Production in yeast of {alpha}-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease

Yasunori Chiba2, Hitoshi Sakuraba3, Masaharu Kotani3, Ryoichi Kase3, Kazuo Kobayashi4, Makoto Takeuchi4, Satoshi Ogasawara5, Yutaka Maruyama5, Tasuku Nakajima5, Yuki Takaoka2 and Yoshifumi Jigami1,2

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial {alpha}-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human {alpha}-galactosidase A ({alpha}-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant {alpha}-GalA contained both neutral (Man8GlcNAc2) and acidic ([Man-P]1–2Man8GlcNAc2) sugar chains. Because an efficient incorporation of {alpha}-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the {alpha}-GalA by a newly isolated bacterial {alpha}-mannosidase. Treatment of the {alpha}-GalA with the {alpha}-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated {alpha}-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated {alpha}-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated {alpha}-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.

Key words: lysosomal disease/mannosidase/mannose-6-phosphate/Saccharomyces cerevisiae/therapeutic glycoprotein


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Lysosomal storage diseases are inborn errors of metabolism resulting from the absence of an enzyme responsible for the hydrolysis of a given substance in lysosomes. Researchers have attempted to develop replacement therapy for lysosomal storage diseases; Gaucher disease (Beutler and Grabowski, 2001Go) was first treated successfully by the glucocerebrosidase from human placenta (Barton et al., 1991Go) and from the recombinant enzyme expressed in Chinese hamster ovary (CHO) cells (Grabowski et al., 1993Go).

Fabry disease is an X-linked recessive glycolipid storage disorder caused by a deficient activity of {alpha}-galactosidase A ({alpha}-GalA) that leads to painful neuropathy and renal, cardiovascular, and cerebrovascular dysfunction (Desnick et al., 2001Go); enzyme replacement therapy for this disease has been developed recently. Expression of {alpha}-GalA in CHO cells had been reported (Ioannou et al., 1992Go, 2001), and a supply of the recombinant enzyme to Fabry model mice resulted in reduced ceramide trihexoside (CTH) storage (Ioannou et al., 2001Go). A clinical trial of replacement therapy with this enzyme has been performed (Eng et al., 2001Go). It was also reported that an infusion of a recombinant {alpha}-GalA from human fibroblasts reduced CTH storage in tissues of patients with Fabry disease (Schiffmann et al., 2000Go, 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, 1987Go) and baculovirus (Coppola et al., 1994Go) were used as the alternative hosts to produce the recombinant {alpha}-GalA in early research attempts. More recently, the methylotrophic yeast Pichia pastoris was also used as a host to produce the {alpha}-GalA (Chen et al., 2000Go). The expression level of {alpha}-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 {alpha}-GalA into human cells, at the nonreduced end. Moreover, P. pastoris produces a ß-mannoside linkage in the associated mannan (Vinogradov et al., 2000Go), 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 {alpha}-GalA by a bacterial {alpha}-mannosidase, to produce the recombinant human {alpha}-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.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Construction of yeast strains to produce human {alpha}-GalA
Because S. cerevisiae sometimes produces the antigenic hypermannosylated sugar chains, we earlier developed an S. cerevisiae YS132–8B strain (Chiba et al., 1998Go) that lacked three genes (OCH1, MNN1, and MNN4) responsible for the biosynthesis of the outer chain of yeast mannan. However, because this strain lacked the ability for mannosylphosphorylation, we constructed a new disruptant HPY21 strain from the KK4 background to delete both the OCH1, which encodes initial {alpha}-1,6-mannosyltransferase, and MNN1, which encodes terminal {alpha}-1,3-mannosyltransferase.

The expression cassette for human {alpha}-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 {alpha}-GalA activity. The recombinant protein was also detected by western blot analysis with polyclonal anti-human {alpha}-GalA antibodies (Ishii et al., 1994Go) (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 ({Delta}och1 {Delta}mnn1 {Delta}mnn4) reported previously (Chiba et al., 1998Go).

Sugar chain structures of the recombinant human {alpha}-GalA produced in HPY21G yeast strain
The recombinant human {alpha}-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). {alpha}-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 concanavalin–Sepharose chromatography followed by chromatography on a Mono Q column. The specific activity of the purified {alpha}-GalA is 1.7 x 106 nmol/h/mg protein, which is almost identical with the recombinant {alpha}-GalA (2 x 106 nmol/h/mg protein) from the insect cells (Ishii et al., 1994Go). 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 {alpha}-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 {alpha}-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., 1996Go), the constitutive expression of MNN4 in HPY21G strain may contribute to the increased level of phosphorylation in sugar chains of recombinant {alpha}-GalA.



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ig. 1. Sugar chain analysis of the recombinant human {alpha}-GalA produced by S. cerevisiae HPY21G cells. Two peaks were observed on a gel filtration chromatogram. The first peak (A) contained mainly acidic sugar chains (MP-M8-PA and (MP)2-M8-PA); the second peak (B) contained a neutral sugar chain (M8-PA). The sugar chain structures were identified by the retention time of authentic samples.

 


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Fig. 2. Structures of the sugar chains abbreviated in the text and in Figure 4.

 
Uncovering nonreducing end mannose attached to Man-6-P residue by a new {alpha}-mannosidase
Tong et al. (1989)Go showed that uncovered Man-6-P residue is important for the {alpha}-GalA to exhibit a high affinity to the Man-6-P receptor because a covered phosphate residue does not show any effective binding with the receptor. Because phosphorylated sugars of S. cerevisiae are capped by mannose residues (Ballou, 1990Go), the terminal mannose residues attached to phosphodiester linkage has to be removed to produce uncovered Man-6-P residues. We first screened commercially available mannosidases, because {alpha}-mannosidase from jackbean (Snaith, 1975Go) digested the phosphodiester linkage of dolichyl-mannosyl phosphate (Herscovics et al., 1975Go), suggesting that {alpha}-mannosidase from jackbean may also digest the phosphodiester-1-{alpha}-mannoside linkage of the sugar chains in S. cerevisiae glycoproteins. In fact, the {alpha}-mannosidase digested the phosphodiester linkage of the fluorescence-labeled sugar chain moiety from S. cerevisiae, but it did not digest the phosphodiester linkage of the sugar chains attached to the recombinant {alpha}-GalA (data not shown). We therefore screened other mannosidases, including {alpha}-1,2-mannosidase from Aspergillus saitoi (Ichishima et al., 1981Go; Inoue et al., 1995Go) and {alpha}-1,6-mannosidase from Xanthomonas sp. (Wong-Madden and Landry, 1995Go); however, no effective enzymes were discovered. Recombinant human {alpha}-mannosidase II (Misago et al., 1995Go; Moremen and Robbins, 1991Go) from yeast was not active in cleaving the linkage of phosphodiester-1-{alpha}-mannoside (data not shown).

Finally, we screened a new bacterium that produces an {alpha}-mannosidase from soil by the enrichment culture on baker’s yeast mannan. We cultured 15 isolated strains with liquid medium containing {alpha}-mannan as a sole carbon source. For first screening, we used alcian blue binding assay (Odani et al., 1996Go). 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 {alpha}-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 {alpha}-GalA. The culture supernatants of strains SO-5 and SO-12 seemed to trim down the sugar chains, because the treated {alpha}-GalAs migrated faster than did the nontreated one on gel electrophoresis (Figure 3). Since {alpha}-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)-{alpha}-D-mannoside and p-nitrophenyl-{alpha}-D-mannoside were digested efficiently by SO-5 supernatant, indicating that the {alpha}-mannosidase produced by SO-5 has a broad specificity. We did not try to further purify the {alpha}-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 {alpha}-mannosidases were secreted in the supernatant, we used the crude supernatant of SO-5 for further treatments of the recombinant {alpha}-GalA.



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Fig. 3. Western blot analysis of the recombinant human {alpha}-GalA before (lane 1) and after treatment by the supernatant of SO-5 (lane 2) and SO-12 (lane 3). The recombinant {alpha}-GalA treated with endo-ß-N-acetylglucosaminidase H (endo-H) was shown in lane 4. Noncontinuous (5–20%) SDS–polyacrylamide gel was used.

 
We analyzed the sugar chain structure of {alpha}-GalA treated by SO-5 supernatant (Figure 4). On the amino column, phosphorylated sugar eluted more slowly than did neutral sugar, and the "uncovered" phosphorylated sugar eluted the slowest. Neutral sugar chains flowed through the column under the condition analyzed. Before treatment with SO-5 supernatant, two peaks corresponding to one and two phosphodiesterized sugar chains were observed at 23 min and 35 min, respectively (Figure 4A). After digestion with SO-5 supernatant, retention times of the above two peaks shifted to 48 min and 56 min, corresponding to one and two phosphomonoesterized sugar chains, respectively (Figure 4B). After isolating each peak product, the components were treated by alkaline phosphatase. One phosphomonoesterized sugar (Figure 4C) is shifted to neutral sugar, eluting at 6 min, corresponding to the sugar chain of Man4GlcNAc2-PA (Figure 4D). Two phosphorylated sugars (Figure 4E) were shifted to neutral sugars, eluting at 7 min and 9 min (Figure 4F), corresponding to the length of Man5GlcNAc2-PA and Man6GlcNAc2-PA, respectively. Figure 5 shows the summary of the deduced sugar chain structure.



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Fig. 4. Sugar chain analysis of the recombinant human {alpha}-GalA. Before and after treatment of the {alpha}-GalA with the supernatant of SO-5, the sugar chain was recovered and labeled as described in Materials and methods. Peaks 1 and 2 indicated by arrows in B were recovered and each peak was treated by alkaline phosphatase. (A) The sugar chain of nontreated {alpha}-GalA; (B) the sugar chain of treated {alpha}-GalA with SO-5; (C) peak 1 fraction before treatment with alkaline phosphatase; (D) peak 1 fraction after treatment with alkaline phosphatase; (E) peak 2 fraction before treatment with alkaline phosphatase; and (F) peak 2 fraction after treatment with alkaline phosphatase.

 


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Fig. 5. Deduced structure of the sugar chain of the recombinant {alpha}-GalA after treatment by SO-5 supernatant.

 
After treatment with SO-5 supernatant, the recombinant {alpha}-GalA was purified by Mono Q column chromatography and its purity was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). This active fraction included the {alpha}-GalA that contained both neutral and acidic N-linked sugar chains. The specific activity was not changed after treatment with SO-5 supernatant (data not shown).

Uptake of the recombinant {alpha}-GalA by Fabry fibroblasts
We investigated the uptake of the recombinant {alpha}-GalA by Fabry fibroblasts in culture (Figure 6) and found that after 18 h incubation both treated and nontreated {alpha}-GalAs were incorporated into the cells. However, the incorporated enzyme activity was three to four times higher in the treated {alpha}-GalA than that in the nontreated one. The deficient enzyme activity in Fabry cells increased in response to the addition of the treated {alpha}-GalA and reached a normal level (about 15–35 nmol/h/mg protein) at a concentration of 1 µg/ml in the culture medium. The uptake of the treated {alpha}-GalA was apparently inhibited by the addition of 5 mM Man-6-P, suggesting that the uptake of the treated {alpha}-GalA may largely depend on the Man-6-P receptor. The time course of {alpha}-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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Fig. 6. Dose-dependency (A) and time course (B) of uptake of the recombinant human {alpha}-GalA by Fabry fibroblasts. (A) Fabry fibroblasts were cultured in culture medium containing recombinant {alpha}-GalA at the concentration of 0.08, 0.57, 1.25, and 7.7 µg/ml. Man-6-P was added into the culture medium at the concentration of 5 mM when necessary. We measured intracellular enzyme activities after 18 h incubation. Closed circles: Treated {alpha}-GalA with SO-5 supernatant was added; open circles: treated {alpha}-GalA with SO-5 supernatant was added in the presence of Man-6-P; closed squares: untreated {alpha}-GalA was added; open squares: untreated {alpha}-GalA was added in the presence of Man-6-P. The original {alpha}-GalA activity in Fabry cells was <1 nmol/h/mg protein, and the average enzyme activity in normal control cells was 15–35 nmol/h/mg protein. The average of three experiments was shown. (B) Fabry fibroblasts were cultured in culture medium containing recombinant {alpha}-GalA , and cells were harvested after 1, 3, 6, and 18 h incubation. The value of incorporated activity after 18 h is indicated as 100%.

 
We also investigated the effect of the incorporated {alpha}-GalA on the degradation of CTH accumulated in Fabry fibroblasts (Figure 7). CTH accumulated in the Fabry fibroblasts was immunostained granularly with anti-CTH monoclonal antibody (Kotani et al., 1994Go), whereas CTH was hardly detected in fibroblast cells from normal control subjects. Double staining with anti-CTH antibody and polyclonal antibodies against lysosome-associated membrane protein-2 revealed that accumulated CTH was localized in lysosomes (data not shown). After the cells were treated with {alpha}-GalA for 18 h, it is likely that the incorporated {alpha}-GalA colocalized with CTH (Figure 7). We confirmed that the incorporated {alpha}-GalA degraded CTH accumulated in the Fabry cells after 5 days of culturing (Figure 7). This was further confirmed by another cell strain sample from an unrelated Fabry family (data not shown).



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Fig. 7. Immunostaining for CTH and {alpha}-GalA in Fabry fibroblasts after uptake of the recombinant human {alpha}-GalA treated with SO-5 supernatant. The cells were cultured for 18 h and 5 days in culture medium including the treated {alpha}-GalA protein (1 µg/ml culture). CTH (upper), stained with an anti-CTH monoclonal antibody (red); {alpha}-GalA (middle), stained with polyclonal anti-{alpha}-GalA antibodies (green); CTH/{alpha}-GalA/Phase contrast (bottom), merged (yellow) with a phase contrast figure.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
To produce the therapeutically effective glycoproteins for lysosomal diseases, the host strain should attach nonantigenic and highly phosphorylated sugar chains to proteins. The extent of mannosylphosphorylation in S. cerevisiae is controlled by the MNN4 gene (Odani et al., 1996Go). MNN4 transcript increases during the stationary phase or under osmotic stress conditions, and the level of mannosylphosphorylation correlates with the level of transcription of MNN4 (Odani et al., 1997Go). The MNN4 gene may function as a positive regulator of mannosylphosphate transferase (Wang et al., 1997Go) (Mnn6 protein). We checked the extent of mannosylphosphorylation in several yeast strains by alcian blue staining (Friis and Ottolenghi, 1970Go) and found that the KK4 strain contained a higher amount of mannosylphosphorylated mannan than did the other strains tested. The promoter region of the MNN4 gene of S. cerevisiae KK4 strain had a mutation, and the Mnn4 protein was produced constitutively in the KK4 strain (Odani, unpublished data). For that reason, we selected the KK4 strain as a candidate for the production of {alpha}-GalA.

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., 1989Go). 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 {alpha}-GalA secreted by HPY21G cells is almost 1:1:1, the {alpha}-GalA seemed to be incorporated effectively into the cell by CI-MPR.

Although we used a low concentration of {alpha}-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 {alpha}-GalA was also tried in methylotrophic yeast P. pastoris (Chen et al., 2000Go). However, they did not treat the recombinant {alpha}-GalA with any {alpha}-mannosidases, and the efficiency of the {alpha}-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 {alpha}-1,3-mannoside at the nonreduced end (Trimble et al., 1991Go). In addition, P. pastoris cells produce ß-mannoside linkage in their mannan (Vinogradov et al., 2000Go), 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., 2000Go; Eng et al., 2001Go). Both of them could decrease plasma and tissue CTH level in patients with Fabry disease, and infusions were well tolerated. Enzyme replacement with {alpha}-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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Yeast strain and culture conditions
S. cerevisiae HPY21 strain (MAT{alpha} och1::LEU2 mnn1::hisG ura3 his1 or his3 trp1 leu2) was constructed from KK4 (Nogi et al., 1984Go) by standard genetic methods. All strains were transformed by the method of Ito et al. (1983)Go. YPAD (2% peptone, 1% yeast extract, 2% glucose, 40 µg/ml adenine sulfate) containing 0.3 M KCl was used for the culture to produce the recombinant {alpha}-GalA. HPY21G was cultured at 30°C for 84 h, and the supernatant was used for the purification and characterization of the recombinant {alpha}-GalA.

DNA constructs
To prepare the human {alpha}-GalA expression vector, we constructed pRS4-GAP-{alpha}-GalA. An open reading frame encoding human {alpha}-GalA was cut with EcoRI from pCXN2Gal (Ishii et al., 1993Go) and was inserted into the EcoRI site of pKT10 vector (Kainuma et al., 1999Go; Tanaka et al., 1990Go). The TDH3 promoter, the open reading frame of {alpha}-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 {alpha}-GalA activity with 4-MU-{alpha}-galactoside as a substrate in the presence of 100 mM N-acetylgalactosamine (Mayes et al., 1981Go). Protein was measured by the method of Bradford (1976)Go. Enzyme activity is expressed as nmol of substrate hydrolyzed in 1 h per mg protein. To measure {alpha}-mannosidase activity, we incubated the reaction mixture (60 µl), containing 5 mM 4-MU-{alpha}-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-{alpha}-mannoside per min at 37°C and pH 7.0. p-Nitrophenyl-{alpha}- or ß-mannoside was used for the substrate specificity analysis of SO-5. The reaction mixture (60 µl), containing 5 mM p-nitrophenyl-{alpha}- 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 {alpha}-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, {alpha}-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, {alpha}-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 A–Sepharose column under conditions reported previously (Ishii et al., 1994Go). 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, {alpha}-GalA was eluted with 0–0.2 M NaCl gradient in the same buffer. The active fraction was recovered, and the purity was estimated by SDS–PAGE.

Sugar chain digestion of {alpha}-GalA by SO-5 supernatant
SO-5 was cultured with mannan medium (2 g {alpha}-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 {alpha}-mannosidase activity in the supernatant was determined. Purified {alpha}-GalA (~100 µg) was incubated at 37°C for 18 h with SO-5 supernatant in which 100 U {alpha}-mannosidase was present. After treatment, the recombinant {alpha}-GalA was purified by Mono Q column chromatography as already described.

Sugar chain analysis
The sugar chains of {alpha}-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., 1998Go). Phosphorylated sugar standards were prepared as described by Wang et al. (1997)Go. Alkaline phosphatase digestion was performed as described elsewhere (Nakayama et al., 1998Go). 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 Ham’s F-10 medium supplemented with 10% fetal calf serum and antibiotics at 37°C in a humidified incubator flushed continuously with 5%CO2–95% air mixture.

Uptake of {alpha}-GalA by Fabry fibroblasts
Fabry fibroblasts were cultured in culture medium containing the desired concentration of the treated or untreated {alpha}-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 {alpha}-GalA assay.

Immunocytochemical analysis of CTH and {alpha}-GalA
To examine the distribution of incorporated {alpha}-GalA and cleavage of the intracellularly accumulated CTH in Fabry cells, we performed immunocytochemical analyses by double staining with rabbit polyclonal anti-{alpha}-GalA antibodies (Ishii et al., 1994Go) (IgG isotype) and a mouse monoclonal anti-CTH antibody (Kotani et al., 1994Go) (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-{alpha}-GalA and anti-CTH antibodies. After washing, they were reacted for 1 h with a fluorescein isothiocyanate–conjugated 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).


    Acknowledgment
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
This work was partly supported by grants from the Tokyo Metropolitan Government; the Japan Society for the Promotion of Science; the Ministry of Health, Labor and Welfare of Japan; and the New Energy and Industrial Technology Development Organization (NEDO) as a part of the Research and Development Projects of Industrial Science and Technology Frontier Program, Japan.


    Abbreviations
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
{alpha}-GalA, {alpha}-galactosidase A; CHO, Chinese hamster ovary; CI-MPR, cation-independent mannose-6-phosphate receptor; CTH, ceramide trihexoside; Man-6-P, mannose-6-phosphate; MES, 2-[N-morpholino]ethanesulfonic acid; MU, 4-methylumbelliferyl; PA, 2-aminopyridine; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: jigami.yoshi@aist.go.jp Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Ballou, C.E. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol., 185, 440–470.[Medline]

Barton, N.W., Brady, R.O., Dambrosia, J.M., Di Bisceglie, A.M., Doppelt, S.H., Hill, S.C., Mankin, H.J., Murray, G.J., Parker, R.I., Argoff, C.E., and others. (1991) Replacement therapy for inherited enzyme deficiency–macrophage-targeted glucocerebrosidase for Gaucher’s disease. N. Engl. J. Med., 324, 1464–1470.[Abstract]

Beutler, E. and Grabowski, G.A. (2001) Gaucher disease. In Valle, D. (ed), The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, pp. 3635–3668.

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254.[CrossRef][ISI][Medline]

Chen, Y., Jin, M., Egborge, T., Coppola, G., Andre, J., and Calhoun, D.H. (2000) Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris. Prot. Expr. Purif., 20, 472–484.[CrossRef][ISI][Medline]

Chiba, Y., Suzuki, M., Yoshida, S., Yoshida, A., Ikenaga, H., Takeuchi, M., Jigami, Y., and Ichishima, E. (1998) Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae. J. Biol. Chem., 273, 26298–26304.[Abstract/Free Full Text]

Coppola, G., Yan, Y., Hantzopoulos, P., Segura, E., Stroh, J.G., and Calhoun, D.H. (1994) Characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A using a baculovirus vector. Gene, 144, 197–203.[CrossRef][ISI][Medline]

Desnick, R.J., Ioannou, Y.A., and Eng, C.M. (2001) Fabry disease. In Valle, D. (ed), The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, pp. 3733–3774.

Eng, C.M., Banikazemi, M., Gordon, R.E., Goldman, M., Phelps, R., Kim, L., Gass, A., Winston, J., Dikman, S., Fallon, J.T., and others. (2001) A phase 1/2 clinical trial of enzyme replacement in Fabry disease: pharmacokinetic, substrate clearance, and safety studies. Am. J. Hum. Genet., 68, 711–722.[CrossRef][ISI][Medline]

Friis, J. and Ottolenghi, P. (1970) The genetically determined binding of alcian blue by a minor fraction of yeast cell walls. C. R. Trav Lab. Carlsberg, 37, 327–341.[ISI][Medline]

Grabowski, G.A., Pastores, G., Brady, R.O., and Barton, N.W. (1993) Safety and efficacy of macrophage targeted recombinant glucocerebrosidase therapy. Pediatr. Res., 33, 139A.

Hantzopoulos, P.A. and Calhoun, D.H. (1987) Expression of the human alpha-galactosidase A in Escherichia coli K-12. Gene, 57, 159–169.[CrossRef][ISI][Medline]

Herscovics, A., Warren, C.D., and Jeanloz, R.W. (1975) Anomeric configuration of the dolichyl D-mannosyl phosphate formed in calf pancreas microsomes. J. Biol. Chem., 250, 8079–8084.[Abstract]

Ichishima, E., Arai, M., Shigematsu, Y., Kumagai, H., and Sumida, T.R. (1981) Purification of an acidic alpha-D-mannosidase from Aspergillus saitoi and specific cleavage of 1, 2-alpha-D-mannosidic linkage in yeast mannan. Biochim. Biophys. Acta, 658, 45–53.[ISI][Medline]

Inoue, T., Yoshida, T., and Ichishima, E. (1995) Molecular cloning and nucleotide sequence of the 1, 2-alpha-D-mannosidase gene, msdS, from Aspergillus saitoi and expression of the gene in yeast cells. Biochim. Biophys. Acta, 1253, 141–145.[ISI][Medline]

Ioannou, Y.A., Bishop, D.F., and Desnick, R.J. (1992) Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion. J. Cell Biol., 119, 1137–1150.[Abstract]

Ioannou, Y.A., Zeidner, K.M., Gordon, R.E., and Desnick, R.J. (2001) Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice. Am. J. Hum. Genet., 68, 14–25.[CrossRef][ISI][Medline]

Ishii, S., Kase, R., Sakuraba, H., and Suzuki, Y. (1993) Characterization of a mutant alpha-galactosidase gene product for the late-onset cardiac form of Fabry disease. Biochem. Biophys. Res. Commun., 197, 1585–1589.[CrossRef][ISI][Medline]

Ishii, S., Kase, R., Sakuraba, H., Fujita, S., Sugimoto, M., Tomita, K., Semba, T., and Suzuki, Y. (1994) Human alpha-galactosidase gene expression: significance of two peptide regions encoded by exons 1–2 and 6. Biochim. Biophys. Acta, 1204, 265–270.[ISI][Medline]

Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol., 153, 163–168.[ISI][Medline]

Kainuma, M., Ishida, N., Yoko-o, T., Yoshioka, S., Takeuchi, M., Kawakita, M., and Jigami, Y. (1999) Coexpression of alpha1, 2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae. Glycobiology, 9, 133–141.[Abstract/Free Full Text]

Kotani, M., Kawashima, I., Ozawa, H., Ogura, K., Ariga, T., and Tai, T. (1994) Generation of one set of murine monoclonal antibodies specific for globo-series glycolipids: evidence for differential distribution of the glycolipids in rat small intestine. Arch. Biochem. Biophys., 310, 89–96.[CrossRef][ISI][Medline]

Mayes, J.S., Scheerer, J.B., Sifers, R.N., and Donaldson, M.L. (1981) Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry’s disease. Clin. Chim. Acta, 112, 247–251.[CrossRef][ISI][Medline]

Misago, M., Liao, Y.F., Kudo, S., Eto, S., Mattei, M.G., Moremen, K.W., and Fukuda, M.N. (1995) Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme. Proc. Natl Acad. Sci. USA, 92, 11766–11770.[Abstract]

Moremen, K.W. and Robbins, P.W. (1991) Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans. J. Cell Biol., 115, 1521–1534.[Abstract]

Nakayama, K., Feng, Y., Tanaka, A., and Jigami, Y. (1998) The involvement of mnn4 and mnn6 mutations in mannosylphophorylation of O-linked oligosaccharide in yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta, 1425, 255–262.[ISI][Medline]

Nogi, Y., Shimada, H., Matsuzaki, Y., Hashimoto, H., and Fukasawa, T. (1984) Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. II. The isolation and dosage effect of the regulatory gene GAL80. Mol. Gen. Genet., 195, 29–34.[ISI][Medline]

Odani, T., Shimma, Y., Tanaka, A., and Jigami, Y. (1996) Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae. Glycobiology, 6, 805–810.[Abstract]

Odani, T., Shimma, Y., Wang, X.H., and Jigami, Y. (1997) Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae. FEBS Lett., 420, 186–190.[CrossRef][ISI][Medline]

Schiffmann, R., Murray, G.J., Treco, D., Daniel, P., Sellos-Moura, M., Myers, M., Quirk, J.M., Zirzow, G.C., Borowski, M., Loveday, K., and others. (2000) Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease. Proc. Natl Acad. Sci. USA, 97, 365–370.[Abstract/Free Full Text]

Schiffmann, R., Kopp, J.B., Austin, H.A., Sabnis, S., Moore, D.F., Weibel, T., Balow, J.E., and Brady, R.O. (2001) Enzyme replacement therapy in Fabry disease. A randomized controlled trial. J. Am. Med. Assoc., 285, 2743–2749.[Abstract/Free Full Text]

Snaith, S.M. (1975) Characterization of jack-bean alpha-D-mannosidase as a zinc metalloenzyme. Biochem. J., 147, 83–90.[ISI][Medline]

Tanaka, K., Nakafuku, M., Tamanoi, F., Kaziro, Y., Matsumoto, K., and Toh-e, A. (1990) IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol. Cell Biol., 10, 4303–4313.[ISI][Medline]

Tong, P.Y., Gregory, W., and Kornfeld, S. (1989) Ligand interactions of the cation-independent mannose 6-phosphate receptor. The stoichiometry of mannose 6-phosphate binding. J. Biol. Chem., 264, 7962–7969.[Abstract/Free Full Text]

Trimble, R.B., Atkinson, P.H., Tschopp, J.F., Townsend, R.R., and Maley, F. (1991) Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris. J. Biol. Chem., 266, 22807–22817.[Abstract/Free Full Text]

Vinogradov, E., Petersen, B.O., and Duus, J.O. (2000) Isolation and characterization of non-labeled and 13C-labeled mannans from Pichia pastoris yeast. Carbohydr. Res., 325, 216–221.[CrossRef][ISI][Medline]

Wang, X.H., Nakayama, K., Shimma, Y., Tanaka, A., and Jigami, Y. (1997) MNN6, a member of the KRE2/MNT1 family, is the gene for mannosylphosphate transfer in Saccharomyces cerevisiae. J. Biol. Chem., 272, 18117–18124.[Abstract/Free Full Text]

Wong-Madden, S.T. and Landry, D. (1995) Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas. Glycobiology, 5, 19–28.[Abstract]





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