enzymatic characterization of mammalian isoQCs. QC and isoQC repre- sent very similar proteins, which are both present in the secretory path- way of cells. The functions of QCs and isoQC probably complement each other, suggesting a pivotal role of pyroglutamate modification for protein and peptide maturation. Abbreviations AD, Alzheimer’s disease; BMGY, buffered glycerol complex medium; BMMY, buffered methanol complex medium; GST, glutathione transferase; IMAC, immobilized metal ion affinity chromatography; pGlu, pyroglutamate; QC, glutaminyl cyclase; TRH, thyrotropin-releasing hormone; TXRF, total X-ray fluorescence. 6522 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS more prone to aggregation, probably contributing to AD pathology [11–14]. A chronic inhibition of QCs has been shown to attenuate AD-like pathology in mouse models, introducing QC activity as a target for drug development [15]. Recently, we have isolated an isoenzyme of human QC (human isoQC) [16]. Database mining led to the identification of a protein of 382 amino acids, which shares a sequence identity with human QC of 45%. In contrast with QC, a signal anchor was identified in isoQC, which putatively mediates the retention of the class II transmembrane protein in the Golgi apparatus. Thus, the protein shares interesting similarity to glyco- syltransferases with regard to subcellular localization, and may act in concert with those in the secretory pro- tein maturation process. This hypothesis is supported by the ubiquitous expression pattern of isoQC, which contrasts with the differential QC expression in glands and neuronal tissue [16]. Because the differences in subcellular localization and tissue distribution might reflect other functions and catalytic specificity, it was the aim of this study to express heterologously and characterize the isoQCs of human and murine (murine isoQC) origin in the methylotrophic yeast Pichia pastoris. The method required extensive optimization of expression, which might have implications for other proteins. Results Expression of human isoQC The isoQC proteins are localized within the Golgi complex in their native forms. The retention in the compartment is mediated by the N-terminus, which includes a membrane anchor directing the nascent pep- tide chain into the secretory pathway (Fig. 1). In order to provoke efficient secretory expression in P. pastoris, the N-terminal region was substituted by the a-factor prepropeptide from Saccharomyces cerevisiae, which should result in the secretion of the protein into the expression medium [17]. The leader sequence thus functions in a similar manner to the signal peptides in native QC proteins (Fig. 1). A coding sequence of six His residues was additionally attached to the 3¢ end of the human isoQC open reading frame, facilitating purification. The expression of this construct should result in a secreted isoQC starting with phenylalanine 48 (isoQC (F48) C-His) (Figs 1 and 2). An isoQC expres- sion construct was generated by applying the vector backbone of pPICZaA, linearized and used for the electroporation of P. pastoris. Unexpectedly, a charac- terization of 50 stably transformed clones for the presence of isoQC activity in the medium revealed only very low concentrations of the recombinant protein (Fig. 3). According to this observation, several sequence modifications were considered in order to improve the solubility and, potentially, the secretion process. In contrast with isoQC, QCs contain one (mouse, rat) or two (human, bovine) sites of N-glyco- sylation. One N-terminal site is conserved in all mam- malian QCs and is also present in a secreted QC from Drosophila melanogaster, all of which were successfully expressed in yeast [4,5,18,19]. Hence, in order to improve the secretion of human isoQC, a potential gly- cosylation site was introduced at position 73 by the exchange of isoleucine with asparagine (isoQC (F48;I73N) , Fig. 2), i.e. resembling the glycosylation site of QC as suggested by a multiple sequence alignment (not shown). A software-based algorithm predicted a high probability of derivatization (http://www.cbs.dtu.dk/ services/NetNGlyc/) of the introduced asparaginyl residue in the modified isoQC. The I73N variant of human isoQC was expressed carrying an N-terminal or C-terminal poly-His fusion tag (isoQC (F48;I73N) N-His, isoQC (F48;I73N) C-His). The comparison of the activity in the expression medium showed an up to 10-fold higher isoQC secretion of the now glycosylated variant in comparison with the unmodified human isoQC (F48) C-His (Fig. 3A). No sig- nificant difference in isoQC activity was observed between expressed proteins with the different poly-His fusions, indicating that the tag did not influence the activity or production process. In addition to the introduction of the N-terminal glycosylation site, a cysteine residue present in human isoQC was exchanged for an alanine. The mutation appears to be conceivable, because the residue is nei- ther conserved in murine isoQC nor in other animal QCs. As the two conserved cysteines have been shown to form a disulfide bond in QC, the third cysteine in human isoQC is potentially free and might therefore interfere with the protein production in yeast as a result of oxidation. However, further improvement of enzyme secretion into the medium was observed fol- lowing expression of the protein (isoQC (F48;I73N;C369A) C-His, isoQC (F48;I73N;C369A) N-His) (Fig. 2). In a final approach to further improve the yield of the secreted protein, the N-terminus of the recombi- nant protein was shortened by 59 amino acids in total. The shortening results in complete deletion of the transmembrane region, which spans amino acid posi- tions 35–52, 34–55 or 40–60 according to predictions of HMMTOP, SUSOI or TMpred [20–22] (all avail- able at http://www.expasy.ch). The plasmid constructs were expressed encoding a human protein containing A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6523 the glycosylation site and the Cys ⁄ Ala mutation (isoQC (E60;I73N;C369A) N-His), or with only one of the modifications, either the glycosylation site (isoQC (E60;I73N) N-His) or the mutated cysteine (iso- QC (E60;C369A) N-His) (Fig. 2). The human isoQC con- structs encoded for an additional poly-His fusion at the N-terminus. The N-terminal shortening of the construct resulted in a 10-fold higher protein yield in comparison with the initial construct (Fig. 3A). As observed previously, the introduction of the glycosyla- tion site further improved the amount of isoQC in the medium. The highest protein yield was finally obtained after expression of isoQC (E60;I73N;C369A) N-His, which revealed a 100-fold higher isoQC concentration in the medium compared with the initial expression con- struct. The broad distribution in activity levels in the expression medium of some constructs (e.g. iso- QC (E60;I73N;C369A) ) are caused by the transformation of yeast. As a result of the integration of the recombinant DNA into the genome of the host, large clonal varia- tions occur with respect to expression, caused, for instance, by multicopy insertion into the genome (Fig. 3A). The results of the determination of the isoQC activ- ity in the medium were corroborated by western blot analysis of the expression medium, applying an isoQC- specific antiserum (Fig. 3A, inset). The most intense immunostaining was observed following the expression of isoQC (E60;I73N;C369A) N-His. From all expressed constructs, isoQC (F48;I73N;C369A) C-His, isoQC (E60;I73N;C369A) N-His and isoQC (E60;I73N) N-His were used for a scale-up of expression in shake Fig. 1. Amino acid sequence alignment of human and murine isoQCs, human QC (hQC), mouse QC (mQC) and Drosoph- ila melanogaster QC (Drome QC). The signal sequences of QC and the signal anchor of isoQCs are highlighted in bold italics. The residues for complexation of zinc ions in the active site (Asp-Glu-His) (bold) and the core structure surrounding the active site, con- taining a conserved disulfide bond (bold and underlined), are conserved in all enzymes. In addition, the secreted QC proteins contain sites of N-glycosylation (bold, italics, under- lined). The position of the introduced glyco- sylation site by mutation of an isoleucine residue in isoQC is shown in bold italics. The alignments were prepared using CLUSTALW at EMBnet-CH (http://www. expasy.ch). Characterization of glutaminyl cyclase isoenzymes A. Stephan et al. 6524 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS flasks. The protein was purified to virtual homogeneity by initial Ni 2+ -immobilized metal ion affinity chroma- tography (IMAC), followed by hydrophobic interac- Cytosolic sequence Membrane anchor Luminal catalytic domain Yeast secretion signal NH 2 COOH isoQC (E 60 ;I73N) COOHNH 2 Native isoQC NH 2 COOH isoQC (F 48 ) NH 2 COOH isoQC (E 60 ; I73N;C369A) NH 2 COOH isoQC (F 48 ;I73N) Ile→ Asn ↓ NH 2 COOH isoQC (F 48 ;I73N;C369A) NH 2 COOH Cys→Ala ↓ isoQC (F 48 ;C369A) COOH isoQC E 61 I74N NH 2 NH 2 COOH isoQC (F 49 ) Human isoQC Murine isoQC Fig. 2. Schematic representation of the different constructs for the secretory expression of human and murine isoQCs in Pichia pastoris. The native isoQC with the cytosolic tail and the membrane anchor is shown for comparison. This N-terminal region is exchanged for the a-leader prepro- sequence of Saccharomyces cerevisiae as a secretion signal. In some constructs, an isoleucine is mutated into an asparagine, generating an N-glycosylation site. Further- more, a third cysteine at the C-terminus of human isoQC is mutated into an alanine. Finally, the constructs differed in terms of their N-terminus, i.e. the N-terminal amino acid corresponding to the open reading frame of isoQC was either a phenylalanine (Phe48 and Phe49 of human and murine QC, respectively) or a glutamic acid residue (position 60 or 61). C - H i s ( F 4 8 ) i s o Q C C - H i s ( F 4 8 ; C 3 6 9 A ) i s o Q C N - H i s ( E 6 0 ; C 3 6 9 A ) i s o Q C N - H is ( F 4 8 ; I7 3 N ) i s o Q C C - H i s ( F 4 8 ; I 7 3 N ) i s o Q C N - H i s ( E 6 0 ; I 7 3 N ) i s o Q C N - H i s ( F 4 8 ;I 7 3 N ; C 3 6 9 A ) is o Q C C -H i s (F 4 8 ; I 7 3 N ;C 3 6 9 A ) i s o Q C N - H i s ( E 6 0 ; I 7 3 N ; C 3 6 9 A ) is o Q C 0 1 2 3 4 5 6 QC-activity (µM·min –1 ) 0.001 0.01 0.1 1 10 Log activity 12345678910 36 kDa A B kDa 250 150 100 75 50 37 25 20 15 51 34 6 2 Fig. 3. Characterization of human isoQC expression in Pichia pasto- ris. (A) Determination of the QC activity in the medium of P. pastoris expressing the different constructs. At least 50 clones of each con- struct were checked with regard to QC activity in the culture medium after small-scale expression. The inset shows western blot analysis of the expression medium and a logarithmic scatter plot of the acti- vity determined for each yeast clone investigated. The logarithmic plot of the QC activity data points to a similar variation of expression after transformation with the different plasmid constructs, which is caused by differences in transcriptional efficacy and insertion events of the expression constructs into the genome of yeast. In western blot analysis, the proteins were visualized using an isoQC antibody. A total amount of 4 lg of protein was applied to each lane. Lane 1, isoQC (F48) C-His; lane 2, isoQC (F48;C369A) C-His; lane 3, iso- QC (E60;C369A) N-His; lane 4, deglycosylated isoQC (F48;I73N) N-His; lane 5, deglycosylated isoQC (F48;I73N) C-His; lane 6, deglycosylated iso- QC (E60;I73N) N-His; lane 7, deglycosylated isoQC (F48;I73N;C369A) N-His; lane 8, deglycosylated isoQC (F48;I73N;C369A) C-His; lane 9, deglyco- sylated isoQC (E60;I73N;C369A) N-His; lane 10, 400 ng of purified and deglycosylated isoQC (F48;I73N;C369A) as a positive control. The degly- cosylated protein migrates at 37 kDa. (B) SDS-PAGE analysis illus- trating the purification of human isoQC (F48;I73N;C369A) C-His after expression in shake flasks. Proteins were visualized by Coomassie staining: lane 1, molecular mass standards (kDa) (Dual Color, Bio- Rad); lane 2, supernatant after expression; lane 3, isoQC-containing fractions after initial affinity chromatography on immobilized Ni 2+ ions; lane 4, human isoQC after hydrophobic interaction chromato- graphy; lane 5, purified protein after desalting. The isoQC protein corresponds to a band migrating between 50 and 70 kDa. The degly- cosylation causes a shift to 37 kDa (lane 6). The upper band (75 kDa) in lane 6 corresponds to the endoglycosidase H f . A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6525 tion chromatography. A purification process illustrated by SDS-PAGE analysis is shown in Fig. 3B. As a result of the competitive inhibition of isoQC and QC by imidazole, the elution was performed with His, which is much less inhibitory to QC and isoQC. Inacti- vation of isoQC, e.g. caused by zinc complexation dur- ing IMAC, was not observed. Typical expression in a final culture volume of 4 L resulted in the isolation of 7 mg of isoQC (E60;I73N) N-His and 14 mg of isoQC (E60;I73N;C369A) N-His variant. Expression of isoQC (F48;I73N;C369A) C-His was carried out in a total volume of 8 L, resulting in the isolation of 7.3 mg of human isoQC protein. The over- all yield of purification was 60%. Expression and purification of murine isoQC On the basis of human isoQC expression in P. pastoris, three different murine isoQC constructs were gener- ated. The introduction of a glycosylation site and the shortening of the N-terminus (isoQC (E61;I74N) N-His) resulted in an increase in secretory expressed murine isoQC, similar to that observed with human isoQC. In addition to the poly-His-tagged proteins, an untagged protein was generated. A comparison of the N-termi- nally His-tagged and untagged protein did not reveal a significant influence on isoQC expression (Fig. 4A). The isoQC (E61;I74N) variant was successfully expressed by fermentation using a 5 L bioreactor, which typically results in the harvesting of 2 L of isoQC-containing medium. Homogeneous protein was obtained after purification, applying two different hydrophobic interaction chromatographic matrices, followed by anion-exchange chromatography and size exclusion chromatography. The purity of murine isoQC was analyzed by SDS-PAGE (Fig. 4B). A typical purifica- tion process resulted in the isolation of 8 mg of murine isoQC protein. Characterization of the substrate and inhibitor specificity In order to rule out an influence of the different modi- fications of human isoQC on substrate conversion, the kinetic parameters for the turnover of H-Gln-bNA (Q-bNA), H-Gln-Glu-OH (QE) and H-Gln-Gln-OH (QQ) and the competitive isoQC inhibitors benzimid- azole and benzylimidazole were analyzed. The evalua- tion of the influence of sequence shortening to glutamic acid 60, glycosylation of the protein and mutation of cysteine 369 into alanine did not show a considerable effect on the kinetic parameters or on the inhibitory specificity (cf. Tables 1 and 2). Accordingly, the substrate and inhibitor specificities of the human isoQC (F48;I73N;C369A) C-His variant were analyzed and compared with the data obtained with a glutathione transferase (GST)-fusion protein, which was expressed in Escherichia coli previously [16]. Interestingly, the substrate specificity of the isoQCs expressed in P. pas- toris and E. coli was virtually identical for the tested substrates, i.e. the highest specificity was obtained for substrates containing hydrophobic residues, e.g. H-Gln-bNA, H-Gln-Tyr-Ala-OH (QYA) or H-Gln- Glu-Tyr-Phe-NH 2 (QEYF) (Table 2). A comparison of N- His ( F49 ) C N - His ( E 61; I7 4N ) i soQ i soQ C (E 61 ;I7 4 N) i s o Q C 0. 0 0. 5 1. 0 1. 5 2. A B 0 QC-activity (µM·min –1 ) kD a 150 100 75 50 37 25 20 1 5 3 4 6 2 0.001 0.01 0.1 1 10 log activity Fig. 4. Expression and characterization of murine isoQC in Pichia pastoris. (A) Determination of QC activity in the medium of P. pas- toris expressing the indicated constructs. The analysis was per- formed as described for human isoQC in Fig. 3. (B) SDS-PAGE illustrating the purification of murine isoQC (E61;I74N) after fermenta- tion. Proteins were visualized by Coomassie staining. Lane 1, molecular mass standards (kDa) (Dual Color, Bio-Rad); lane 2, supernatant after expression; lane 3, isoQC-containing fractions after initial hydrophobic interaction chromatography applying expanded bed absorption; lane 4, isoQC after hydrophobic inter- action chromatography; lane 5, isoQC after anion exchange chroma- tography; lane 6, isoQC after desalting and deglycosylation of the protein. isoQC corresponds to a protein between 50 and 70 kDa. The deglycosylated protein migrates at 37 kDa and the endoglycosi- dase H f is at 75 kDa. Characterization of glutaminyl cyclase isoenzymes A. Stephan et al. 6526 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS the k cat ⁄ K m values of both human isoQC variants, however, revealed a prominent difference. The specific- ity constant obtained with isoQC expressed in P. pas- toris was two- to three-fold higher for almost every substrate tested, suggesting a potential influence of the expression system or of the isoQC fusion construct. As a result of the very similar Michaelis constants for the isoQCs expressed in E. coli and P. pastoris, the influ- ence on substrate specificity was mainly caused by k cat . Similar to K m , no significant difference was observed in the K i values between isoQCs expressed in yeast and E. coli (Table 2). Presumably, the expression and puri- fication of isoQC in P. pastoris resulted in efficient recovery of the active protein. The relative substrate specificity of murine isoQC was similar to that of the human enzyme (Table 3). Most substrates, however, were more efficiently con- verted into products by the murine protein, which con- trasts with the related QCs [5]. The higher proficiency was caused by the lower Michaelis constants and higher turnover numbers. In order to further characterize the activity of mur- ine isoQC in comparison with QC, the pH dependence of catalysis was assessed under first-order conditions, i.e. at [S] << K m . Both enzymes displayed a pH opti- mum of k cat ⁄ K m of between 7.5 and 8 (Fig. 5). The kinetic data of the pH dependence were evaluated by applying a model, which considers three dissociating groups, one of the substrate and two of the enzyme. The pK a value of the applied substrate H-Gln-AMC (6.83 ± 0.01) has been determined previously and matches well with the acidic inflection points of the pH dependences of isoQC and QC. The nonsymmetric character of the bell-shaped curve was calculated assuming two dissociating groups of the enzyme. Eval- uation of these data resulted in pK a values of 9.5 ± 0.3 and 8.2 ± 0.4 for murine isoQC and 9.0 ± 0.2 and 8.3 ± 0.3 for murine QC. Apparently, all dissociating groups, which influence the catalytic process in QC and isoQC, are conserved, supporting an identical catalytic mechanism. Characterization of metal dependence and structural elements The animal QCs and isoQCs share a structural relationship to bacterial double-zinc aminopeptidases [23–25]. Although the coordinating residues of the aminopeptidases are also conserved in QCs, it has been shown that only one zinc ion is bound to QC [5,26]. Therefore, murine isoQC without an affinity tag was analyzed for the presence of transition metal ions using total X-ray fluorescence (TXRF) spectroscopy. Table 1. Kinetic parameters of substrate conversion by different human isoQC variants. The substrates are displayed in the three-letter code of amino acids. Reactions were carried out in 0.05 M Tris ⁄ HCl, pH 8.0, at 30 °C. ND, not determined. Compound isoQC (F60;I73N;C369A) N-His isoQC (F60;I73N;C369A) N-His deglycosylated isoQC (F60;I73N) N-His K m (mM) k cat (s )1 ) K i (mM) K m (mM) k cat (s )1 ) K i (mM) K m (mM) k cat (s )1 ) K I (mM) Substrates H-Gln-bNA 0.034 ± 0.003 9 ± 2 ND 0.039 ± 0.007 7 ± 1 ND 0.058 ± 0.003 12 ± 1 ND H-Gln-Gln-OH 0.19 ± 0.01 5.7 ± 0.2 0.16 ± 0.02 4.6 ± 0.3 0.19 ± 0.003 6.2 ± 0.1 H-Gln-Glu-OH 1.10 ± 0.04 4.9 ± 0.2 0.89 ± 0.03 4.3 ± 0.2 0.79 ± 0.02 4.9 ± 0.1 Inhibitors Benzimidazole 0.24 ± 0.02 0.22 ± 0.03 0.27 ± 0.02 Benzylimidazole 0.0080 ± 0.0002 0.009 ± 0.001 0.010 ± 0.001 A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6527 A typical spectrum is displayed in Fig. 6A. Three inde- pendently prepared enzyme samples showed a signifi- cantly increased zinc concentration. The averaged zinc content in murine isoQC was 0.99 ± 0.38 moles of zinc per mole of enzyme, clearly supporting stoichiom- etric zinc binding of murine isoQCs. Accordingly, full reactivation of the murine isoQC apo-enzyme was obtained in the presence of equimolar zinc concentra- tions (Fig. 6B). In addition to zinc, reactivation was also obtained with cobalt ions, also suggesting an equi- molar stoichiometry of metal to protein necessary for activity. With regard to zinc content and reactivation, therefore, the isoQCs are very similar to QCs. A cata- lytic role of the transition metal ion is also suggested by a comparison of the far-UV CD spectrum of murine isoQC and its apo-enzyme (Fig. 6C). The CD Table 3. Kinetic evaluation of peptide substrates by mouse isoQC. Protein was expressed in Pichia pastoris. a ND, not determined. Compound Murine isoQC Murine QC K m (mM) k cat (s )1 ) K i (mM) k cat ⁄ K m (mM )1 Æs )1 ) k cat ⁄ K m (mM )1 Æs )1 ) K i (mM) Substrates H-Gln-bNA 0.032 ± 0.003 17.48 ± 0.97 3.55 ± 0.13 b 554 ± 47 550 ± 30 c 1.77 ± 0.18 c H-Gln-AMC 0.030 ± 0.006 6.98 ± 0.35 ND 311 ± 27 125 ± 4 c 5.9 ± 0.7 c H-Gln-Gln-OH 0.092 ± 0.005 8.66 ± 0.37 95 ± 6 213 ± 8 c H-Gln-Glu-OH 0.47 ± 0.04 7.79 ± 0.44 16 ± 2 36 ± 1 c H-Gln-Gly-OH 0.16 ± 0.01 4.57 ± 0.12 28 ± 2 30 ± 2 c H-Gln-Gly-Pro-OH 0.102 ± 0.006 11.4 ± 0.4 111 ± 7 ND H-Gln-Tyr-Ala-NH 2 0.058 ± 0.004 22.9 ± 0.9 394 ± 21 ND H-Gln-Phe-Ala-NH 2 0.060 ± 0.006 24.1 ± 0.5 403 ± 49 ND H-Gln-Glu-Tyr-Phe-NH 2 0.029 ± 0.003 11.78 ± 0.61 413 ± 46 ND H-Gln-Glu-Asp-Leu-NH 2 0.16 ± 0.01 6.4 ± 0.1 104 ± 4 ND Inhibitors Imidazole 0.103 ± 0.027 0.16 ± 0.01 c Benzimidazole 0.124 ± 0.004 0.192 ± 0.003 c Methylimidazole 0.052 ± 0.005 0.023 ± 0.001 c Benzylimidazole 0.0039 ± 0.0003 0.0064 ± 0.0007 c Cysteamine 0.069 ± 0.006 0.042 ± 0.002 c N-Dimethylcysteamine 0.027 ± 0.003 0.029 ± 0.002 c a Reactions were carried out in 0.05 M Tris ⁄ HCl, pH 8.0, at 30 °C. b Substrate inhibition. c Data from [5]. Table 2. Kinetic parameters of substrate conversion by recombinant human isoQC obtained from different host systems. a Compound isoQC (F48;I73N;C369A) C-His GST-isoQC E. coli K m (mM) k cat (s )1 ) K i (mM) k cat ⁄ K m (mM )1 Æs )1 ) k cat ⁄ K m (mM )1 Æs )1 ) K i (mM) Substrates H-Gln-bNA 0.035 ± 0.001 3.4 ± 0.1 1.57 ± 0.09 b 229 ± 22 93 ± 7 c 1.47 ± 0.07 c H-Gln-AMC 0.030 ± 0.006 1.07 ± 0.03 4.47 ± 0.91 b 103 ± 29 63 ± 6 c 5.73 ± 0.81 c H-Gln-Gln-OH 0.11 ± 0.01 2.7 ± 0.2 54 ± 5 25 ± 4 c H-Gln-Glu-OH 0.61 ± 0.06 2.6 ± 0.2 9 ± 1 4 ± 0.6 c H-Gln-Gly-OH 0.36 ± 0.05 1.65 ± 0.04 9 ± 2 4 ± 0.3 c H-Gln-Gly-Pro-OH 0.23 ± 0.02 4.0 ± 0.1 38 ± 3 19 ± 1 c H-Gln-Tyr-Ala-NH 2 0.08 ± 0.02 7.7 ± 0.4 207 ± 57 66 ± 13 H-Gln-Phe-Ala-NH 2 0.10 ± 0.02 7.5 ± 0.3 117 ± 34 33 ± 2 H-Gln-Glu-Tyr-Phe-NH 2 0.040 ± 0.004 3.3 ± 0.1 123 ± 6 110 ± 21 H-Gln-Glu-Asp-Leu-NH 2 0.16 ± 0.01 6.4 ± 0.1 55 ± 5 10 ± 1 Inhibitors Imidazole 0.235 ± 0.013 0.219 ± 0.0009 c Benzimidazole 0.250 ± 0.005 0.199 ± 0.008 c Methylimidazole 0.082 ± 0.003 0.079 ± 0.0047 c Benzylimidazole 0.0062 ± 0.0002 0.0073 ± 0.0005 c a Reactions were carried out in 0.05 M Tris ⁄ HCl, pH 8.0, at 30 °C. b Substrate inhibition. c Data from [16]. Characterization of glutaminyl cyclase isoenzymes A. Stephan et al. 6528 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS spectra were virtually identical between isoQC, apo- isoQC and QC. Thus, the loss of the metal ion does not result in large structural rearrangements. The virtually identical spectra further support the strong similarity between QC and isoQC globular domains. Finally, we characterized whether the conserved cysteines in human isoQC form a disulfide bond, as described for human QC [4]. The characterization of the disulfide status of the protein by SDS-PAGE clearly suggests disulfide bond formation (Fig. 7). Therefore, the structurally conserved features of isoQC and QC proteins not only contain metal complexation and general fold, but also the formation of a disulfide bond close to the active center of the protein. In addition to the two conserved cysteines, human isoQC contains a third cysteine residue in the C-ter- minal part of the protein (Fig. 1). The presence of a third cysteine might imply the formation of dimers in the secretory pathway because, in an oxidative envi- ronment, cysteine does not usually appear unbound. In the case of isoQC, it should be noted that the cysteine residue is not conserved in murine (Fig. 1), bovine (UniProt: Q0V8G3) and macaque (UniProt: Q4R942) isoQCs. Initial expression of murine and human isoQCs in human HEK293 cells and an accompanying western blot analysis involving a reducing or nonreducing sample preparation did not reveal the formation of homo- or heterodimers (not shown). The data thus imply that dimerization involv- ing a covalent interaction does not occur in the Golgi complex. Discussion In a first approach to characterize the recently discov- ered isoQCs from mouse and humans, we aimed to C Wavelength in nm 200 220 240 260 Molar ellipticity (degrees·cm²·dmol –1 ) –15 000 –10 000 –5000 0 5000 10 000 15 000 20 000 25 000 m-isoQC apoenzyme m-isoQC reactivated mQC A Counts 2000 4000 Photon energy, keV 0246810121416 0 m-isoQC Cl P Ca Cu Zn Zn Br B Ratio metal to enzyme 024 Activity (µM·min –1 ) 0 2000 4000 6000 Zinc Cobalt Manganese Nickel Calcium Fig. 6. Spectroscopic analysis of murine isoQC. (A) TXRF spectrum of a murine isoQC preparation. IsoQC was dissolved in 10 m M Tris ⁄ HCl, pH 7.6. The evaluation of the measurements revealed equimolar amounts of zinc bound to the enzyme. (B) Reactivation of murine apo-isoQC with different divalent metal ions. The enzyme was inactivated by 1,10-phenanthroline and subjected to dialysis against Chelex-treated buffer. Reactivation was carried out by the addition of different concentrations of transition metal ions to the inactivated protein. The ratios of the concentrations of transition metal and apo-enzyme are indicated on the x-axis. (C) CD spectro- scopic analysis of murine isoQC, murine QC and murine apo-isoQC. The protein was dissolved in 10 m M potassium phosphate buffer, pH 6.8. There was virtually no difference in the spectra between the apo- and holo-enzymes and murine QC, supporting a low influence of the active site zinc on the structure of isoQC. pH 68 k cat /K m (mM –1 ·s –1 ) 0 20 40 60 80 100 120 140 160 180 200 220 240 260 m-isoQC mQC Fig. 5. pH dependence of murine isoQC catalysis. Determination of the specificity constant k cat ⁄ K m for the conversion of H-Gln-AMC by purified murine isoQC (h) and QC (O), determined under first- order rate law conditions ([S] << K m ). The substrate concentration was 0.005 m M and the reactions were carried out at 30 °C in a buf- fer consisting of 0.2 M Tris ⁄ HCl, 0.1 M Mes and 0.1 M acetic acid. A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6529 express and characterize the proteins in P. pastoris in order to compare the catalytic properties with those of the sister enzyme QC. In contrast with E. coli, P. pas- toris has been shown to exert many post-translational modifications, such as N-glycosylation and disulfide formation [27–31], some of which have been shown to be present in animal and plant QCs [4,5,19,32] and may also be important for the expression of isoQCs. On the basis of these results, we aimed to obtain the secretory expression of murine and human isoQCs in P. pastoris. In the native state, isoQC is expressed as a class II transmembrane protein, which is anchored by an N-terminal signal peptide in the membrane of the endoplasmic reticulum and retained in the Golgi appa- ratus. In order to obtain secreted protein, the short cytosolic tail, including the major part of the mem- brane anchor, was deleted and substituted by an a-leader prepro sequence of yeast, which should direct the protein efficiently into the secretory pathway. Although the remaining protein shares 51% sequence identity with the mature QCs, isoQC expression resulted in media virtually devoid of QC activity. In contrast with the QC proteins, human and murine iso- QCs lack a conserved N-terminal site for N-glycosyla- tion (Fig. 1). It is known that glycosylation may lead to an increase in protein solubility [33] and decreased aggregation propensity [34]. Furthermore, deglycosyla- tion of human QC resulted in protein precipitation (results not shown). Therefore, we introduced an N-glycosylation site into isoQC for heterologous expression. Glycosylation resulted in a significant increase in isoQC activity in the medium. Although the reason for the improvement was not investigated in detail, an increase in protein solubility and, in turn, a decrease in hydrophobic interactions between the protein might be a primary cause. Consistent with this hypothesis, an enzymatic deglycosylation of the isoQC protein resulted in a dramatic decrease in solubility to about 1 mgÆmL )1 , whereas the glycosylated protein did not precipitate up to 30 mgÆmL )1 (not shown). In addition to glycosylation, the N-terminal truncation to glutamic acid 60/61 of the proteins further improved the expression of human and murine isoQCs. Based on sequence comparisons, the finally deleted region corre- sponds mainly to unstructured parts of the protein, which might also exert an influence on efficient protein production. The sequential optimization of the protein construct used for expression ended in a 100-fold improvement of the protein yield, which was secreted into the medium of the cells and could be purified by two- to three-step protocols. The secretory expression in yeast facilitates an efficient purification process, because yeast – in spite of a fully developed secretory machinery – secretes only a few proteins into the extra- cellular space [35]. Because the heterologous protein reaches a high specific activity in the expression med- ium, secretion can be regarded as a separation step, allowing efficient recovery of the protein of interest, even without an affinity tag, as shown here for murine isoQC. Moreover, the expression of proteins in the secretory pathway facilitates the appropriate formation of post- translational structural elements, such as disulfide bonds. As shown in this study, the two cysteine resi- dues, which are conserved in murine and human isoQCs, form a disulfide bond, which is reminiscent of the disulfide bond formation in human QC. Appar- ently, the disulfide bridge is an evolutionary conserved structural element of QC and isoQC (Fig. 1). Accord- ing to the crystal structure of human QC [25], the cysteine residues are close to the active site, probably exerting a stabilizing effect on the flexibility in that region. The high degree of sequence similarity between the isoQCs and their sister enzyme QC was finally mir- rored by the characterization of the secondary struc- ture, metal dependence and catalytic activity. A virtually identical catalytic activity was demonstrated by the comparison of murine isoQC and murine QC, which were both expressed in P. pastoris. The data clearly suggest that the active site of both enzymes has a very similar structure, forming identical secondary interactions with the substrate to facilitate binding and turnover. Apparently, the core features of both pro- teins, i.e. the active site and the general fold, are iden- tical, a hypothesis which is also supported by an alignment of the proteins, which shows a high degree of conservation of the inner core structures between QC and isoQC and a weaker similarity in the connect- ing loops (Fig. 1). The far-UV CD spectra of QC and isoQC are shown to be identical, confirming the kDa 1 534 1098762 50 37 Fig. 7. Analysis of disulfide formation in human isoQC expressed in Pichia pastoris. Lanes 1 and 10, molecular mass standards (kDa); lanes 2, 3, 8 and 9, sample prepared under reducing conditions (5% b-mercaptoethanol); lanes 4–7, samples prepared under non- reducing conditions. Electrophoresis (15% gels) was performed at a constant voltage of 200 V for 1.5 h, and protein was visualized by Coomassie staining. Characterization of glutaminyl cyclase isoenzymes A. Stephan et al. 6530 FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS above-mentioned assumptions. According to the pro- posed high degree of similarity, isoQCs and QCs con- tain one zinc ion in the active site, which is responsible for the catalytic activity of the enzyme. As shown here by titration experiments, zinc can be replaced by cobalt, which results in a less active enzyme. Similar results have been reported previously for human QC [24]. The results of the titration experiments clearly suggest that the binding of one transition metal ion is necessary and sufficient for the exertion of full enzy- matic activity, and that the metal ion does not exert a direct effect on the structure of the protein. Thus, with regard to the recombinant expression strategy introduced in the present study, it appears that the various mutagenic changes to the isoQCs did not exert a relevant influence on the catalytic activity of the soluble, heterologous proteins. However, a potential influence of the deletion of the N-terminal signal anchor in isoQC cannot be fully excluded. In its native state, the protein is a membrane-bound enzyme of the Golgi complex, and membrane anchoring might potentially affect substrate turnover, perhaps by prox- imity to the membrane or other interacting proteins. The present results thus mirror well the catalytic potential of the globular domain, especially in relation to the sister enzyme QC, but cannot be translated into the in vivo situation without caution. The characterization of QC and isoQC revealed that evolution apparently resulted in globular proteins with a very similar catalytic power, virtually identical sub- strate specificity and similar subcellular localization within the secretory pathway. Most likely, the proteins had a common ancestor. It still remains unclear, however, whether the proteins are responsible for the conversion of the same substrates, i.e. pGlu-modified proteins and ⁄ or peptide hormones and pGlu-modified amyloid peptides in neurodegenerative disorders. The localization of QC and isoQC in the secretory pathway enables the conversion of secretory proteins by both enzymes – QC is probably transported within the regulated pathway [8] and isoQC exerts its function as a resident enzyme of the Golgi apparatus [16]. Although the localization of both proteins appears to be virtually identical at first glance, the presence of QC in vesicles of the regulated secretory pathway might indicate the responsibility of QC for the conversion of substrates requiring extensive post-translational processing, e.g. the neuropeptide TRH [36–38]. The liberation of the substrate in secretory vesicles requires the presence of QC in the same compartment, because N-terminal pGlu formation represents a finishing reaction in the post-translational maturation of these hormones. In contrast, many proteins do not require such processing of the precursor; the N-terminal glutaminyl residue is directly generated by signal pepti- dase cleavage in the endoplasmic reticulum, e.g. in ribonuclease or a-amylase. These proteins are also likely to be secreted via the constitutive pathway from the Golgi complex. Therefore, the primary converting enzyme of these proteins might be isoQC. Taken together, it appears that the liberation of N-terminal glutamine in the secretory pathway results inevitably in N-terminal pGlu formation. The broad and similar substrate specificity of QC and isoQC might therefore be important for the conversion of these different substrates. Finally, the similar cellular distribution of two proteins with virtually identical specificity, as shown here, might also point to an overall important role of pGlu protein formation for physiology. The elucidation of the physiological function might have implications for drug development, as a partial complementation of QC and isoQC might compensate for the side-effects of potential, isoform-specific drug candidates. Indeed, it is likely that the protein func- tions of QC and isoQC complement each other, as QC knockout mice do not exhibit an apparent phenotype (S. Schilling et al., unpublished results). In summary, the first detailed heterologous expres- sion and characterization study of mammalian isoQCs was accomplished. Expression was mainly optimized by the insertion of an artificial glycosylation site into the isoQC protein, resulting in efficient protein secre- tion by the yeast P. pastoris, resembling the subcellular localization in the native tissue of origin. These results might have implications for the expression of other mammalian proteins, which display a high tendency to aggregation and are, therefore, difficult to express. The isolation of the isoQC protein represents a basis for structural investigations and drug candidate profiling. Materials and methods Materials The E. coli strain DH5a was applied for all plasmid con- struction and propagation; P. pastoris strain X-33 (AOX1, AOX2) was used for the expression of the different isoQC variants. Yeast was grown, transformed and analyzed according to the manufacturer’s instructions (Invitrogen, Karlsruhe, Germany). The glutaminyl peptides were obtained from Bachem (Bubendorf, Switzerland) or synthe- sized as described elsewhere [39]. Recombinant pyroglutamyl aminopeptidase from Bacillus amyloliqefaciens was purchased from Qiagen (Hilden, Germany) and glutamic dehydrogenase from Fluka (Seelze, Germany). The imidaz- ole derivatives were purchased from Sigma-Aldrich A. Stephan et al. Characterization of glutaminyl cyclase isoenzymes FEBS Journal 276 (2009) 6522–6536 ª 2009 Probiodrug AG. Journal compilation ª 2009 FEBS 6531 [...]... Bateman RCJ (2004) Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site BMC Biol 2, 2 Characterization of glutaminyl cyclase isoenzymes 24 Schilling S, Niestroj AJ, Rahfeld J-U, Hoffmann T, Wermann M, Zunkel K, Wasternack C & Demuth HU (2003) Identification of human glutaminyl cyclase as a metalloenzyme: inhibition by imidazole derivatives and heterocyclic chelators... catalytic properties, and competitive inhibitors of the zinc-dependent murine glutaminyl cyclase Biochemistry 44, 13415–13424 6 Song I, Chuang CZ & Bateman RCJ (1994) Molecular cloning, sequence analysis and expression of human pituitary glutaminyl cyclase J Mol Endocrinol 13, 77–86 7 Pohl T, Zimmer M, Mugele K & Spiess J (1991) Primary structure and functional expression of a glutaminyl cyclase Proc... buffer were applied to the TXRF quartz glass sample support and dried under IR radiation Afterwards, 5 lL of diluted Se aqueous standard solution (internal standard; Sigma-Aldrich) was added to each sample and dried again The X-ray fluorescence signal was collected for 100 s For all determinations, an Extra II TXRF module containing molybdenum and tungsten primary X-ray sources (Seifert, Ahrensburg, Germany),... and 38 mA For CD spectroscopic analysis, the proteins were dialyzed against buffer containing 10 mm NaH2PO4 CD spectra of murine QC and murine isoQC were acquired with a Jasco J-715 spectrapolarimeter using quartz cuvettes of 0.1 cm path length The mean of 10 wavelength scans between 190 and 260 nm was calculated, and the spectra were corrected by subtraction of the buffer spectra The apo-enzymes and. ..Characterization of glutaminyl cyclase isoenzymes A Stephan et al (Taufkirchen, Germany) The low-salt Luria–Bertani medium, required for the propagation of E coli, and the buffered glycerol complex medium (BMGY) and buffered methanol complex medium (BMMY), required for the propagation of yeast, were prepared according to the ‘Pichia protocols’ (Invitrogen) Cloning procedures The different human and murine isoQC... expression vector pPICZaA (Invitrogen) using the EcoRI and NotI restriction sites The primers used for cloning are listed in Table 4 The PCR fragment for the human isoQC(F48) C-His was generated using primer 5 (sense) and primer 6 (antisense) Mutagenesis primers 1 and 2 were used for the insertion of the glycosylation site, and primer 3 (sense) and primer 4 (antisense) were applied for the mutation... Journal compilation ª 2009 FEBS 6533 Characterization of glutaminyl cyclase isoenzymes A Stephan et al TXRF and CD spectroscopy Acknowledgement Murine isoQC was desalted by size exclusion chromatography using a Sephadex G-25 desalting column (1.0 · 10 cm), which was equilibrated in 10 mm Tris ⁄ HCl, pH 7.6 The isoQC-containing fractions were collected and the protein was concentrated to approximately 3... site-directed mutagenesis was performed according to standard PCR techniques, followed by digestion of the parent DNA using DpnI (Quik-Change II site-directed mutagenesis kit; Stratagene, La Jolla, CA, USA) C- and N-terminal His-tags were introduced by primer pairs 5 ⁄ 6 and 7 ⁄ 8, respectively Finally, a human isoQC starting with glutamic acid 60 and bearing an N-terminal His-tag was generated by applying... codon 74 (Ile) using primer 3 as sense and primer 4 as antisense The sequences of all expression constructs were verified Transformation of P pastoris and mini-scale expression Plasmid DNA was amplified in E coli DH5a and purified according to the recommendations of the manufacturer (Qiagen); 20–30 lg of DNA (in vector pPICZaA) were linearized with PmeI, precipitated and dissolved in deionized water; 1–5... five column volumes, and eluted in a reversed flow direction with 50 mm phosphate buffer, pH 6.8 Fractions exhibiting isoQC were pooled and applied to a HiPrep desalting column (2.6 · 10 cm), which was equilibrated with 50 mm Bis-Tris, pH 6.8, 100 mm NaCl The purification was analyzed by SDS-PAGE and the protein content was determined according to the methods of Bradford [40] or Gill and von Hippel [41] . Mammalian glutaminyl cyclases and their isoenzymes have identical enzymatic characteristics Anett Stephan 1 , Michael. Recently, isoenzymes of mammalian QCs have been identified. In order to gain further insight into the biochemical characteristics of iso- QCs, the human and murine