65) expressed in yeast

Journal of Biotechnology 97 (2002) 183– 190 www.elsevier.com/locate/jbiotec

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast Jonathan C. Tong, Ian R. Mackay, Judy Chin, Ruby H.P. Law, Karine Fayad, Merrill J. Rowley * Department of Biochemistry and Molecular Biology, Monash Uni6ersity, Clayton Vic. 3800, Australia Received 12 November 2001; received in revised form 11 March 2002; accepted 27 March 2002

Abstract Background and aims: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to g-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1 – 101 of GAD67 to a human cDNA for amino acids 96–585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. Methods: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. Results: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), Km of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at − 80 °C, or in lyophilized form at −20 °C. Conclusions: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Recombinant; Pyridoxal phosphate (PLP); GAD; Enzyme; Yeast; Type 1 diabetes

1. Introduction

* Corresponding author. Tel.: +61-3-9905-1438; fax: + 613-9905-4699. E-mail address: [email protected] (M.J. Rowley).

Glutamic acid decarboxylase (GAD) catalyses the conversion of glutamate to the inhibitory neurotransmitter g-aminobutyric acid (GABA), and performs other intracellular metabolic functions (Martin and Rimvall, 1993). GAD is present pre-

0168-1656/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 0 6 0 - 3

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dominantly in brain and pancreatic b-islet cells (Erlander et al., 1991). The identification of the gene for feline GAD (Benson et al., 1989) was followed by recognition of two functionally different isoforms, GAD65 and GAD67, that diverge in sequence particularly in the first 100 N-terminal residues, are hydrophobic (GAD65) or hydrophilic (GAD67), and are located in cell membranes (GAD65) or in the cytosol (GAD67) (Bu et al., 1992). Serum autoantibodies to GAD, particularly GAD65, were identified in 1988 by Solimena and colleagues by Western immunoblot (Solimena et al., 1988) in the rare neurological disease Stiff-Man Syndrome which can be associated with diabetes, and subsequently in spontaneously occurring type 1 diabetes mellitus (Baekkeskov, 1986). This led to an upsurge of interest in GAD. Native brain GAD (Rowley et al., 1992), and subsequently recombinant forms of GAD65 prepared in various expression systems, Escherichia coli (Hao and Schmit, 1993), yeast (Powell et al., 1996; Law et al., 1998; Papakonstantinou et al., 2000; Hampe et al., 2001), baculovirus (Mauch et al., 1993), and rabbit reticulocyte lysate (Rowley et al., 1996), were developed for use in diagnostic assays (Zimmet et al., 1993; Tuomi et al., 1993), and also for use as tolerogenic ‘vaccines’ to assess prevention of diabetes in non-obese diabetic (NOD) mice (Petersen et al., 1994; Pleau et al., 1995) preparatory to use in humans (Schernthaner, 1995). However, the preparation of highly purified recombinant GAD65 is difficult, as the N-terminal region of GAD65 is very hydrophobic. To overcome this we designed a hybrid construct in which the Nterminal hydrophobic region GAD65 (amino acids 1–96) is replaced by the more hydrophilic counterpart of the GAD67 (amino acids 1– 101, Teoh et al., 1998). This molecule can be readily expressed from yeast strains Saccharomyces cere6isiae and Pichia pastoris (Law et al., 1998; Papakonstantinou et al., 2000), and purified by affinity chromatography. The resulting GAD67/ 65 hybrid was shown to react similarly to mammalian brain GAD by immunoprecipitation using sera from patients with type 1 diabetes (Law et al., 1998). In this report, we assessed the biochemical properties of the recombinant GAD67/65,

and compared the results with published data from preparations of brain GAD containing mixed isoforms of GAD65 and GAD67.

2. Materials and methods

2.1. Expression and purification of rGAD67 /65 in Saccharomyces cere6isiae Human gene constructs were used to encode rGAD67/65 which was expressed from S. cere6isiae strain YRD-15 (MATa ura3 his3 leu2 ) from the vector pMONBC6 as described previously (Law et al., 1998). The phosphoglycerate kinase promoter (PGK1 ) was used to drive a high-level constitutive expression of protein, in growth media containing 10% glucose, 1% yeast extract, 20 mg ml − 1 uracil, 20 mg ml − 1 histidine, 0.12% (NH4)2SO4, 0.1% KH2PO4, 0.07% MgCl2, 0.05% NaCl, 0.01% CaCl2 and 0.005% FeCl3. rGAD67/ 65 was prepared by affinity purification on a GAD-1 monoclonal antibody column (Law et al., 1998). rGAD67/65 was stored in 30% (w/v) glycerol at − 20 °C unless otherwise noted. The yeast cells were lysed by vortexing the cells with equal volumes of glass beads (425–600 mm diameter) and lysis buffer (50 mM KH2PO4, 1 mM EDTA, 1 mM aminoethylisothiouronium bromide (AET), 20 mM pyridoxal phosphate (PLP), 10 mM 2-mercaptoethanol (2-ME), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM glutamate, pH 7.2); and the rGAD65/67 was purified from clarified lysate by affinity chromatography using the monoclonal antibody GAD1, as described previously (Law et al., 1998). All buffers contained 1 mM AET, 20 mM PLP, and 10 mM glutamate to maintain enzyme activity unless otherwise stated.

2.2. Measurement of rGAD67 /65 enzyme acti6ity The procedure for measuring rGAD67/65 enzymatic activity was based on that described previously (Rowley et al., 1992; Law et al., 1998; Papakonstantinou et al., 2000). The enzyme was incubated with the substrate 10 mM L-glutamate containing 100 000 cpm of 3H-glutamate in 50

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mM KH2PO4, 1 mM 2-ME, 1 mM PMSF, 0.2 mM PLP, and 1 mM AET in a final volume of 130 ml. The pH of the final reaction mixture was 7.2. The standard incubation was performed for 30 min at 37 °C. Activity was quantified using a spin column chromatography procedure that uses the anion exchange resin AG1-X8 (Bio-Rad, Hercules, CA) to separate 3H-GABA from the more highly acidic substrate 3H-glutamate. Reactions were stopped after 30 min with 20 ml of 0.25 M H2SO4. Control reactions were stopped with 20 ml of 0.25 M H2SO4 prior to the addition of L-glutamate. All reactions were performed in duplicate with tests and controls included for each run.

2.3. Effect of 6ariation in pH, inorganic phosphate concentration, substrate concentration and time on enzyme acti6ity The effect of pH on enzyme activity was tested over a range of pH 4– 12 by adding 10 M KH2PO4, or 10 M KHPO4 to the total reaction mix as appropriate. The final pH was determined using Universalindikator pH sticks with pH ranges from pH 0 to 14, 4.0 to 7.0, 6.5 to 10.0 (Merck, Darmstadt, Germany) immediately prior to the 30 min incubation at 37 °C. The effects of changes in concentration of inorganic phosphate were assessed at a range of concentrations from 0.1 to 1.0 M. Various glutamate concentrations from 0 to 20 mM were assessed by addition of unlabelled L-glutamate, with all assays containing 100 000 cpm of 3H-glutamate. The time-course in the reaction was stopped by the addition of 20 ml of 0.25 M H2SO4 after incubation at 37 °C for 0, 30, 60 and 120 min.

2.4. Preparation of PLP-free rGAD67 /65 Although GAD is a PLP-dependent decarboxylase, GAD67 binds PLP more strongly than does GAD65 (Martin et al., 2000). We observed that rGAD67/65 prepared by the standard procedure from yeast lysate was enzymatically active even when tested using a reaction buffer that lacked PLP (unpublished data). To study the requirement of rGAD67/65 for PLP, we prepared PLPfree GAD using a yeast lysate that had been

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dialyzed against PLP-free lysis buffer, and purified using buffers that lacked PLP. The enzyme activity of this GAD preparation was then tested before and after the addition of PLP in the reaction buffer.

2.5. Enzyme stability of rGAD67 /65 at different temperatures The effect of temperature on the retention of enzyme activity of rGAD67/65 was measured using a single preparation that had been separated into aliquots and stored at − 80, − 20, 4, 22 and 37 °C for 2, 4, 20, 72 h, 7, 15 days, 1, 2, 3 or 4 months, and on lyophilized rGAD67/65 stored at − 20 °C for up to 3 months. All preparations were routinely stored in 30% glycerol at −20 °C, except for the aliquots stored at − 80 °C and lyophilized samples.

3. Results rGAD67/65 was purified by affinity chromatography from the cell lysate of recombinant S. cere6isiae and the yield is about 400 mg l − 1 of cell culture. The specific enzyme activity is between 15.2 and 30.2 nK at and the protein concentration is about 100 mg ml − 1 depending on the preparations.

3.1. Effects of 6ariation in pH, phosphate concentration, substrate concentration and time on enzyme acti6ity The effects of changing pH on the activity of the enzyme were examined over a pH range of 4–12. In each of three separate experiments, enzyme activity was greatest at pH 7.4, and was lowest at pH 5 and below and at pH 10 and above (Fig. 1A). This loss was not related to changes in the phosphate concentration, after adjustment of the pH with either 10 M KH2PO4, or 10 M KHPO4, since the enzyme activity increased with increasing concentrations of inorganic phosphate at a constant pH of 6.8, the normal pH of the reaction (Fig. 1B). There was a linear increase in activity of rGAD67/65 with increasing substrate

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linear increase in the amount of 3H-GABA produced with increasing time over a 2 h period at 37 °C (data not shown).

3.2. Effects of PLP on enzyme acti6ity Standard preparations of rGAD67/65 from yeast lysates were enzymatically active even when tested using a reaction buffer that lacked PLP. Accordingly, to examine whether PLP was essential for enzyme activity, GAD was prepared from a yeast lysate that had been derived using a PLP-free lysis buffer. The ensuing PLP-free GAD preparation had no detectable enzyme activity. Enzyme activity was restored by the addition of PLP to the reaction mixture (Table 1).

3.3. Enzyme stability: effects of temperature and time

Fig. 1. Enzyme activity of recombinant GAD67/65 expressed as counts per minute (cpm) of: (A) changes in pH of total reaction mix; (B) changes in inorganic phosphate concentration of total reaction mix (test and control reactions are represented by squares and triangles, respectively); and (C) concentration of the substrate, L-glutamate. cpm refers to release of 3H-GABA.

concentration, reaching a maximum at 10 mM (Fig. 1C). From a Lineweaver– Burk double reciprocal plot, the Km of rGAD67/65 for L-glutamate was 1.3 mM at pH 7.4 and 37 °C. There was a

The enzyme activity of a single preparation of rGAD67/65 stored in 30% glycerol at −20 °C for 4 months was used as a control. Enzyme activity of rGAD67/65 stored at − 80, 4, 22 and 37 °C expressed as a percentage of the initial activity of the preparation is shown in Table 2a. There was a constant or apparent increase in activity over 1 month when stored in 30% glycerol at − 20 °C and a slow decrease in activity thereafter (Table 2b). Lyophilized rGAD67/65 retained 76% of the initial specific enzyme activity over a 3 month period compared with 42% for rGAD67/65 stored in 30% glycerol at − 20 °C (Table 2c).

Table 1 Effects of PLP on enzyme activity of recombinant GAD67/65 Enzyme activity (nmol min−1)

Specific activity (nmol min−1 mg−1)

No PLP in reaction mix rGAD67/65a 60 rGAD67/65 83

0.00 0.20

0.44 1200

PLP in reaction mix rGAD67/65a 60 rGAD67/65 83

0.13 0.28

1100 1700

Enzyme condition

a

Protein concentration (mg ml−1)

rGAD67/65 produced in PLP-free buffers and subsequently dialyzed to remove all traces of PLP.

N.D. N.D. 94 9 1 N.D. N.D. N.D. N.D. 91 93 N.D. 1249 7

−80 °C N.D. 699 6 729 7 5892 45 93 N.D. 50 9 0.1 N.D. 509 9 N.D.

4 °C N.D. 54 9 3 49 9 2 33 9 0.1 8 9 0.2 59 1 N.D. N.D. N.D. N.D.

22 °C

Residual enzyme activity of rGAD67/65 at specified temperature (%)*

1.0 1.3 1.4 1.6 1.2 1.6 0.8 0.9 0.7 0.7 0.7 0.6

Enzyme activity (nmol min−1) 510 680 740 840 630 840 420 470 370 370 370 310

Specific enzyme activity (nmol min−1 mg−1) 100 134 146 165 124 165 83 93 72 72 72 62

Residual activity (%)

29 9 6 26 9 2 591 N.D. N.D. N.D. N.D. N.D. N.D. N.D.

37 °C

155 94 94 94

155 60 54 88

10.9 4.6 5.7 2.8

Stored at −20 °C

Stored at −20 °C

Lyophilized

Enzyme activity (nmol min−1)

Protein concentration (mg ml−1)

10.9 4.3 5.3 4.3

Lyophilized

3530 2450 3030 1490

Stored at −20 °C

3530 3520 4900 2670

Lyophilized

Specific enzyme activity (nmol min−1 mg−1)

Values are mean of triplicates 9S.D. *, Percentage of initial specific enzyme activity. N.D., Not determined.

0 15 30 90

Days

(c) In 30% (w/6) glycerol at −20 °C and lyophilized rGAD67 /65 stored at −20 °C, reconstituted and held at 4 °C o6ernight

0 2 28 30 32 35 65 82 89 96 124 131

Duration of storage (days)

(b) In 30% (w/6) glycerol at −20 °C with a protein concentration of 95.8 vg ml−1

2h 4h 20 h 3 days 7 days 15 days 1 month 2 months 3 months 4 months

Duration of storage

100 69 86 42

Stored at −20 °C

Residual activity (%)

100 97 139 76

Lyophilized

(a) According to temperature and time of storage. Residual enzyme reacti6ity at a gi6en time point is expressed as a percentage of the acti6ity obtained from the starting material. rGAD67 /65 was stored in the presence of 30% (w/6) glycerol for all samples except for the −80 °C samples

Table 2 Enzyme activity of rGAD67/65

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4. Discussion The construction of various recombinant molecular forms of GAD65 has simplified the large-scale expression and purification of this molecule. Our rGAD67/65 construct has advantages by reason of removal of the highly hydrophobic N-terminal sequence of 100 residues which adheres tightly to membranes (Christgau et al., 1992; Solimena et al., 1993, 1994; Shi et al., 1994), whilst the major functional domain of the protein, and capacity to react with specific antibodies are retained (Teoh et al., 1998). However, detailed enzymatic studies on recombinant forms of GAD are lacking. Here we show that rGAD67/65 has enzymatic properties mostly comparable with those reported for GAD prepared from human brain (Blindermann et al., 1978); the optimal pH of rGAD67/65 is slightly higher (7.4 vs. 6.8) and the Km for glutamate is identical (1.3 mM). We observed that the enzyme activity of GAD was stimulated by increasing the concentration of inorganic phosphate, in accord with Porter and Martin (1988) who showed that while ATP acts as a strong competitive inhibitor of activation of GAD, inorganic phosphate may stimulate its activation at low concentrations (1– 5 mM) but inhibit it at higher concentrations. However, in contrast to their finding that higher concentrations of phosphate were inhibitory, we found increases in enzyme activity at inorganic phosphate concentrations up to 1 M. It has been suggested that due to the stabilization of an intermediate complex from inactive apoGAD (GAD without PLP cofactor bound) to active holoGAD (GAD with bound PLP cofactor) by a low inorganic phosphate concentration through ionic interactions, and that a high concentration of inorganic phosphate can compete with the phosphate groups of PLP for the binding site on the enzyme (Chen et al., 1998). In other words, the intermediate complex provides a favorable configuration for the formation of a Schiff base, whereby the o-amino group of the lysine at the active site reacts with the aldehyde group of PLP, so leading to a large conformational change by the enzyme, and a resulting increase in stability (Chen et al., 1998). Our results suggest that free

phosphate in solution at concentrations up to 1 M is insufficiently competitive at blocking the binding site on the enzyme for the phosphate groups of the co-factor PLP. In order to investigate whether the binding of PLP is affected by substituting the first 100 amino acids of GAD65 with GAD67, we studied the binding characteristics of PLP to recombinant GAD67/65. Studies of GAD cofactor interactions by Kaufman and colleagues (Kaufman et al., 1991) showed that, in mouse brain extracts, almost all of the GAD67 component, which is distributed throughout neurons, is saturated with PLP and thus present in an active holoenzyme form whereas only about half of the GAD65, which is concentrated mainly in axon terminals, exists as active holoenzyme and half is present as inactive apoenzyme. Hence apoGAD65 may act as a GAD reservoir in the synaptic terminals and be available for regulation by ATP and inorganic phosphate through their ability to influence strongly the association of PLP to GAD, with association rates decreased by ATP and increased by inorganic phosphate (Martin, 1987; Kaufman et al., 1991). Our experimental data suggest that yeast-expressed rGAD67/65 has PLP binding characteristics that are similar to those of GAD65, in that the addition of PLP increases enzyme activity, presumably by converting apoenzyme to holoenzyme. Only limited studies on the stability of GAD have been performed; data on enzyme stability at temperatures below 0 °C are even lacking until now. Studies of enzymatic activities for a number of rat brain enzymes showed that GAD was one of the least stable enzymes among the several studied, including choline acetyltransferase, glycerol-3-phosphate dehydrogenase, glutamine synthetase, lactate dehydrogenase and 2%,3%-cyclic nucleotide phosphohydrolase (Ritchie et al., 1986). We found that when recombinant GAD was stored at 4 °C, the enzyme activity declined to 58% of the control value by 72 h, and when stored at 22 °C, activity declined to 33% of the control value by 72 h. These results are similar to those for GAD from rat brain in which enzyme activity declined to 65% of the control value at 4 °C for 72 h, and to 29% of the control value at

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25 °C for 72 h (Ritchie et al., 1986). When kept at − 20 °C, recombinant GAD67/65 activity was stable for 35 days after which it began to decline to 72% of the control value by 4 months. At − 80 °C, rGAD67/65 retained its activity for at least 4 months. Recombinant GAD67/65 was more stable at −20 °C when lyophilized than when kept in glycerol. After 3 months at − 20 °C, activity of lyophilized rGAD67/65 declined to 76% of control compared with 42% when kept in 30% glycerol. Accordingly we recommend storage of recombinant GAD at − 80 °C without glycerol. Where this is not possible, we recommend lyophilized storage of the enzyme at −20 °C. In conclusion, we have demonstrated that, first, yeast rGAD67/65 is comparable with human brain GAD as an enzyme; second, rGAD67/65 is activated in the presence of concentrations of inorganic phosphate of at least up to 1 M; third, the PLP interaction with rGAD67/65 is similar to that of GAD65; and fourth, rGAD67/65 is stable over a period of 4 months at −80 °C with no detectable loss of activity. These data should be useful for studies that involve comparison of different preparations or types of recombinant GAD in future research or therapeutic applications.

Acknowledgements We thank Professor Paul Zimmet, Dr Mark Myers and Dr Theo Papakonstantinou for their collaborative assistance and helpful discussions. JT is the inaugural holder of the Angelo Alberti Memorial Postgraduate Scholarship for Diabetes Research. We are grateful to the International Diabetes Institute and to Autogen Ltd. for their part support of this work.

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