The yeast S.cerevisiae is a useful host for the production of high molecular weight therapeutics such as growth factors, cytokines etc. These secretory proteins undergo posttranslational modifications including limited proteolysis, folding, disulfide bond formation, phosphorylation and glycosylation. High level expression of numerous heterologous proteins has been achieved in prokaryotes such as Escherichia coli. However, the products may have aberrant disulfide bonds and no protein-glycosylation. Therefore, yeast is a preferable host for the production of glycoproteins such as human erythropoietin (Elliott et al., 1989) and alpha-1-antitrypsin (Moir and Dumais, 1987).
Glycosylation has essential functions in all eukaryotic cells. In both yeast and mammals, the transfer of precursor oligosaccharides from lipid intermediates to asparagine (Asn) residues in the Asn-X-Thr/Ser signal initiates the biosynthesis of the sugar chains. Subsequently, a series of enzymes in the ER, Golgi apparatus and vacuole process these glycans to the mature forms (Kornfeld and Kornfeld, 1985; Kukuruzinska et al., 1987; Moremen et al., 1994). Glycosidases and glycosyltransferases convert typical Glc3Man9GlcNAc2 glycans to Man8GlcNAc2 glycans and then process them to mannan-type glycans in yeasts, or hybrid- and complex-type glycans in mammals. The first step towards the formation of complex-type glycans is catalyzed by the Golgi enzyme GnT-I. The enzyme takes Man5GlcNAc2 N-glycan as a substrate, and catalyzes the transfer of a GlcNAc from UDP-GlcNAc to the terminal [alpha]1-3 mannose residue which is linked to the [beta]-mannose of the N-glycan core. After the GnT-I and [alpha]-mannosidase II steps, the biosynthesis pathways for mammalian complex-glycans diversify from bi- to tetra-antennary oligosaccharides.
The GnT-I genes have been cloned from rabbit (Sarkar et al., 1990), human (Kumar et al., 1990), mouse (Kumar et al., 1992), rat (Fukada et al., 1994), chicken (Warren et al., 1992), and Xenopus laevis (Mucha et al., 1995). GnT-I is a type II membrane protein which is localized in the medial-Golgi (Burke et al., 1994). GnT-I shares typical membrane orientation with glycosyltransferases that have been cloned. It has a short cytoplasmic tail, a transmembrane signal/anchor domain, a stem region at the N-terminal and a lumenal catalytic domain at the C-terminal. The transmembrane region is essential for the localization of GnT-I in the Golgi apparatus (Tang et al., 1992). The GnT-I gene has been successfully expressed in E.coli (Kumar et al., 1992) and Aspergillus nidulans (Kalsner et al., 1995). Here, we describe the expression and characterization of GnT-I in S.cerevisiae. The GnT-I expressed in yeast is also active in vitro. The C-terminal catalytic region faces the lumenal side, and it is distributed in the ER, Golgi apparatus, and vacuole, probably because the transmembrane region of GnT-I has been removed in the yeast ER. We are now working on the biosynthesis of Man5GlcNAc2 sugar chains in S.cerevisiae to serve an acceptor substrate for the GnT-I expressed in the same yeast cells.
Expression of a rat GnT-I cDNA in S.cerevisiae
We constructed a rat GnT-I expression plasmid (pGNI-8) based on the YEp13K harboring the GPD promoter, PGK terminator, and the LEU2 marker as described in Materials and methods (Figure
Figure 1. Construction of the expression vector for rat GnT-I cDNA. The multicopy plasmid pSY114 was used for the insertion of the entire open reading frame encoding rat GnT-I. The 5[prime]-end of the gene was modified by polymerase chain reaction (PCR) amplification in order to introduce a HindIII restriction site at the ATG translation start signal (Materials and methods). Each number in the figure shows the position of base pairs relative to the initiation codon. The TAG at 1350 base pairs terminates translation. Abbreviations for restriction enzymes are as follows; H, HindIII; Sm, SmaI; Sl, SalI.
Figure 2. Subcellular localization of GnT-I in yeast cells expressing the rat GnT-I cDNA at different levels. (A) Southern hybridization and Western blot analyses of yeast cells expressing the rat GnT-I cDNA. GN-1, GN-2, and GN-3 are yeast cells transformed with the integration plasmid pGNI-9 digested with AflII (GN-1 and GN-2) or BstEII (GN-3). YCp and YEp indicate yeast cells transformed with pRS315/GnT-I and pYO325/GnT-I, respectively; 1.0 × 107 cells were homogenated with glass beads and subjected to DNA electrophoresis for Southern hybridization. For Western analysis, the samples were suspended and boiled in a sample buffer for SDS-PAGE and subjected to SDS-PAGE on 10% (w/w) gel. The probe for the Southern hybridization was a 1.5 kb HindIII/SmaI fragment of the rat GnT-I cDNA. GnT-I was detected by Western blotting using an anti-GnT-I polyclonal antibody. (B) Subcellular distribution of GnT-I activity in yeast cells expressing the rat GnT-I cDNA. GnT-I and GDPase activities were assayed as described in Materials and methods. The control indicates yeast cells transformed with pRS305 digested with BstEII. CL, Crude lysate; LSP, low speed pellet; HSP, high speed pellet; HSS, high speed supernatant. Subcellular localization of the GnT-I enzyme
We performed subcellular fractionation experiments upon the three yeast cell lines with different copy numbers of GnT-I gene as described in Materials and methods to determine whether overproduction causes mislocalization of the products. Figure
GN-2 cells were grown in SD medium, converted to spheroplasts, homogenized and subjected to differential centrifugation. Figure three shows the distribution of marker enzymes for the ER and Golgi apparatus in the three fractions LSP, HSP, and HSS. Overproduction of GnT-I had minimal effect on the distribution of these marker enzymes (data not shown). Although the LSP and HSP fractions were crude mixtures of membranes, it should be noted here that the ER marker Sec12p was preferentially distributed in the LSP, and GDPase for the Golgi marker was not. The Sec12p was not detected in the crude lysate fraction probably by proteolysis. The GnT-I product and activity were recovered in the LSP and HSP fractions. Split bands of GnT-I were observed in the LSP fraction. These bands will be mentioned below. The LSP fraction was further subjected to sucrose bilayer centrifugation (1.2/1.5 M) to fractionate the ER from the vacuole, as described in Materials and methods (Figure Orientation of GnT-I in the yeast membranes
GnT-I is a type II membrane protein localized in the Golgi apparatus in mammals. We checked whether the C-terminus of GnT-I is located in the lumen or cytoplasm in yeast. If the catalytic domain of GnT-I is lumenal in yeast organella, as in mammals, its C-terminal domain should be non-susceptible to trypsin. We also expressed GnT-I cDNA with a c-Myc tag at the C-terminus of GnT-I in yeast (pGNI-8-Myc). The GnT-I/c-Myc product was detected with anti-c-Myc or anti-GnT-I antibody. In the absence of the detergent Triton X-100, GnT-I was not degraded during 30 min incubation at 0°C, while in the presence of the detergent, GnT-I was degraded to a 45 kDa fragment (Figure
Figure 3. Subcellular localization of GnT-I in GN-2 cells. Lysed spheroplasts from 1 × 107 cells of GN-2 were subjected to differential centrifugation as described in Materials and methods and analyzed by Western blot analysis using the anti-GnT-I and Sec12p antibodies, respectively. Sec12p (Nakano et al., 1988) and GDPase (Abeijon et al., 1988) were used as markers for the ER and Golgi apparatus, respectively. The enzyme activities of GnT-I and GDPase were assayed as described in Materials and methods. The enzyme activity is shown as nmol/min/1010 cells. Abbreviations for the fractions are as described in Figure 2.
According to the SDS-PAGE results shown in Figures
Figure 4. Sucrose bilayer fractionation of the LSP fraction in the GN-2 cells. Eighty percent of the LSP fraction was resuspended in 50 µl of lysis buffer and layered on the top of a 1.2/1.5 M sucrose gradient. After an 85,000 × g spin for 1 h, 10 equivalent fractions were removed and subjected to Western blot analysis and a GDPase assay. Sec12p (Nakano et al., 1988), Vma1p (Hirata et al., 1990), and GDPase (Abeijon et al., 1988) were used as markers for the ER, vacuole, and Golgi apparatus, respectively. For Western blot analysis (GnT-I, Vma1p, and Sec12p), samples derived from 1 × 107 of cells were used. GDPase was assayed as described in Materials and methods. The enzyme activity is shown as nmol/min/1010 cells.
Figure 5. Susceptibility of GnT-I in the membrane fraction to tryptic digestion. The homogenate from yeast cells (YPH500P/pGNI-8-Myc) was treated with or without trypsin in the absence or presence of Triton X-100 (TX-100) on ice. After the indicated times (0-30 min), the tryptic action was stopped by the addition of trypsin inhibitor and the samples were analyzed by SDS-PAGE followed by immunoblotting with the anti-c-Myc (c-Myc) and anti-GnT-I (GnT-I) antibodies, respectively. Arrowheads indicate the mature 51 kDa form and 45 kDa fragment of GnT-I/c-Myc.
We have described here the expression and localization of the rat GnT-I cDNA in the yeast S.cerevisiae. The GnT-I enzyme expressed in the yeast is active in vitro, but its location differs from that of mammals. The GnT-I enzyme is localized throughout the membranes in the secretory pathway, including the ER, Golgi apparatus, and vacuole. This is probably because signal peptidases in the yeast ER cleave off the membrane anchor region of the GnT-I protein. To retain GnT-I in the Golgi apparatus, we have constructed the OCH1/GnT-I chimeric gene which is under the control of the GAL1 promoter, and characterized the gene product. It is localized mainly in the LSP fraction containing the ER and vacuole (unpublished observations). This result is consistent with results that the overexpressed OCH1 gene product is mainly localized in the vacuole (Chapman and Munro, 1994). We are currently constructing a vector that expresses a low level of the OCH1/GnT-I product to locate it in the Golgi apparatus.
Several mammalian glycosyltransferases have been expressed and characterized in the yeast S.cerevisiae. The human [beta]-1,4-galactosyltransferase (GalT) has been expressed in yeast, but it was localized in the ER (Krezdorn et al., 1994). Schwientek and Ernst (1994) showed that the MNT1/GalT chimeric gene products can be expressed effectively and localized in the yeast Golgi apparatus.
The [alpha]-2,6-sialyltransferase for Asn-linked sugar chains is localized in the ER, but not in the Golgi apparatus of yeast (Krezdorn et al. 1994). However, the chimeric gene product, a transmembrane anchor region of [alpha]-2,6-sialyltransferase fused to yeast invertase Suc2p, is localized in the yeast Golgi apparatus, suggesting that the transmembrane anchor region of [alpha]-2,6-sialyltransferase also functions as a Golgi retention signal in yeast (Schwientek et al., 1995). The discrepancy between these results is probably due to the quality control in the yeast ER, including folding and glycosylation in the case of expression of the full-length [alpha]-2,6-sialyltransferase. Alternatively, it is possible that the chimera with a yeast gene supports the efficient expression and folding of foreign gene products in yeast.
It has been reported that the GnT-I cDNA is expressed in E.coli (Kumar et al., 1992), Asperugillus nidulans (Kalsner et al., 1995) and Arabidopsis thaliana (Gomez and Chrispeels, 1994). The GnT-I products thus expressed are active in vitro. The GnT-I expressed in A.thaliana is shows in vivo activity, because it complements a mutation of A.thaliana lacking Asn-linked complex type glycans. In A.nidulans, GnT-I is active in vitro, but does not function in vivo, because there were no Endo H-resistant glycoproteins nor GalT-dependent incorporation of galactose into glycoproteins (Kalsner et al., 1995). A.nidulans may have no precursor sugar chains to act as a substrate for GnT-I and/or no UDP-GlcNAc transporter (Abeijon et al., 1996) to import UDP-GlcNAc into the Golgi apparatus lumen. As S.cerevisiae has no acceptor substrate for GnT-I and no UDP-GlcNAc transporter, S.cerevisiae resembles A.nidulans. Genetic engineering and gene disruption are easier to perform in S.cerevisiae than in A.nidulans, thus we believe that yeast is a preferable host for the production of mammalian-type glycoproteins. Now we are planning to introduce [alpha]1,2-mannosidase, UDP-GlcNAc:[alpha]-6-d-mannoside [beta]-1,2-N-acetylglucosaminyltransferase II, sialyltransferases, and other glycosyltransferases, sugar-nucleotide transporters and sugar-nucleotide synthesis enzymes into yeast if they are necessary for the production of glycoproteins with human compatible sugar chains. Organisms and growth conditions
The protease-deficient S.cerevisiae strains used in this study were EHA-1C (Mat[alpha] leu2-3, 112 pep4-3) (Nakayama et al., 1992) obtained from Dr. Y. Jigami (National Institute of Bioscience and Human Technology) and YPH500P (Mat[alpha] ura3-52 lys2-801 ade2-101 trp1-[Delta]63 his3-[Delta]200 leu2-[Delta]1 [Delta]pep4::ADE2; Yoshida et al., 1994). Yeast cells were grown at 30°C in SD medium (Rose et al., 1990). Transformation of yeast cells was carried out as described previously (Rose et al., 1990). Plasmid construction
GnT-I cDNA was isolated from rat liver by the screening of a cDNA library (Fukada et al., 1994). For heterologous expression in S.cerevisiae, we used the GPD promoter and the PGK terminater of pSY114 (~10 kb), which is based on YEp13K (Sone et al., 1988; Yamano et al., 1994). This plasmid also has a LEU2 yeast selective marker. The 5[prime]-end of the gene was modified by amplification by the polymerase chain reaction (PCR) to introduce a HindIII restriction site at the ATG translation start signal. The GnT-I cDNA was inserted into the GPD/PGK cassette between the HindIII and SmaI sites (Figure Southern blot analysis
DNA was prepared from the transformants as described by Rose et al. (1990) and Southern blot analysis was carried out. These DNA samples were digested with SalI and blotted onto nylon transfer membrane using 0.4 N NaOH. The 1.0 kb probe DNA, which is derived from the GPD promoter region of pSY114, was labeled with 32P using Megaprime DNA labeling systems (Amersham). The membrane was subjected to x-ray film autoradiography. The intensity of each band derived from the GPD/GnT-I/PGK cassette was measured using the Bioimaging analyzer (BAS2000, Fuji Film). The chromosomal GPD gene was used as a standard for calculating the intensity. GnT-I assays
GnT-I activity was measured as described previously (Schachter et al., 1989), except for the use of a pyridylaminated (PA) oligosaccharide as a substrate. The assay mixture contained 100 mM MES (pH 6.1), 100 mM GlcNAc, 5 mM AMP, prewarmed 0.5% BSA, 20 mM MnCl2, 10 mM UDP-GlcNAc, 2 µM PA oligosaccharide, and yeast extract in a total volume of 50 µl. The substrate oligosaccharide, Man[alpha]1-6(Man[alpha]1-3)Man[alpha]1-6(Man[alpha]1-3)Man[beta]1-4GlcNAc[beta]1-4GlcNAc-PA (PA017), was purchased from Takara Shuzo. The assay mixture was incubated at 37°C for 60 min. The reaction was stopped by boiling for 2 min, filtered through a Ultrafree C3GV microtube (Millipore), and subjected to HPLC analysis.
The Tsk-gel Amide-80 column ([phis]4.6 × 250 mm, Toso) was equilibrated with a solvent containing acetonitril and 3% acetic acid, 10 mM triethanolamine (pH 7.3) at a ratio of 70/30 (v/v). The assay products were eluted isocratically at a rate of 1 ml/min using the same solvent and were detected by the fluorescence of the PA tag; [lambda]ex 320 nm to [lambda]m 400 nm. The structure of the reaction product was identified by proton-NMR. Determination of protein concentration
Protein concentration was determined using the bicinchoninic acid reagent (Pierce) with bovine serum albumin as a standard. Polyacrylamide gel electrophoresis and Western blot analysis
Proteins were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. The gels were electrophoretically blotted onto PVDF membranes (Millipore). The membranes were probed with a rabbit polyclonal antibody raised against the recombinant rat GnT-I fusion protein or with a mouse monoclonal c-Myc antibody (9E10) purchased from Santa Cruz Biotechnology Inc. The rabbit and mouse antibodies were detected using an ECL detection system (Amersham) with a goat anti-rabbit and anti-mouse IgG coupled to horseradish peroxidase (HRP), respectively. Subcellular fractionation of yeast cell lysate
Yeast cells were grown to a density of 1 × 107 cells/ml in 500 ml of SD medium. The cells were converted to spheroplasts as described previously (Nishikawa and Nakano, 1991). The spheroplasts were resuspended in 10 ml of ice-cold lysis buffer (10 mM triethanolamine acetate (pH 7.5), 250 mM sorbitol, 2 µg/ml antipain, 2 µg/ml chymostatin, 3 µg/ml pepstatin A, 2 µg/ml leupeptin, 1 mM benzamidine-HCl, 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonylfluoride (PMSF)) and were immediately homogenized six times for 1 min period with 1 min intervals on ice using a motor-driven 30 ml Potter-Elvehjem homogenizer (Wheaton). The lysate was centrifuged three times at 500 × g for 5 min to remove unbroken spheroplasts. The supernatant was subjected to centrifugation at 10,000 × g for 15 min at 4°C in an RT100.3 rotor (Beckman) to yield a low-speed pellet (LSP) and a low-speed supernatant (LSS) fraction. The LSS fraction was further centrifuged at 100,000 × g for 60 min at 4°C in an RT100.3 rotor to yield a high-speed pellet (HSP) and a high-speed supernatant (HSS) fraction.
The LSP fraction was subjected to differential centrifugation as described by Gaynor et al. (1994). Eighty percent of the LSP fraction was resuspended in 50 µl of lysis buffer and layered on a sucrose bilayer consisting of 900 µl of 1.5 M sucrose and 900 µl of 1.2 M sucrose in lysis buffer. After an 85,000 × g spin for 1 hr, 10 equivalent fractions were removed and subjected to Western blot analysis and were assayed for GDPase. Anti-Sec12p and anti-Vma1p antibodies were used in Western blotting to detect marker proteins in the ER and vacuole, respectively. These antibodies were kindly provided by Drs. Nakano and Anraku, respectively. GDPase activity was assayed as a marker enzyme for the Golgi apparatus as described previously (Abeijon et al., 1989). Orientation of GnT-I in the yeast membrane
The susceptibility of GnT-I in the membrane fractions to tryptic digestion was examined as described previously (Nakano et al., 1988). The yeast cell homogenate, which was prepared as described above, was treated with or without 0.5 mg/ml trypsin in the absence or presence of 0.1% Triton X-100 on ice. After the indicated time (0-30 min), the tryptic digestion was stopped by the addition of 1.1 mg/ml soybean trypsin inhibitor (Sigma). Each sample equivalent to 1.0 OD600 of cells was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting with anti-c-Myc or anti-GnT-I antibody.
We thank Drs. Nakano, Ohya, and Anraku (University of Tokyo) for providing antibodies against Sec12p and Vma1p; Dr. Jigami (National Institute of Bioscience and Human Technology) for providing EHA-1C strain; and Dr. Miura for technical assistance for Southern blot analysis. This work was performed as a part of the R&D Project of Industrial Science and Technology Frontier Program supported by NEDO (New Energy and Industrial Technology Development Organization).
Asn, asparagine; ER, endoplasmic reticulum; GalT, UDP-galactose:N-acetylglucosamine [beta]-1,4-galactosyltransferase (EC 2.4.1.38/90); GnT-I, UDP-N-acetylglucosamine:[alpha]-3-d-mannoside b-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101); PA, pyridylaminated; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecylsulfate; SD, synthetic minimum dextrose.
2To whom correspondence should be addressed: KIRIN Brewery Co., Ltd., Central Laboratories for Key Technology, 1-13-5, Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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