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CONSTRUCTION OF EXPRESSION SYSTEM FOR HUMAN ₁-ACID GLYCOPROTEIN IN PICHIA PASTORIS AND EVALUATION OF ITS DRUG-BINDING PROPERTIES

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

(Received May 4, 2004; Accepted June 25, 2004)

	Abstract

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Human ₁-acid glycoprotein (hAGP) is a plasma glycoprotein that functions as a major carrier of basic ligands. This is the first report of the recombinant hAGP (rhAGP). In this study, rhAGP was expressed in the methylotropic yeast Pichia pastoris (GS115) using the expression vector, pPIC9, and then purified by anionic exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight of rhAGP was much lower than that of hAGP, because of the difference in glycan chain content. Results of glycopeptidase F digestion suggest that the peptide moiety of rhAGP was the same as that of hAGP. The results of circular dichroism spectra measurement indicated that rhAGP predominantly formed a ß-sheet-rich structure that was the same as that of hAGP and typical of the lipocalin family. From the experiments using AGP-binding drugs (chlorpromazine, warfarin, and progesterone) and quinaldine red as a probe for the binding site, it was indicated that rhAGP also had the same ligand-binding capacity and binding site structure as hAGP. These findings strongly suggest that this recombinant hAGP (rhAGP) is very useful for the exploration of the ligand-binding site and biological function of hAGP.

The main activity of hAGP is the binding of basic drugs and steroid hormones in plasma (Kremer et al., 1988; Baumann et al., 1989; Treuheit et al., 1992). It is very important to evaluate the binding site on hAGP molecules to understand the pharmacokinetics of these drugs. Previous studies have proposed that several amino acid residues were involved in these binding sites (Kremer et al., 1988; Halsall et al., 2000; Kopecky et al., 2003), but their detailed positions are not clear. These residue positions have not been examined using mutants, mainly because of the lack of an established hAGP expression system. Dente et al. (1988) reported the expression of rhAGP in the cultured cell line and in transgenic mice, but there was no evidence of purified rhAGP.

In the present study, we used the methylotropic yeast Pichia pastoris as the expression host (Sreekrishna et al., 1988; Cregg and Higgins, 1995; Romanos, 1995) for construction of the hAGP expression system, because of its ability to grow to very high cell density (Cregg and Higgins, 1995) while producing alcohol oxidase at up to 30% of its total soluble protein when fully induced (Cregg et al., 1989); it also secretes very little of its own native protein, simplifying purification of heterologous secreted protein (Barr et al., 1992). Furthermore, Escherichia coli and mammalian cells may not be suitable for the AGP expression system for the following reasons. 1) Carbohydrate chains of hAGP have an important role in aqueous solubility. However, E. coli can produce only nonglycosylated protein because it lacks an endoplasmic reticulum and Golgi apparatus. 2) In mammalian cells, quantities of recombinant proteins expressed are generally less than in P. pastoris.

	Materials and Methods

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Materials. hAGP, chlorpromazine, and progesterone were purchased from Sigma-Aldrich (St. Louis, MO). Potassium warfarin was donated by Eisai Co. (Tokyo, Japan). Restriction enzymes, E. coli JM109, the DNA ligation kit and the DNA polymerase Premix Taq (EX Taq version), and glycopeptidase F (GPF) were obtained from Takara Biotechnology Co. (Kyoto, Japan). The DNA sequencing kit was obtained from Applied Biosystems (Tokyo, Japan). The Pichia expression kit was purchased from Invitrogen (Carlsbad, CA). DEAE Sephacel, Phenyl Sepharose Fast Flow, and Sephadex G-75 superfine were purchased from Amersham Biosciences Inc. (Piscataway, NJ).

Strain and Plasmid. E. coli JM109 was used as the host strain for constructing hAGP/pPIC9. pPIC9 contains the alcohol oxidase I promoter, His⁺ selectable marker, and prepro--mating factor secretion signal derived from Saccharomyces cerevisiae. hAGP cDNA (AGP-A gene) was a gift from Kyowa Hakko Co. (Tokyo, Japan). P. pastoris (GS115) was selected as the host strain for expression (Cregg et al., 1989).

Medium. E. coli JM109 was grown in Luria broth (Ausubel et al., 1990) containing ampicillin (50 mg/ml). Buffered glycerol-complex medium (BMGY; 1% yeast extract, 2% peptone, 0.1 M potassium phosphate, 1.34% yeast nitrogen base, 4 x 10^-5% biotin, and 1% glycerol; pH 6.0) and buffered methanol-complex medium (BMMY; same as BMGY except that 0.5% methanol was used instead of glycerol) were used for growing P. pastoris and producing rhAGP, respectively. Minimal dextrose (MD) agar (1.34% yeast nitrogen base, 4 x 10^-5% biotin, 1% dextrose) was used for screening of His⁺ transformants, and minimal methanol (MM) agar (same as MD except that 0.5% methanol was used instead of dextrose) was used for methanol utilization (Mut) screening.

Construction of Expression Vector. A 549-base pair DNA fragment encoding hAGP was amplified by PCR using hAGP cDNA (template) and the following oligonucleotide primers: 5' GGACTAGTCTCGAGAAAAGACAGATCCCATTGTGTGCC-3 (5' XhoI) and 5' GCGGAATTCCTAGGATTCCCCCTCCTCCTG-3 (3' EcoRI). The PCR reaction mixture (final volume, 50 µl) contained the following: 50 ng of template, 1 µl of primers (20 pmol), 25 µl of Premix Taq (0.05 unit/µl) containing 4 mM Mg²⁺, and 0.4 mM deoxynucleotide. The mixture was subjected to denaturation at 96°C for 5 min and 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min, using a 9600 DNA Thermal Cycler (PerkinElmer Life and Analytical Sciences, Boston, MA). The PCR product was purified using a QIAGEN kit (QIAGEN, Valencia, CA), and after digestion with XhoI and EcoRI, it was cloned into the XhoI and EcoRI sites of pPIC9, generating hAGP/pPIC9 (Fig. 1). Portions of hAGP/pPIC9 were sequenced using a 5' XhoI primer.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Expression construct for rhAGP in Pichia pastoris (GS115).

Transformation of P. pastoris. P. pastoris GS115 (His^-) was transformed with hAGP/pPIC9 digested with BglII. Approximately 20 µg of linearized plasmid DNA was used for electroporation in 0.2-cm cuvettes, using a Gene Pulser (Bio-Rad, Hercules, CA) at 1.5 kV, 25 µF, and 200 W. Immediately after pulsing, 1 ml of cold 1 M sorbitol was added to the cuvettes. Cells were plated onto MD agar for the selection of His⁺ transformants. To screen for methanol utilization, each colony on the MD plate was first spotted onto MM agar and then onto a new MD plate. After 48 h, Mut^S and Mut⁺ colonies were identified.

Screening for rhAGP. Colonies for selection of GS115/Mut^S and Mut⁺ [hAGP/pPIC9] were inoculated from MD plates to 5 ml of BMMY and incubated for 3 days at 30°C with shaking. Methanol was added every 24 h to a final concentration of 0.5%. Secretion of rhAGP into the culture medium was monitored using 12.5% SDS-PAGE and Coomassie Blue staining. hAGP was used as a reference standard.

Expression of rhAGP. In the growth phase, a Mut^S colony was proliferated in 100 ml of BMGY in a 1-liter flask at 30°C with shaking. In the induction phase, the growth-phase cells were harvested by centrifugation (1500g, 10 min, 20°C), and cell pellets were resuspended in 1 liter of BMMY in a 3-liter flask at 30°C with shaking. The cells were then grown for an additional 96 h. Methanol was added to a final concentration of 0.5% every 24 h to maintain induction.

Purification of rhAGP. The growth medium was separated from the yeast by centrifugation (6000g, 10 min, 4°C), and the secreted rhAGP was isolated from the medium as follows (Fig. 2). The medium was brought to 65% saturation with ammonium sulfate at room temperature. The temperature was then lowered to 4°C, and the pH was adjusted to 4.0. After shaking for 12 h, the precipitated protein was collected by centrifugation (12,000g, 60 min, 4°C) and resuspended in distilled water. Dialysis was performed for 48 h at 4°C against 100 volumes of distilled water, followed by a further 24 h of dialysis against 100 volumes of 10 mM Tris-HCl buffer (pH 7.4). Then, the solution was loaded onto a column of DEAE Sephacel. rhAGP was eluted with a linear gradient of 0 to 1 M NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluted rhAGP was loaded onto a column of Phenyl Sepharose Fast Flow. Finally, rhAGP was purified using Sephadex G-75 superfine.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Flow chart for purification of rhAGP.

CD Measurement. The CD spectra were measured using a Jasco Model J-720 spectropolarimeter (Jasco, Tokyo, Japan) at 25°C. The data were expressed as mean residue ellipticity, []. The protein concentration was 0.5 mg/ml for the far-UV CD measurements in 20 mM sodium phosphate buffer (pH 7.4). Cells with 1-mm and 10-mm lightpaths were used for the far- and near-UV CD measurements, respectively.

Western Blot analysis. rhAGP was subjected to 12.5% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. Immunoreactive protein was detected using polyclonal antibodies against hAGP raised in Japanese white rabbits (Tissot et al., 1990). Primary antibody/antigen complexes were conjugated to horseradish peroxidase and developed using the Bio-Rad HRP substrate.

Deglycosylation of hAGP and rhAGP. The deglycosylation of hAGP and rhAGP was performed using GPF. After denaturing with 1% SDS and 2-mer-captoethanol, 25 mg of denatured AGPs were incubated with 1 mU of GPF at 37°C and pH 8.6 for 12 h. Treated and untreated proteins were analyzed by electrophoresis.

Fluorescence Measurement of Quinaldine Red. Fluorescence of quinaldine red was measured using a Jasco FP-770 fluorometer. AGP was dissolved at 10 µM in appropriate buffers. For the measurements of quinaldine red fluorescence, the excitation wavelength was 495 nm, and emission was monitored from 550 to 650 nm. Quinaldine red concentration was varied between 1 and 30 µM in AGP solution (10 µM), because it was reported that hAGP had a single binding site for quinaldine red, which was also the site for hAGP-binding drugs (Imamura et al., 1993, 1994). Spectra were recorded immediately after mixing.

Ligand Binding Assay. Drug-binding parameters were calculated using the tryptophan fluorescence quenching method (Nishi et al., 2002). We obtained a fluorometric titration curve by plotting the tryptophanyl fluorescence intensity of AGP (excitation = 295 nm) using a Jasco FP-770 fluorometer. The drug concentration was varied between 0.1 and 60 µM in 2 ml of AGP solution (10 µM). The fluorescence intensity was corrected for the change in AGP concentration with the change in volume. Based on the fluorometric titration curve, straight lines were drawn to represent the lowest and highest concentrations of the drug. The intersection point of these lines was used to obtain the number of binding sites and the binding constant, using eqs. 1 to 6:

(1)

(2)

(3)

(4)

(5)

(6)

where [P_t] and [P_f] are the concentrations of total and free protein, respectively, [D_t] and [D_f] are the concentrations of total and free drug, respectively, n is the number of binding sites, and Q_f is the quenching fraction.

	Results

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Construction of pPIC9 Expression Vector. To secrete rhAGP in culture medium, hAGP cDNA was inserted into a multicloning site on pPIC9 expression vector (Fig. 1). Heterologous proteins fused to the downstream signal sequence (-factor sequence) are cleaved in the sequence Glu-Lys-Arg-X by the KEX 2 endopeptidase, which cleaves on the carboxyl side of dibasic residues (Julius et al., 1984). In S. cerevisiae, it has been observed that the Glu-Ala repeats adjacent to the KEX 2 cleavage site are not necessary for cleavage by KEX2 (Brake et al., 1984).

Screening of Secreting Clones. For transformation of hAGP/pPIC9 into P. pastoris, the plasmid was linearized by digestion with BglII. His⁺ transformants (GS115/His⁺) that appeared on the MD plate were spotted onto a MM plate to score for Mut⁺ and Mut^S. For protein concentration analysis and electrophoresis, 20 colonies of Mut⁺ and 10 colonies of Mut^S, respectively, were monitored in 5 ml of BMMY at 30°C for 3 days. Average protein concentration of Mut^S were higher than that of Mut⁺ (Fig. 3). In electrophoresis of Mut^S, bands other than rhAGP were very faint (data not shown). Based on these results, the Mut^S clone that expressed rhAGP most abundantly was selected for large-scale culture.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Screening of secreted colonies (MutS and Mut+).

Physical Characterization. rhAGP was purified using anionic exchange (DEAE Sephacel), hydrophobic interaction (Phenyl Sepharose Fast Flow), and gel filtration (Sephadex G-75 superfine) chromatography. Although results of SDS-PAGE and Western blotting showed a single band, its molecular weight was different from that of hAGP (Fig. 4, A and B). This difference seemed to derive from the difference in molecular weight per glycan chain of each AGP molecule, because it has been reported that most foreign protein secreted from P. pastoris is not subjected to extensive glycosylation (mannosylation). Furthermore, it was observed that bands for rhAGP and hAGP treated with GPF appeared at the same position, indicating that the peptide moiety of rhAGP was identical to that of hAGP (Fig. 4C).

View larger version (23K): [in this window] [in a new window]   FIG. 4. SDS-PAGE and Western blot analysis of hAGP (lane 2) and rhAGP (lane 3). A, SDS-PAGE of hAGP and rhAGP. B, Western blot analysis of hAGP and rhAGP using hAGP polyclonal antibody. C, SDS-PAGE of hAGP and rhAGP treated with GPF. Lane 1 represents a molecular weight marker.

Structural Characteristics. Conformational structure of rhAGP was evaluated by the far- and near-UV CD spectra. The far-UV CD spectrum of rhAGP at pH 7.4 and 25°C had a minimum at 217 nm, consistent with the abundance of ß-sheet structure (Fig. 5A). On the other hand, the near-UV CD spectrum for rhAGP generated the significant decrease of CD intensity without changing the spectrum pattern compared with that of hAGP (Fig. 5B). These results suggested that rhAGP has formed the same secondary structure as that of hAGP, and the difference in type of glycan chain resulted in the minor change of the tertiary structure of rhAGP.

View larger version (12K): [in this window] [in a new window]   FIG. 5. Far-(A) and near-(B) UV CD spectra of hAGP (solid line) and rhAGP (dashed line).

Ligand-Binding Characteristics. It is known that hAGP has the binding sites for acidic and basic ligands and steroid hormones, respectively, and these sites overlap each other (Maruyama et al., 1990). To investigate the binding capacity of rhAGP to three drugs, chlorpromazine, warfarin, and progesterone, the values of n, the number of binding site, and K_a, association constant, were calculated using the tryptophan fluorescence quenching method (Nishi et al., 2002). Figure 6 shows the titration curves of tryptophanyl fluorescence intensity using chlorpromazine as a typical example. Significant differences in drug-binding properties of these three drugs between hAGP and rhAGP were not observed (Table 1). This result indicated that a ligand-binding capacity of rhAGP was similar or equivalent to that of hAGP.

View larger version (14K): [in this window] [in a new window]   FIG. 6. Quenching of tryptophanyl fluorescence of AGP by chlorpromazine. Symbols represent hAGP (filled circles) and rhAGP (open circles), respectively.

It has recently been shown in our laboratories that quinaldine red binds strongly and selectively to hAGP and then emits the fluorescence (Imamura et al., 1993, 1994). Therefore, to obtain preliminary information on the binding site on rhAGP, we examined the effect of chlorpromazine, warfarin, and progesterone on the fluorescence of quinaldine red bound to hAGP and rhAGP (Fig. 7). As shown in Fig. 7, in both hAGP and rhAGP, all drugs caused significant decreases in the fluorescence of quinaldine red in the order chlorpromazine > warfarin > progesterone. These results indicated that rhAGP had almost the same drug-binding site structure as that of hAGP.

View larger version (12K): [in this window] [in a new window]   FIG. 7. Displacement of quinaldine red by AGP-binding drug. The experiments were performed at a quinaldine red concentration of 10 µM, and hAGP (A) and rhAGP (B) concentration of 10 µM. Drugs used in this experiment were as follows: progesterone (squares), warfarin (triangles), and chlorpromazine (circles).

	Discussion

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hAGP is a plasma protein that contains 183 amino acid residues, five glycan chains, and a largely ß-sheet structure in aqueous solution (Halsall et al., 2000). This structural pattern is common to proteins in the lipocalin family (Flower et al., 2000), but detailed structure and biological functions remain obscure.

Of the numerous activities of hAGP that have been described, the most common is binding of ligands, particularly therapeutic drugs. For example, hAGP binds thalidomide and thereby affects the drug's immunomodulatory activity against tumor necrosis factor- (Turk et al., 1996). hAGP is reportedly involved in intracellular events, such as controlling thrombocytic agglutinability, controlling bacterial englobement, extension during engrafting, and inhibiting lymphocyte growth (Kremer et al., 1988; Baumann et al., 1989; Turk et al., 1996). Although many functions of hAGP are reported, it is not known which part of the hAGP molecule is involved in each function. One of the major reasons for this is the lack of an established rhAGP expression system.

In this study, we constructed the first hAGP expression system that uses the yeast Pichia pastoris. Two phenotypes, Mut^S and Mut⁺, were observed in the screening process after linearized hAGP/pPIC9 digested with BglII was inserted into P. pastoris (GS115) by a single crossover event. From the level and pattern of rhAGP expression, Mut^S strains were selected, although it has been reported that Mut⁺ strains of P. pastoris produce high levels of biomass in fermentation and, for S. cerevisiae, secretion is localized to the growing bud tip (Schekman and Novick, 1982; Digan et al., 1989).

The prepro--mating factor leader sequence derived from S. cerevisiae allows effective secretion and processing of rhAGP in P. pastoris. Cleavage of the leader is apparently mediated via KEX 2 activity in P. pastoris. In the secretory pathway of yeast, the signal peptide is removed by a peptidase (Blobel, 1977), and folding occurs in the endoplasmic reticulum with the assistance of accessory proteins including disulfide isomerase (Freedman, 1989). P. pastoris seems to have an advantage in the glycosylation of secreted proteins in that it does not appear to hyperglycosylate proteins, unlike S. cerevisiae. Both yeasts mainly produce N-linked glycosylation of the high-mannose type. However, the length of the oligosaccharide chains added post-translationally to proteins is much shorter in P. pastoris (average, 8-14 mannose residues per side chain) than in S. cerevisiae (50-150 mannose residues) (Grinna and Tschopp, 1989), and very little O-linked glycosylation has been observed in P. pastoris.

In the experiments for the drug-binding function, the order in the degree of the quenching of tryptophanyl fluorescence (data not shown) and displacement of quinaldine red for hAGP and rhAGP was as follows: chlorpromazine > warfarin > progesterone. This could be due to a slight difference in the binding region of the drugs, since rhAGP has almost the same binding capacity and number of binding sites as that of hAGP (Table 1). The binding region of chlorpromazine may be in the neighborhood of tryptophan residues and may overlap with that of quinaldine red to an extent greater than that between quinaldine red and progesterone. Since quinaldine red and chlorpromazine belong to the basic drug group, they may interact with hAGP and rhAGP at the basic drug-binding region.

Whereas there are some reports that oligosaccharide chain and sialic acid influence the structure of hAGP (Sebankova et al., 1999) and the binding of some drugs to hAGP (Friedman et al., 1986; Shiono et al., 1997), our data indicate that the types of glycan chain attached to hAGP and rhAGP do not greatly affect its ligand-binding properties, despite minor changes of tertiary structure. It is known that some biological functions of hAGP were strongly linked to glycoform, including sialic acid (Sialyl Lewis^x) (Fournier et al., 2000), and others to peptide moiety (Boutten et al., 1992; Van Molle et al., 1997). Therefore, rhAGP, studied here, is fully used for drug binding studies, but it may or may not be used for the investigation of the biological functions.

The present results indicate that rhAGP produced in P. pastoris is very useful for evaluation of structural and functional properties of hAGP. Therefore, mutants prepared using this expression system may bring a lot of information about structural and drug-binding properties. However, in most individuals, hAGP exists as a mixture of two or three genetic variants: A variant and the mixture of F1/S variant, and more than 20 substitutions of amino acid residues between these variants were found (Yuasa et al., 1987; Eap and Baumann, 1989). It has been reported that there are differences in the binding capacity of some ligands between these hAGP variants (Herve et al., 1998), although the structure of these variants is almost the same in physiological condition (Kuroda et al., 2003). In the present study, we used F1/S variant coded by AGP-A gene, not A variant coded by AGP-B/B', for expression of hAGP because, in blood, F1/S variant comprises about 70% of whole AGP (Yuasa et al., 1987; Eap and Baumann, 1989). Of course, the expression of A variant should be investigated, and this study is currently underway.

	Footnotes

 
doi:10.1124/dmd.104.000513.

ABBREVIATIONS: AGP, ₁-acid glycoprotein; hAGP, human AGP; rhAGP, recombinant hAGP; CD, circular dichroism; GPF, glycopeptidase F; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; MD, minimal dextrose; MM, minimal methanol; Mut, methanol utilization; Mut⁺, high methanol utilization; Mut^S, slow methanol utilization; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

Address correspondence to: Masaki Otagiri, Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan. E-mail: otagirim{at}gpo.kumamoto-u.ac.jp

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