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J Biol Chem, Vol. 273, Issue 41, 26298-26304, October 9, 1998


Production of Human Compatible High Mannose-type (Man5GlcNAc2) Sugar Chains in Saccharomyces cerevisiae*

Yasunori ChibaDagger , Misa SuzukiDagger , Satoshi YoshidaDagger , Aruto YoshidaDagger , Hiroshi IkenagaDagger , Makoto TakeuchiDagger §, Yoshifumi Jigami, and Eiji Ichishimaparallel

From the Dagger  Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., Yokohama, Kanagawa 236-0004, Japan, the  National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305-0046, Japan, and the parallel  Department of Bioengineering, Faculty of Engineering, Sohka University, Hachiohji, Tokyo 192-0003, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for alpha -1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro alpha -1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an alpha -1,2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man5GlcNAc2. This is the first report of a recombinant S. cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Saccharomyces cerevisiae is useful for the production of recombinant proteins of biological interest because of the established expression system, and it can be easily grown in large quantities. Moreover, yeast share the early steps of the mammalian Asn-linked glycosylation pathway. However, the mature Asn-linked oligosaccharides of yeast are mannan glycans and are highly antigenic against mammals. Thus, it would be necessary to eliminate the antigenicity of the sugar chains when recombinant therapeutic glycoproteins are produced in yeast.

Several genes concerned with the biosynthesis of yeast sugar chains have been cloned, and the glycosylation pathway of yeast has been clarified. The OCH1 gene encodes an alpha -1,6-mannosyltransferase that initiates alpha -1,6-polymannose outer chain formation on the Asn-linked inner oligosaccharide Man8GlcNAc2 in S. cerevisiae (1). MNN1 has been proposed as the structural gene for the alpha -1,3-mannosyltransferase that elongates the outer chain and the inner core oligosaccharide (2, 3). The Delta och1 mnn1 double mutant accumulated a single oligosaccharide moiety, Man8GlcNAc2, a high mannose-type structure (1). This mutant may be useful to produce recombinant therapeutic glycoproteins without any antigenicity toward humans.

On the other hand, some glycoproteins of therapeutic value require complex-type sugar chains for their efficacy. Erythropoietin (EPO),1 a hematopoietic glycoprotein factor produced in the kidney, has three complex-type Asn-linked sugar chains and one mucin-type sugar chain. It is reported that the composition and structure of each sugar chain affected the biological activity, the efficiency of secretion, and had profound effects on the half-life of EPO in the blood circulation (4). It seems that the most active form of the EPO molecule requires tetra-antennary Asn-linked sugar chains (5) with full sialylation, to prevent serum clearance by the action of the hepatic asialoglycoprotein binding protein (6, 7). When EPO was expressed in the Delta och1 mnn1 mutant yeast, the recombinant EPO should have high mannose-type oligosaccharides, which are trapped by the mannan-binding proteins of serum, liver, and macrophages, or excreted in the urine through the kidney because of their small size.

From the viewpoint of glycotechnology, we are trying to construct the mammalian-type glycosylation system in S. cerevisiae as a host to produce glycoprotein therapeutics (Fig. 1). The first aim of this research was to convert the mannan-type sugar chain of S. cerevisiae to a Man5GlcNAc2 sugar chain, because it is an intermediate for hybrid- and complex-type sugar chains. However the Delta och1 mnn1 mutant can only produce the Man8GlcNAc2 structure (1). Further trimming of the mannose residues by alpha -1,2-mannosidase requires alpha -mannosidase I. Several alpha -1,2-mannosidases have been isolated from mammals, yeast, and fungi (8), and some mammalian alpha -1,2-mannosidase genes have been cloned (9, 10). During preparation of the manuscript, it was reported that a truncated soluble form of the human alpha -1,2-mannosidase IB was expressed as a secreted protein in Pichia pastris (11). The S. cerevisiae alpha -1,2-mannosidase gene (MNS1) has been cloned (12) and expressed in S. cerevisiae. However, this enzyme only removes a specific single mannose residue from Man9GlcNAc2 and produces Man8GlcNAc2. The Aspergillus alpha -1,2-mannosidase gene (msdS) has also been cloned and has been expressed successfully in yeast cells as a chimeric gene with the signal sequence of the aspergillopepsin I gene from Aspergillus saitoi (13, 14). The recombinant alpha -1,2-mannosidase activity was secreted into the culture medium, indicating that the products of the msdS gene had passed through the yeast secretion pathway. Therefore, alpha -1,2-mannosidase could be used as a tool to produce the mammalian-type sugar chains in the yeast if this enzyme was retained in the endoplasmic reticulum (ER) or Golgi apparatus.


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  		Fig. 1.   Strategy for genetic manipulation of S. cerevisiae, and the comparison of the N-glycosylation pathway in mammalian cells and S. cerevisiae.

In yeast cells, the His-Asp-Glu-Leu (HDEL) C-terminal sequence of proteins acts as a retention/retrieval signal for the endoplasmic reticulum (ER) (15). Proteins with an HDEL sequence are bound by a membrane-bound receptor (Erd2p) (16, 17) and then enter a retrograde transport pathway for return to the ER from the Golgi apparatus. In this study, the expression of the A. saitoi alpha -1,2-mannosidase in the ER was demonstrated by adding "HDEL" to the C terminus of the alpha -1,2-mannosidase open reading frame. The introduced alpha -1,2-mannosidase was also shown to convert Asn-linked oligosaccharides into Man5GlcNAc2, the intermediate form for hybrid- and complex-type sugar chains, in mutant yeast cells with disruptions in three of the original mannosyltranferase genes (OCH1, MNN1, and MNN4).

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Yeast Strain and Culture Conditions-- The enzyme activity and the localization of the HDEL-tagged MsdSp was determined in S. cerevisiae pep4 disrupted YPH500 cells (MATalpha ura3-52 lys2-801 ade2-101 trp1-Delta 63 his3-Delta 200 leu2-Delta 1 pep4::ADE2) (18). YS132-8B (MATalpha och1::LEU2 mnn1::URA3 mnn4::LYS2 leu2-Delta 1 ura3-52 trp1-Delta 1 lys2-801AM his3-Delta 200 ade2-101OC), which had been constructed by standard genetic methods (19), were used to analyze the Asn-linked sugar chains of carboxypeptidase Y (CPY) or mannoproteins. All strains were transformed by the method of Ito et al. (20). Transformants were selected on synthetic minimal dextrose (SD) medium with auxotrophic supplements.

DNA Constructs-- For preparation of the HDEL-tagged MsdSp, the tag sequence was introduced by amplifying the 0.6-kilobase region between the HindIII site and the stop codon of the msdS gene with the following mutation primers: 5'-TGCGCCCGGAAGTGATTGAA-3' and 5'-CCTACAATTCGTCGTGTGTACTACTCACCCGCACTGG-3'. The polymerase chain reaction product was subcloned into pCR-Script Amp SK(+) (Stratagene) and digested with HindIII and NotI. The coding region of the C-terminal domain of pGAM1, an expression plasmid for A. saitoi alpha -1,2-mannosidase (13), was substituted with the recovered 0.6-kilobase HindIII-NotI fragment to create the pGAMH1 plasmid. The msdS and insertion sequence were confirmed by DNA sequencing.

alpha -1,2-Mannosidase Assay-- Man6GlcNAc2 oligosaccharide was obtained from Seikagaku Co. (Tokyo, Japan) and labeled with 2-aminopyridine (21). Pyridylaminated oligosaccharide (Man6GlcNAc2-PA) was purified by gel filtration (TOYOPEARL HW-40, 1.6 × 73 cm, Tosoh Corp., Japan) and the purity confirmed by reversed-phase HPLC using an ODS-80TM column (0.46 × 15 cm, Tosoh Corp.).

Yeast cell extracts were prepared as described below. Yeast cells were cultured on SD medium lacking tryptophan. The cell density was determined at 600 nm using a 10-mm cuvette. The pelleted cells were washed with deionized water, resuspended in extraction buffer (0.1 M sodium acetate buffer (pH 5.0) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100), and vortex mixed with acid-washed glass beads (425-600 µm diameter). Soluble cell extract was separated from cell debris by centrifugation and assayed for activity. Samples containing 10-100 µg of protein were incubated for 30 min with 150 pmol of Man6GlcNAc2-PA in 0.1 M sodium acetate buffer (pH 5.0) at 37 °C. The assay was stopped by boiling, and the sample was filtrated using an Ultrafree-MC centrifugal filter unit (0.22 µm pore size low-binding Durapore, Millipore). The filtrates were analyzed by HPLC with a Hitachi F-1050 fluorescence spectrophotometer, using an ODS-80TM column (4.6 × 150 mm). The solvent and elution conditions used are as described by Kondo et al. (21). One unit of the enzyme was defined as the amount of enzyme that was required to liberate 1 µmol of mannose from Man6GlcNAc2-PA per min at 30 °C and pH 5.0.

Marker Enzyme Assay-- NADH cytochrome P-450 reductase, a marker enzyme for the ER, guanosine diphosphatase, a marker enzyme for the Golgi apparatus, and glucose-6-phosphate dehydrogenase, a cytosol marker, were assayed as described (22-24), respectively.

Western Blot Analysis-- Rabbit anti-MsdSp and rabbit anti-glucose-6-phosphate dehydrogenase antisera were obtained from Sawaday Technology (Tokyo, Japan). Mouse anti-CPY monoclonal antibody 10A5-B5, mouse anti-alkaline phosphatase monoclonal antibody 1D3-A10, and mouse anti-dolichol phosphate mannose synthase monoclonal antibody 5C5-A7 were purchased from Molecular Probes, Inc. (Eugene, OR).

Samples containing 1 µg of protein were subjected to SDS-PAGE. SDS-PAGE was carried out using the buffer system of Laemmli (25) in 10% gel. Electroblotting of the fractionated proteins onto polyvinylidene difluoride membrane (Millipore Corp.) was carried out by the method of Towbin et al. (26), and detection was performed essentially according to the method of Hsu et al. (27).

Subcellular Fractionation-- Cells were grown in SD medium and were converted to spheroplasts by the method of Vita et al. (28). The following procedures were also performed at 4 °C. The spheroplasts were harvested by centrifugation. The spheroplasts were resuspended in a hypoosmotic lysis buffer (0.25 M sorbitol, 10 mM triethanolamine (pH 7.2), 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, antipain (2 µg/ml), chymostatin (2 µg/ml), pepstatin A (3 µg/ml), leupeptin (2 µg/ml)) and homogenized for up to 20 times using a glass tissue homogenizer. The lysate was centrifuged at 220 × g for 5 min to remove unlysed spheroplasts. The 220 × g supernatant (CL) was centrifuged at 10,000 × g for 15 min to separate the low speed pellet (LSP) and supernatant fractions. The supernatant was centrifuged at 100,000 × g for 80 min to separate the high speed pellet (HSP) and supernatant fractions (HSS). The LSP and HSP were resuspended by sonication on ice in lysis buffer containing 1% Triton X-100. Aliquots of the LSP, HSP and HSS fractions were used to assay alpha -1,2-mannosidase, NADH cytochrome P-450 reductase, guanosine diphosphatase, and glucose-6-phosphate dehydrogenase activities and were also subjected to Western blot analyses.

Aliquots (200 µl) of the resuspended LSP fraction were placed on top of four 1.8-ml 1.2/1.5 M discontinuous sucrose gradients containing 10 mM triethanolamine (pH 7.2). After centrifugation at 80,000 × g in an RT-100T Beckman Ultracentrifuge at 4 °C for 65 min, 200 µl each were collected from the top of each gradient and pooled. Aliquots of the pools were resuspended as described above and were subjected to Western blot analysis.

Endo-beta -N-acetylglucosaminidase H Treatment-- Recombinant endo-H was purchased from Genzyme Co. (Boston, MA). Yeast cell extracts were prepared as described above. Aliquots (containing 15 µg of proteins) of cell extracts were brought to 50 µl of 50 mM sodium citrate buffer (pH 6.0) containing 0.1% SDS and 1 mM PMSF and denatured at 100 °C for 5 min. After dilution with 50 mM sodium citrate buffer (pH 6.0) containing 1 mM PMSF, 0.5 milliunits of endo-H was added to the sample and incubated for 16 h at 37 °C. Samples that substituted buffer for endo-H were used as negative controls. The samples containing 1 µg of protein were subjected to SDS-PAGE and analyzed by Western blotting with mouse anti-CPY antibody.

Purification of CPY-- p-Aminobenzylsuccinic acid was purchased from Sigma. CNBr-activated Sepharose 4B was obtained from Amersham Pharmacia Biotech, and glycyl-tyrosine was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CPY produced in YS132-8B cells harboring pG3 or pGAMH was purified using affinity column chromatography. The affinity gel was prepared by coupling the specific inhibitor, p-aminobenzylsuccinic acid, via an azo linkage to Sepharose-glycyl-tyrosine as described by Johansen et al. (29). Before use, the column (1.0 × 3.0 cm) was equilibrated with 10 mM MES buffer (pH 5.0). S. cerevisiae YS132-8B and transformed YS132-8B cells were cultivated in SD medium containing 0.3 M sorbitol at 30 °C and harvested at stationary phase. The culture was centrifuged at 220 × g for 5 min, and the pellet was disrupted as described above. The yeast cell lysate was applied to the column and washed extensively with 500 ml of 1 M NaCl in 10 mM sodium acetate buffer (pH 4.3). Elution was performed with 10 mM phosphate buffer (pH 7.0). The eluate was concentrated by Centricon-10 (Nihon Millipore Ltd., Japan) and applied to SDS-PAGE. The purified CPY was lyophilized and subjected to Asn-linked oligosaccharide analysis.

Preparation of Mannoprotein-- S. cerevisiae YS132-8B and transformed YS132-8B cells were cultivated in SD medium containing 0.3 M sorbitol at 30 °C and harvested at mid-log phase. Mannoproteins were extracted by hot citrate buffer (0.1 M citrate buffer, pH 7.0) followed by precipitation with ethanol (30). The precipitates were further purified by a Concanavalin A-agarose column (0.8 × 2 cm, Honen Corp., Japan), which was equilibrated with Con A buffer (0.1 M Tris-HCl (pH 7.2) containing 0.15 M NaCl, 1 mM MnCl2, and 1 mM CaCl2). The column was eluted by the Con A buffer containing 0.2 M alpha -methyl-D-mannoside. The eluted fractions were dialyzed against water and lyophilized.

HPLC Analysis of Asn-linked Oligosaccharides on CPY and Mannoproteins-- N-glycanase was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). CPY or mannoproteins were dissolved in 100 mM sodium phosphate buffer (pH 7.2) containing 0.5% SDS and 50 mM 2-mercaptoethanol and then denatured at 100 °C for 5 min. After dilution with 100 mM sodium phosphate buffer and addition of 0.5% Nonidet P-40, 2.5 units of N-glycanase was added to the sample and incubated for 16 h at 37 °C. Liberated Asn-linked oligosaccharides were separated from the salts and peptides using an AG 501-X8 mixed bed resin (Bio-Rad), and from Nonidet P-40 using Bio-Beads S-X8 (Bio-Rad). Reductive pyridylamination and structural analyses of the purified oligosaccharides were carried out essentially according to the method of Kondo et al. (21). Pyridylaminated (PA-) oligosaccharides were analyzed by HPLC using a size-fractionation column (TSKgel Amide-80, 4.6 × 250 mm, Tosoh Corp.) and a reversed-phase column (TSKgel ODS-80TM, 4.6 × 150 mm). Authentic PA-oligosaccharides and PA-glucose oligomer were purchased from Takara Shuzo Co. (Kyoto, Japan).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Expression of the alpha -1,2-Mannosidase-- Measurement of alpha -1,2-mannosidase activity by the Nelson-Somogyi method was attempted but was not sensitive enough to detect the amount of enzyme present. We have developed a new assay for alpha -1,2-mannosidase using a fluorescent oligosaccharide. PA-oligosaccharide made it possible to assay picomole per minute-ordered enzyme activity. For the assay of the alpha -1,2-mannosidase activity in vitro, we used Man6GlcNAc2-PA oligosaccharide as a substrate. The optimal assay conditions, such as enzyme concentration, reaction time, and substrate concentration were determined as described under "Experimental Procedures."

A soluble form of A. saitoi alpha -1,2-mannosidase was constructed with the HDEL ER retention/retrieval signal sequence at the C terminus. This construct was subcloned into the multicopy plasmid pG3, termed pGAMH1, and was used to transform S. cerevisiae YPH500 cells. alpha -1,2-Mannosidase activity that converted Man6GlcNAc2-PA substrate into Man5GlcNAc2-PA was observed in the cell extracts of the recombinant yeast with the pGAMH1 vector (Fig. 2B), whereas there was no such activity in the extract of the recombinant yeast transfected with the pG3 vector only (Fig. 2A). The activity of ER alpha -1,2-mannosidase and vacuole alpha -mannosidase in yeast were not detected under these assay conditions. Four milliunits of the enzyme activity was recovered from a 500-ml yeast culture.


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  		Fig. 2.   In vitro alpha -1,2-mannosidase assay of cell extracts of the recombinant yeasts. The cell extract was incubated with 10 µM pyridylaminated 6-oligomannose type sugar chain (Man6GlcNAc2-PA) at 30 °C in a final volume of 15 µl for 30 min. After incubation, the alpha -1,2-mannosidase activity was measured by HPLC analysis. Chromatograms of the reaction product of the yeast cell extract harboring null vector (panel A) and harboring alpha -1,2-mannosidase-HDEL expression plasmid (panel B) were displayed. Peaks 1, Man6GlcNAc2-PA; peak 2, Man5GlcNAc2-PA.

Localization of the alpha -1,2-Mannosidase-- To determine the localization of the expressed alpha -1,2-mannosidase in yeast, we investigated the subcellular distribution of the enzyme. Fig. 3A illustrates the protocol for the fractionation of the yeast cells.


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  		Fig. 3.   Subcellular fractionation of the recombinant alpha -1,2-mannosidase. Wild-type (YPH500) spheroplasts harboring pGAMH1 were osmotically lysed. A, lysed spheroplasts were subjected to differential centrifugation as described under "Experimental Procedures." CL, LSP, HSP, and HSS fractions were subjected to Western blotting and enzyme assays. ppt, precipitate. B, Western blot analysis was carried out using various yeast protein antibodies.

The alpha -1,2-mannosidase activity was localized primarily in the LSP fraction (77%) (Table I). The LSP fraction also contained 69% of NADPH cytochrome P-450 reductase (ER marker) (31, 32). In contrast, most of the guanosine diphosphatase (70%), a Golgi marker (23), was found in the HSP fraction. Kex2p (33), a late Golgi marker, was split into the HSP and HSS fractions. The cytosol marker, glucose-6-phosphate dehydrogenase, was detected mainly in the HSS fraction (76%).

                              
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Table I
Subcellular distribution of the recombinant alpha -1,2-mannosidase

The Western blot pattern also showed that the ER marker protein (dolichol phosphate mannose synthase) and the vacuolar membrane protein (alkaline phosphatase) were localized in the LSP fraction, whereas both CPY, which is a soluble protein in the vacuole, and cytosolic glucose-6-phosphate dehydrogenase were fractionated in HSS fraction (Fig. 3B). The introduced alpha -1,2-mannosidase gene products were detected in the LSP fraction. Because it is known that the LSP fraction contained the vacuole in addition to the ER (34), discontinuous sucrose density centrifugation was performed to determine whether the expressed alpha -1,2-mannosidase was localized in the ER or the vacuole (Fig. 4). Alkaline phosphatase, a vacuolar marker enzyme, was distributed to fractions 1-3, the most light density fraction. In contrast, dolichol phosphate mannose synthase, an ER marker, was distributed to fractions 4-6. This result indicated that the vacuole and ER are well separated from each other in this system. The signals of the alpha -1,2-mannosidase appeared around fraction 5. The results strongly suggested that the alpha -1,2-mannosidase with the HDEL-tag is mainly localized in the ER.


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  		Fig. 4.   Distribution of alpha -1,2-mannosidase and marker proteins after discontinuous sucrose density centrifugation. Sucrose gradient was fractionated into nine fractions. Fraction 1 came from the top (the lightest fraction) to the bottom (the heaviest fraction).

Oligosaccharide Structures of the Recombinant Triple Mutant Yeast-- The apparent molecular mass of the CPY produced in the recombinant yeasts was analyzed on SDS-PAGE followed by Western blot analysis. YS132-8B, which has disrupted OCH1, MNN1, and MNN4 genes, will not have any outer mannosyl chains on its glycoproteins. As shown in Fig. 5, the CPY from YS132-8B carrying the null vector gave a single signal with an apparent molecular mass of 62 kDa on SDS-PAGE. However the CPY from YS132-8B harboring the pGAMH1 plasmid gave an additional signal below the original one, indicating that the sugar chains of the CPY have been trimmed by the introduced alpha -1,2-mannosidase. Treatment of each cell lysate with endo-H gave a single signal of an N-deglycosylated CPY (Fig. 5, third and fourth lanes).


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  		Fig. 5.   Western blot analysis of the carboxypeptidase Y from the recombinant yeast cell extracts. S. cerevisiae YS132-8B strain (Delta och1 Delta mnn1 Delta mnn4, triple mutant) was used as a host. First and third lanes, from YS132-8B harboring the null vector; second and fourth lanes, from YS132-8B harboring the expression plasmid, pGAMH1. Endo-H digestion resulted in the shift of the signals corresponding to the deglycosylated form (third and fourth lanes).

The oligosaccharide structures of glycoproteins produced in these yeasts were analyzed using CPY as a model glycoprotein. While the sugar chains of CPY produced in the yeast with the null vector were eluted at the Man8GlcNAc2-PA position on the amide column (Fig. 6A, graph a), those produced in the yeast with the pGAMH1 plasmid showed four peaks at positions corresponding from Man5GlcNAc2-PA to Man8GlcNAc2-PA, respectively (Fig. 6A, graph b). The molar ratio of each glycoform was Man5GlcNAc2-PA:Man6GlcNAc2-PA:Man7GlcNAc2-PA:Man8GlcNAc2-PA = 27:22:22:29. The fraction eluted at the position corresponding to Man5GlcNAc2-PA (indicated with a open arrow in Fig. 6A) was pooled and subjected to reversed-phase chromatography. Only one peak was observed at the same position as authentic Manalpha 1-3[Manalpha 1-3(Manalpha 1-6)Manalpha 1-6]Manbeta 1-4GlcNAcbeta 1-4GlcNAc-PA (Fig. 6B); this is the smallest structure of mammalian-type high-mannose sugar chains.


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  		Fig. 6.   Analysis of Asn-linked oligosaccharides in the triple mutant strain YS132-8B. A, chromatogram of the sugar chains of CPY on HPLC using a TSKgel Amide-80 column. Graph a, from YS132-8B harboring null vector; graph b, from YS132-8B harboring the expression plasmid, pGAMH1. B, the peak indicated by the open arrow in panel A was pooled and subjected to HPLC using a TSKgel ODS-80TM column. Graph a, standard sugar chain of Manalpha 1-3[Manalpha 1-3(Manalpha 1-6)Manalpha 1-6]Manbeta 1-4GlcNAcbeta 1-4GlcNAc-PA. Graph b, the pooled fraction in panel A. C, chromatogram of the sugar chains of mannoproteins on HPLC using a TSKgel Amide-80 column. Graph a, from YS132-8B harboring null vector; graph b, from YS132-8B harboring the expression plasmid, pGAMH1. The elution times of authentic PA-sugar chains were indicated by arrows. M5, Man5GlcNAc2-PA; M6, Man6GlcNAc2-PA; M7, Man7GlcNAc2-PA; M8, Man8GlcNAc2-PA.

Besides CPY, we also investigated the oligosaccharide structures of cell wall mannoproteins. As shown in Fig. 6C, the mannoproteins produced in the yeast with the pGAMH1 plasmid contained Man5GlcNAc2. The molar ratio of each glycoform in mannoproteins was Man5GlcNAc2-PA:Man6GlcNAc2-PA:Man7GlcNAc2-PA:Man8GlcNAc2-PA = 10:13:16:61.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

alpha -Mannosidase I digests alpha -1,2-mannosidic linkages and converts Man8GlcNAc2 oligosaccharide into Man5GlcNAc2. This is the first step in the biosynthesis of hybrid-type and complex-type sugar chains from high mannose-type sugar chains. There are several successive enzymatic reactions necessary to complete complex-type structures. N-Acetylglucosaminyltransferase (GnT)-I, alpha -mannosidase II, GnT-II, beta -1,4-galactosyltransferase, etc. work in succession in mammalian cells. Since the alpha -1,2-mannosidase acts upstream in the biosynthetic pathway of oligosaccharides, it must be located either in the ER or the early Golgi apparatus to reconstruct this system in yeast. We have already succeeded in expressing the A. saitoi alpha -1,2-mannosidase as a chimeric protein with a transmembrane domain of Och1p (data not shown). Although Och1p resides in the early Golgi apparatus of yeast (35), the expressed chimeric enzyme was localized not only in the Golgi apparatus, but also in the ER and the cytosol fractions. We could not detect any Man5GlcNAc2 sugar chain structure in the recombinant yeast (data not shown). Evidence suggested that the mislocalization of the chimeric alpha -1,2-mannosidase prevented the trimming of sugar chains in the yeast. In this study, we attempted to localize the alpha -1,2-mannosidase to the yeast ER using a retention/retrieval signal.

There has been several retention/retrieval systems proposed to date. Some of these systems require a transmembrane domain and/or a cytoplasmic tail. Xaa-Xaa-Arg-Arg (XXRR; X is any amino acid) in the N-terminal cytoplasmic region and Lys-Lys-Xaa-Xaa (KKXX), in the C-terminal cytoplasmic domain of membrane proteins are known as retrieval signals for the ER. It has also been demonstrated that the N-terminal 16 amino acids of the alkaline phosphatase in the cytoplasmic tail contain a vacuolar sorting signal in S. cerevisiae (36). Lussier et al. reported that an N-terminal cytoplasmic domain was necessary for Kre2p to correctly localize in the Golgi apparatus and that the entire Kre2p cytoplasmic tail plus the transmembrane domain and 36 amino acids in the luminal stem region were required to localize a Pho8p reporter protein in the yeast Golgi apparatus (35). These results suggested that there is no accurate signal for the retention of exogenous membrane proteins in the ER or Golgi apparatus of yeast. Therefore, we constructed an expression vector with an HDEL signal for the transfer of soluble alpha -1,2-mannosidase proteins from the Golgi apparatus to the ER.

In yeast, two alpha -mannosidases have been found, and these have different substrate specificity and pH optima to the A. saitoi alpha -1,2-mannosidase. S. cerevisiae ER alpha -mannosidase (Mns1p) cannot act on Man6GlcNAc2 oligosaccharide, and vacuolar alpha -mannosidase (Ams1p) cannot act at pH 5.0. Whereas A. saitoi alpha -1,2-mannosidase can remove the alpha -1,2-linked mannose of Man6GlcNAc2 oligosaccharide at pH 5.0. Based on these facts, we have developed an assay method that specifically detects A. saitoi alpha -1,2-mannosidase activity.

The subcellular fractionation experiments indicated that the product of the msdS gene was mainly localized in the LSP fraction (Fig. 3), which includes the ER, vacuole, and plasma membrane. However, it is unlikely that MsdSp was localized in the vacuole, because the signal distribution of MsdSp was quite different from that of CPY, which is the vacuolar marker (Fig. 3). Furthermore, MsdSp will never be anchored at the plasma membrane because it is a soluble protein. The fractionation in the sucrose discontinuous gradients also showed that the signal distribution of the product of the msdS gene did not match with that of the vacuole but with that of the ER.

CPY was chosen as one of the reporter glycoproteins to analyze the glycosylation phenotype of the genetically constructed yeast, because it has four Asn-linked oligosaccharides of known structure (37), and it has an established purification method (29). The triple mutant strain (YS132-8B) used in this study lacks three of the yeast mannosyltransferase activities, and the elongation of N-glycan is terminated at the Man8GlcNAc2 structure (1), which is a substrate for the alpha -1,2-mannosidase. Structural analysis of the CPY sugar chains produced in the mutant yeast harboring the pGAMH1 plasmid showed that the introduced alpha -1,2-mannosidase digested the sugar chains up to Man5GlcNAc2 (Fig. 6). The fact that the mannoproteins of the yeast with pGAMH1 vector also had the Man5GlcNAc2 structure suggests that the introduced alpha -1,2-mannosidase could digest the oligosaccharide chains of secretary proteins. The observed lower molar ratio of Man5GlcNAc2 in mannoproteins might be because of the cell harvesting period; the mannoproteins were recovered at mid-log phase of the culture, whereas CPY was done at stationary phase. Because there were also intermediates ranging from Man8GlcNAc2 to Man6GlcNAc2 both on CPY and mannoproteins, the expression level of the introduced alpha -1,2-mannosidase seemed not to be sufficient for complete trimming of each sugar chain. It might be more suitable to produce therapeutic glycoproteins using a vector with an inducible promoter, such as CUP1 or GAL1 promoter. We could induce production of a target protein after stationary phase, where alpha -1,2-mannosidase would be sufficiently expressed to convert all of the sugar chains to Man5GlcNAc2.

In this study, S. cerevisiae was manipulated to produce Man5GlcNAc2 N-glycan. Increasing the efficiency of the alpha -1,2-mannosidase reaction remains to be done. Furthermore, the Man5GlcNAc2 N-glycan is a hybrid and a complex-type intermediate, the latter of which is better suited and more effective for human therapeutics. We have already succeeded in expressing GnT-I, GnT-II, and beta -1,4-galactosyltransferase activities in yeast, but to make hybrid- and complex-type sugar chains in yeast cells, co-expression of GnT-I and alpha -1,2-mannosidase is required and is an object of our future research.

    ACKNOWLEDGEMENTS

We thank Dr. Y. Shimma for providing the YS132-8B strain and S. Ogino for valuable assistance with this research. We are also grateful to Dr. M. Zimbo for helpful suggestions.

    FOOTNOTES

* This work was supported by 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.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Tel.: 81-45-788-7200; Fax: 81-45-788-4047.

The abbreviations used are: EPO, erythropoietin; ER, endoplasmic reticulum; CPY, carboxypeptidase Y; endo-H, endo-beta -N-acetylhexosaminidasePA, 2-aminopyridinePMSF, phenylmethylsulfonyl fluorideGnT-, N-acetylglucosaminyltransferaseSD, synthetic minimal dextroseCL, crude lysateLSP, low speed pelletHSP, high speed pelletHSS, supernatant fractionPAGE, polyacrylamide gel electrophoresisHPLC, high performance liquid chromatographyMES, 2-morpholinoethansulfonic acid.
    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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