2 Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan; 3 Department of Clinical Genetics, Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo 113-8613, Japan; 4 Central Laboratory, Kirin Brewery Co., Ltd., Yokohama 236-0004, Japan; and 5 Division of Applied Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Received on May 16, 2002; revised on August 7, 2002; accepted on August 13, 2002
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Abstract |
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Key words: lysosomal disease/mannosidase/mannose-6-phosphate/Saccharomyces cerevisiae/therapeutic glycoprotein
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Introduction |
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Fabry disease is an X-linked recessive glycolipid storage disorder caused by a deficient activity of -galactosidase A (
-GalA) that leads to painful neuropathy and renal, cardiovascular, and cerebrovascular dysfunction (Desnick et al., 2001
); enzyme replacement therapy for this disease has been developed recently. Expression of
-GalA in CHO cells had been reported (Ioannou et al., 1992
, 2001), and a supply of the recombinant enzyme to Fabry model mice resulted in reduced ceramide trihexoside (CTH) storage (Ioannou et al., 2001
). A clinical trial of replacement therapy with this enzyme has been performed (Eng et al., 2001
). It was also reported that an infusion of a recombinant
-GalA from human fibroblasts reduced CTH storage in tissues of patients with Fabry disease (Schiffmann et al., 2000
, 2001). Because these recombinant enzymes are produced in mammalian cells, production of the recombinant enzyme is expensive and a careful monitoring for viral infection is essential.
Escherichia coli (Hantzopoulos and Calhoun, 1987) and baculovirus (Coppola et al., 1994
) were used as the alternative hosts to produce the recombinant
-GalA in early research attempts. More recently, the methylotrophic yeast Pichia pastoris was also used as a host to produce the
-GalA (Chen et al., 2000
). The expression level of
-GalA is considerable in these hosts; however, the N-linked sugar chains of these glycoproteins have not been analyzed carefully and may not contain mannose-6-phosphate (Man-6-P) residues, which are essential for the incorporation of
-GalA into human cells, at the nonreduced end. Moreover, P. pastoris produces a ß-mannoside linkage in the associated mannan (Vinogradov et al., 2000
), which is antigenic in humans. In the approach detailed in this article, we used Saccharomyces cerevisiae in which two genes were disrupted in relation to the N-linked mannan biosynthesis, in combination with the in vitro treatment of the recombinant
-GalA by a bacterial
-mannosidase, to produce the recombinant human
-GalA, which shows a three- to fourfold higher uptake into the Fabry fibroblast cells, mainly due to the exposure of Man-6-P residues at the nonreduced end of the sugar chains.
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Results |
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The expression cassette for human -GalA gene under the S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase promoter was introduced into the TRP1 locus of HPY21 strain, and the resulting strain was called HPY21G. Whereas no activity was detected in the culture supernatant from HPY21 host strain, the supernatant from HPY21G transformant showed
-GalA activity. The recombinant protein was also detected by western blot analysis with polyclonal anti-human
-GalA antibodies (Ishii et al., 1994
) (data not shown). The amount of the recombinant protein was about 100 µg in 1-L culture, which is the same level as that of the triple disruptant cells (
och1
mnn1
mnn4) reported previously (Chiba et al., 1998
).
Sugar chain structures of the recombinant human -GalA produced in HPY21G yeast strain
The recombinant human -GalA was purified as described in Materials and methods. Culture filtrate was concentrated by ultrafiltration and dialyzed sufficiently against 150 mM sodium phosphate buffer (pH 4.6).
-GalA activity was recovered by 2-[N-morpholino]ethanesulfonic acid (MES) buffer (pH 6.0) containing 4 M NaCl via Blue-Sepharose chromatography. The active fraction was further purified by concanavalinSepharose chromatography followed by chromatography on a Mono Q column. The specific activity of the purified
-GalA is 1.7 x 106 nmol/h/mg protein, which is almost identical with the recombinant
-GalA (2 x 106 nmol/h/mg protein) from the insect cells (Ishii et al., 1994
). The purified proteins were used for the structural analysis of sugar chains. Each sugar chain was labeled with 2-aminopyridine (PA) and analyzed by amino column chromatography (Figure 1) after the removal of excess PA by gel filtration. The
-GalA contained not only neutral-type (nonphosphorylated) (Figure 1B) but also one- and two-phosphorylated acidic-type sugar chains (Figure 1A, also see Figure 2). The ratio of the nonphosphorylated, one-phosphorylated, and two-phosphorylated sugars of
-GalA secreted by HPY21G cells is almost 1:1:1. Because the ratio of neutral oligosaccharides to two acidic ones in mannoproteins from YS125-15B strain, which lacks OCH1, MNN1, and KRE2 genes, was reported to be 1:1.3:0.3 (Odani et al., 1996
), the constitutive expression of MNN4 in HPY21G strain may contribute to the increased level of phosphorylation in sugar chains of recombinant
-GalA.
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Finally, we screened a new bacterium that produces an -mannosidase from soil by the enrichment culture on bakers yeast mannan. We cultured 15 isolated strains with liquid medium containing
-mannan as a sole carbon source. For first screening, we used alcian blue binding assay (Odani et al., 1996
). After yeast cells were treated by each bacterial culture supernatant, the cells were recovered, washed, and stained with alcian-blue solution. Because the alcian blue binds to negatively charged cell wall, the higher staining of cells might be an indication of uncovering of phosphate residue by the treatment with
-mannosidase. Because two strains (SO-5 and SO-12) were selected as candidates for the uncovering enzyme producer, we examined the culture supernatants ability to digest the sugar chains of the recombinant
-GalA. The culture supernatants of strains SO-5 and SO-12 seemed to trim down the sugar chains, because the treated
-GalAs migrated faster than did the nontreated one on gel electrophoresis (Figure 3). Since
-GalA treated by SO-12 supernatant showed the same apparent molecular mass as that digested by endo-ß-N-acetylglucosaminidase H (Figure 3, lane 3 and 4), it was likely that the shift of mobility was due to the endo-glycosidase produced by SO-12 strain. In contrast, both 4-methylumbelliferyl (MU)-
-D-mannoside and p-nitrophenyl-
-D-mannoside were digested efficiently by SO-5 supernatant, indicating that the
-mannosidase produced by SO-5 has a broad specificity. We did not try to further purify the
-mannosidase because the supernatant of SO-5 did not contain any significant activities of either ß-mannosidase or endo-ß-N-acetylglucosaminidase. Although it is unclear how many
-mannosidases were secreted in the supernatant, we used the crude supernatant of SO-5 for further treatments of the recombinant
-GalA.
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Uptake of the recombinant -GalA by Fabry fibroblasts
We investigated the uptake of the recombinant -GalA by Fabry fibroblasts in culture (Figure 6) and found that after 18 h incubation both treated and nontreated
-GalAs were incorporated into the cells. However, the incorporated enzyme activity was three to four times higher in the treated
-GalA than that in the nontreated one. The deficient enzyme activity in Fabry cells increased in response to the addition of the treated
-GalA and reached a normal level (about 1535 nmol/h/mg protein) at a concentration of 1 µg/ml in the culture medium. The uptake of the treated
-GalA was apparently inhibited by the addition of 5 mM Man-6-P, suggesting that the uptake of the treated
-GalA may largely depend on the Man-6-P receptor. The time course of
-GalA incorporation into Fabry cells is shown in Figure 6B. The enzyme was rapidly incorporated into the cells, indicating the 33% of total activity within 1 h incubation and the highest and constant activity at 6 h incubation.
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Discussion |
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The relationship between the number of Man-6-P residues and affinity to cation-independent Man-6-P receptor (CI-MPR) has been reported (Tong et al., 1989). The dissociation constant for a high-mannose oligosaccharide containing two Man-6-P residues was 300 times lower than that for pentamannosyl phosphate, suggesting that the uptake of the glycoproteins into the cell depends on the contents of Man-6-P residues in a sugar chain. Because the ratio of the nonphosphorylated, one-phosphorylated, and two-phosphorylated sugars of
-GalA secreted by HPY21G cells is almost 1:1:1, the
-GalA seemed to be incorporated effectively into the cell by CI-MPR.
Although we used a low concentration of -GalA enzyme (1 µg/ml in culture), the enzyme almost completely degraded the CTH that was accumulated in the Fabry cells. A production of human
-GalA was also tried in methylotrophic yeast P. pastoris (Chen et al., 2000
). However, they did not treat the recombinant
-GalA with any
-mannosidases, and the efficiency of the
-GalA incorporation into human fibroblasts was low. Although the sugar chain structure of P. pastoris is almost identical to that of S. cerevisiae, P. pastoris contains no terminal
-1,3-mannoside at the nonreduced end (Trimble et al., 1991
). In addition, P. pastoris cells produce ß-mannoside linkage in their mannan (Vinogradov et al., 2000
), which is antigenic in humans. In that case, a disruption of the gene encoding ß-mannosyltransferase will be necessary to produce therapeutic glycoproteins in P. pastoris.
Although enzyme replacement therapy may not be applicable to diseases affecting the central nervous system due to the presence of blood-brain barrier, this therapy will be effective for many lysosomal diseases without brain defects by selecting the desired genes transformed into S. cerevisiae. The sugar chain of the products is humanized and nonantigenic, and the products could be free from contamination of infectious agents between mammals. A large-scale cultivation of S. cerevisiae cells to serve as therapeutic glycoproteins would have the great advantage of being much less costly than the cultivation of mammalian cells. Two recombinant enzymes have been applied to clinical trial; one is agalsidase beta (Fabrazyme), produced by CHO cells, and the other is agalsidase alpha (Replagal), produced by human fibroblast cells (Schiffmann et al., 2000; Eng et al., 2001
). Both of them could decrease plasma and tissue CTH level in patients with Fabry disease, and infusions were well tolerated. Enzyme replacement with
-GalA is likely to improve the prognosis of Fabry disease, although the uptake of the enzyme into glomerular epithelium and endomyocardium should be improved. Furthermore, efforts should be directed to producing the enzyme more economically, because enzyme replacement therapy requires a large amount of enzyme to fill the constant and continuous need by patients. Our production technology using yeast will be useful for providing such enzymes more affordably than the current technology.
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Materials and methods |
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DNA constructs
To prepare the human -GalA expression vector, we constructed pRS4-GAP-
-GalA. An open reading frame encoding human
-GalA was cut with EcoRI from pCXN2Gal (Ishii et al., 1993
) and was inserted into the EcoRI site of pKT10 vector (Kainuma et al., 1999
; Tanaka et al., 1990
). The TDH3 promoter, the open reading frame of
-GalA and the TDH terminator were extracted by BamHI and inserted into the BamHI site of integration vector pRS404 (BD Biosciences Clontech, Palo Alto, CA). The vector was cut with Bst1107I and used for yeast transformation, which integrated the expression cassette into the TRP1 locus of the genome of S. cerevisiae. The correct integration was confirmed by PCR.
Enzyme assays
We assayed -GalA activity with 4-MU-
-galactoside as a substrate in the presence of 100 mM N-acetylgalactosamine (Mayes et al., 1981
). Protein was measured by the method of Bradford (1976)
. Enzyme activity is expressed as nmol of substrate hydrolyzed in 1 h per mg protein. To measure
-mannosidase activity, we incubated the reaction mixture (60 µl), containing 5 mM 4-MU-
-mannoside in 0.1 M potassium phosphate buffer (pH 7.0), at 37°C for 30 min. The reaction was stopped with 0.7 ml 0.2 M glycine buffer (pH 10.7), and we measured free 4-MU using a fluorometer (excitation: 375 nm, emission: 450 nm). One unit of the enzyme was defined as the amount of the enzyme required to liberate 1 µmol of 4-MU from 4-MU-
-mannoside per min at 37°C and pH 7.0. p-Nitrophenyl-
- or ß-mannoside was used for the substrate specificity analysis of SO-5. The reaction mixture (60 µl), containing 5 mM p-nitrophenyl-
- or ß-mannoside in 0.1 M potassium phosphate buffer (pH 7.0), was incubated at 37°C for 30 min. The reaction was stopped with 0.7 ml 0.2 M glycine buffer (pH 10.7), and the absorbance of free p-nitrophenol was measured at 415 nm using a spectrophotometer.
Purification of -GalA
The following procedures were performed at 4°C, and all column materials were purchased from Amersham Biosciences Corporation (Piscataway, NJ). The culture supernatant of HPY21G was concentrated and desalted by ultrafiltrate module SLP-1053 (Asahi-Chemical, Tokyo, Japan). The sample was dialyzed to 25 mM MES buffer (pH 6.0) and applied to a HiLoad 16/10 Q Sepharose HP column equilibrated with the same buffer. After the column was washed, -GalA was eluted with 0.2 M NaCl in the same buffer. The fractions containing the enzyme activity were pooled, the pH was adjusted to 4.6 by HCl, and then the fractions were applied to a Blue-Sepharose column equilibrated with BLUE buffer (0.15 M sodium acetate buffer, pH 4.6). After the column was washed,
-GalA was eluted with 4 M NaCl in the 0.1 M MES buffer (pH 6.0).
The fractions containing the enzyme activity were pooled and applied to a concanavalin ASepharose column under conditions reported previously (Ishii et al., 1994). The fractions containing the enzyme activity were pooled again and applied to a HiTrap Q column equilibrated with 25 mM MES buffer (pH 6.0). After the column was washed,
-GalA was eluted with 00.2 M NaCl gradient in the same buffer. The active fraction was recovered, and the purity was estimated by SDSPAGE.
Sugar chain digestion of -GalA by SO-5 supernatant
SO-5 was cultured with mannan medium (2 g -mannan, 500 mg (NH4)2SO4, 400 mg MgSO4 · 7H2O, 20 mg Fe2SO4, 60 mg CaCl2 · 2H2O, 1 g yeast extract, 7.54 g K2HPO4, 2.32 g KH2PO4/L) at 37°C for 24 h, and
-mannosidase activity in the supernatant was determined. Purified
-GalA (~100 µg) was incubated at 37°C for 18 h with SO-5 supernatant in which 100 U
-mannosidase was present. After treatment, the recombinant
-GalA was purified by Mono Q column chromatography as already described.
Sugar chain analysis
The sugar chains of -GalA was digested by glycopeptidase F (Roche Diagnostics, Tokyo, Japan). The method of labeling with PA as well as neutral oligosaccharide analysis by high-performance liquid chromatography were done as described elsewhere (Chiba et al., 1998
). Phosphorylated sugar standards were prepared as described by Wang et al. (1997)
. Alkaline phosphatase digestion was performed as described elsewhere (Nakayama et al., 1998
). Separation of phosphorylated sugars on an amino column was done with solvent A (acetonitrile: 40 mM triethanolamine acetate [pH 7.0] = 6:4) and solvent B (acetonitrile : 625 mM triethanolamine acetate [pH 7.0] = 2:8). After injection, the concentration of solvent B was increased from 0% to 100% for 50 min, then the concentration remained at 100% for 10 min. The sugar chain structures corresponding to the main peaks were identified by the retention time of authentic samples.
Cell culture
Cultured skin fibroblasts from two independent Fabry patients from unrelated Fabry families and normal subjects were established and maintained in our laboratory. The cells were cultured in Hams F-10 medium supplemented with 10% fetal calf serum and antibiotics at 37°C in a humidified incubator flushed continuously with 5%CO295% air mixture.
Uptake of -GalA by Fabry fibroblasts
Fabry fibroblasts were cultured in culture medium containing the desired concentration of the treated or untreated -GalA with SO-5 supernatant in the presence or absence of 5 mM Man-6-P. After 18 h culture, the cells were mechanically harvested and sonicated, and the homogenate was used for intracellular
-GalA assay.
Immunocytochemical analysis of CTH and -GalA
To examine the distribution of incorporated -GalA and cleavage of the intracellularly accumulated CTH in Fabry cells, we performed immunocytochemical analyses by double staining with rabbit polyclonal anti-
-GalA antibodies (Ishii et al., 1994
) (IgG isotype) and a mouse monoclonal anti-CTH antibody (Kotani et al., 1994
) (IgG isotype). The cultured Fabry fibroblasts grown on Lab-Tek chamber slides (Nunc; Naperville, IL) were fixed with ice-cold 2% paraformaldehyde in phosphate buffered saline, pH 7.4, for 10 min, followed by blocking with 5% bovine serum albumin in phosphate buffered saline for 1 h. The cells were incubated for 1 h with the anti-
-GalA and anti-CTH antibodies. After washing, they were reacted for 1 h with a fluorescein isothiocyanateconjugated goat anti-rabbit IgG F(ab')2 (1:100 diluted; Jackson ImmunoResearch; West Grove, PA) and a rhodamine-conjugated donkey anti-mouse IgG F(ab')2 (1:100 diluted; Jackson ImmunoResearch). We examined the stained cells and obtained a phase contrast figure under a microscope (Axiovert 100M; Carl Zeiss; Oberkochen, Germany) equipped with a confocal laser scanning imaging system (LSM510; Zeiss).
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Acknowledgment |
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Abbreviations |
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Footnotes |
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References |
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