Glycosylation is one of the major posttranslational modification. This modification plays a key role in the biological function of native glycoproteins in eucaryotic cells. Two representing structures of oligosaccharides are N-glycan attached to asparagine residues and O-glycan attached to serine or threonine residues in a peptide backbone. In spite of remarkable differences in final structures between mammals and lower eucaryotes such as Saccharomyces cerevisiae, they share a common oligosaccharide core intermediate in the ER (reviewed in Lehle and Tanner, 1995; Trimble and Verostek, 1995; Orlean, 1997). Therefore, yeast has been used to study the molecular mechanisms of this common glycosylation pathway (Lehle and Tanner, 1995; Eckart and Bussineau, 1996).
Yeast has also been used as a production host of recombinant proteins. However, one of the major drawbacks for yeast as a host cell is the lack of further biosynthetic pathways for complex type oligosaccharides formation. This sometimes alters the biological function and/or immunogenicity of particular proteins (Schultz et al., 1987; Ballou, 1990; Baenziger, 1994). In order to clarify the biological function of diverse oligosaccharides and to construct a desirable yeast expression system for glycoproteins of therapeutic use, we have been manipulating the glycosylation system of S.cerevisiae by introducing mutations responsible for oligosaccharide biosynthesis. So far we have constructed [Delta]och1mnn1 double mutant cells which produce Man8GlcNAc2, high mannose-type (ER-core) oligosaccharides (Nakayama et al., 1992; Nakanishi-Shindo et al., 1993), and [Delta]och1[Delta]mnn1[Delta]mnn4 triple mutant cells (Odani et al., 1996) which produce less yeast specific mannosylphosphorylated Man8GlcNAc2. The next stage for sugar structure remodeling is to add the complex type oligosaccharides to this ER-core intermediate.
As one such example, Schwientek et al. introduced human [beta]1,4 galactosyltransferase ([beta]1,4 GalT) in an alg1 mutant strain (Schwientek et al., 1996). This strain produced Gal[beta]1,4-GlcNAc[beta]1,4-GlcNAc at the nonpermissive temperature, suggesting S.cerevisiae could import UDP-Gal into the Golgi lumen. However, it remained unsolved how UDP-Gal was transported into the Golgi lumen. Recently, we have reported in vivo galactose incorporation to mannoproteins by expressing the Schizosaccharomyces pombe [alpha]1,2 GalT gene in the S.cerevisiae with mnn1 background strain R16B (Roy et al., 1998). Furthermore, we have detected the presence of UDP-Gal transport activity in this transformed S.cerevisiae R16B strain in in vitro assay. Therefore, we reasoned that this endogenous activity allowed UDP-Gal to be transported from cytoplasm to the Golgi lumen where galactose transfer takes place. However, unlike S.pombe, only 15-20% of the total cells were galactosylated in this strain. Thus we hypothesized that not only the production and localization of GalT but also the efficient supply and its transport of UDP-Gal should be the key for the effective in vivo galactosylation in S.cerevisiae.
Recently, complementary DNAs for the human and S.pombe UDP-Gal transporters have been cloned and characterized (Ishida et al., 1996; Miura et al., 1996; Tabuchi et al., 1997; Yoshioka et al., 1997). Other nucleotide sugar transporter cDNAs including the murine CMP-sialic acid transporter have also been cloned in the past few years (Abeijon et al., 1996; Eckhardt et al., 1996; Dean et al., 1997; Ma et al., 1997). Of these, human UDP-Gal transporter and murine CMP-sialic acid transporter have been heterologously expressedin S.cerevisiae in active form to demonstrate in vitro that these cDNAs encode the transporter per se, but not regulatory proteins (Berninsone et al., 1997; Sun-Wada et al., 1998). Nonetheless, there is no in vivo study to demonstrate whether the expressed foreign sugar nucleotide transporter functions in S.cerevisiae or not.
In this report, we have first developed the in vivo neo-galactosylation system in S.cerevisiae. Using this system, we have then analyzed the limiting factor(s) for galactosylation which includes the expression level of S.pombe [alpha]1,2 GalT (Chappell and Warren, 1989; Chappell et al., 1994) and the effect of UDP-Gal transporter from human origin (Ishida et al., 1996). This is the first report to demonstrate that the human UDP-Gal transporter expressed in S.cerevisiae functions for the in vivo galactosylation and to clarify that this UDP-Gal transporter rather than [alpha]1,2 GalT is the major limiting factor for efficient galactosylation in S.cerevisiae. We have also addressed the in vivo target acceptor of S.pombe [alpha]1,2 GalT using this heterologous system and demonstrated that this enzyme transfers galactose to both N- and O-linked oligosaccharides. A further characterization indicates that this [alpha]1,2 GalT transfers galactose to O-mannosyl-Ser/Thr and generates Gal-Man-Ser/Thr structure. Coexpression of [alpha]1,2 galactosyltransferase and UDP-galactose transporter genes in S.cerevisiae
To provide galactosyltransferase activity in S.cerevisiae, the gma12+ gene encoding S.pombe [alpha]1,2-galactosyltransferase (Chappell et al., 1994) was overexpressed in strain W303-1A that was designated as MKY1. The product of gma12+ was already confirmed to retain its activity after efficient localization at the Golgi membranes in S.cerevisiae (Roy et al., 1998). To provide a high level of UDP-Gal transporter activity, human UGT2 cDNA (Ishida et al., 1996) was expressed under the control of the strong TDH3 promoter in W303-1A to construct MKY3. In S.cerevisiae exogenous galactose is converted to UDP-Gal by the endogenous enzymes, encoded by GAL1, GAL10, and GAL7 gene clusters located in the right arm of chromosome II, whose expression is induced by galactose (St. John and Davis, 1981). Therefore, the strains were grown in galactose containing media to supply UDP-Gal as a donor substrate. The galactose incorporation to the cell surface was judged by monitoring the fluorescence intensity using FITC conjugated [alpha]-galactose specific lectin, GS I-B4. The results are summarized in Table I. Neither strains MKY1 nor MKY3 were stained. When both genes were coexpressed in a multicopy plasmid ~50% of MKY5 cells were stained, indicating an increased galactose incorporation into cell surface glycoproteins compared to our previous results (15-20% of total cells; Roy et al., 1998). Microscopic views of the wild type W303-1A and strain MKY5 are shown in Figure
Figure 1. FITC conjugated GS I-B4 lectin staining of W303-1A and isogenic strains harboring [alpha]1,2 GalT and UDP-Gal transporter. W303-1A; parental strain (A), MKY5; W303-1A harboring multicopy plasmids, YEpUGAPgma12+ and pUGT2 (B), and MKY14; chromosomal integration of gma12+ and hUGT2 genes in the [Delta]mnn1 background (C). They were grown in either YP (A and C) or synthetic (B) media containing galactose and stained with FITC-GS I-B4. Light micrograph, Nomarski (left), and fluorescence (right) are shown.
Table I. Overexpression of gma12+ gene impairs cell growth and morphology
Figure 2. Western immunoblot analysis of hUGT2 protein. Microsomal fractions were prepared from Had-1 cells harboring UGT2 (lane 1) and the various yeast constructs; W303-1A (lane 2), MKY5 (lane 3), MKY13 (lane 4) and MKY14 (lane 5) as described in Materials and methods. Equal amounts (40 µg) of microsomal proteins were loaded on SDS-PAGE (12%), subjected to immunoblot analysis using anti-UGT2 antibody. The bar on the right identifies the position where UGT2 migrates. Molecular mass markers are shown on the left in kilodaltons.
In the course of our experiments, we noticed that when gma12+ was overexpressed in a multicopy plasmid, yeast cells showed a slow growth and abnormal morphology with multibudded and elongated forms (Figure
Figure 3. The effects of gma12+ and hUGT2 dosage on cell morphology. Light micrographs of various strains grown to mid-logarithmic phase in galactose containing synthetic media are shown. MKY1; overexpression of gma12+ (A), MKY3; overexpression of hUGT2 (B), MKY5; co-overexpression of gma12+ and UGT2 (C), and MKY6; single copy expression of gma12 + (D). Magnification is the same for all strains. [Delta]mnn1 provides a better acceptor for galactosylation in S.cerevisiae
In strain MKY8 ~50% of the cells showed a galactose incorporation into their cell surface. One reason for the partial incorporation of galactose may be due to the limitation of suitable acceptor structure for S.pombe [alpha]1,2-GalT in S.cerevisiae. When gma12+ gene was expressed in S.cerevisiae, the enzyme showed a higher activity towards mannoproteins isolated from mnn1, which lacks [alpha]1,3 mannosyltransferase, than those isolated from wild type or mnn2 strains, which lacks first [alpha]1,2 linked mannose, in vitro (Roy et al., 1998). To examine whether mannoproteins lacking [alpha]1,3 linked terminal mannose serve as better acceptors for [alpha]1,2 galactosylation in vivo, the MNN1 gene that encodes terminal [alpha]1,3 mannosyltransferase was disrupted in the MKY6 background. While the resulting strain MKY12 harboring [Delta]mnn1 and URA3::gma12+ did not show any lectin staining (Table I), strain MKY13 in which a multicopy plasmid pUGT2 was introduced into MKY12 showed approximately 60-70% of the cells staining by FITC-lectin (Table I). A direct effect of [Delta]mnn1 mutation was confirmed by comparing the fluorescence intensity between strains MKY8 and MKY13 (50% versus 65% staining; Table I). Taken together, these results indicate that mannoproteins lacking terminal [alpha]1,3 mannosyl residue indeed provide a better acceptor for Gma12p. The necessity of coexpression of [alpha]1,2 GalT and hUGT2 for galactosylation (MKY12 versus MKY13) are consistent with the previous result (MKY1 versus MKY5), independent from the supply of preferable acceptor by [Delta]mnn1 mutation.
Integration of both [alpha]1,2 GalT and hUGT2 to the yeast chromosome allowed us to grow the cells in nonselective YP media (MKY14). When strain MKY14 thus constructed was grown in YPG, 80% of the cells were uniformly stained by FITC-conjugated GS I-B4 (Table I, Figure
The fluorescence intensity of each strain was quantitatively evaluated by FACS analysis (Figure
Figure 4. FACS analyses of various constructs. Cells grown in either synthetic or YP media containing galactose were stained with FITC conjugated lectin GS I-B4. Fluorescence intensity was measured by FACS as described in Materials and methods. W303-1A (A) grown in YPG and strains MKY5 (B), MKY8 (C), and MKY13 (D) grown in synthetic media containing galactose are shown. MKY14 grown in either YPD (E) or YPG (G) are also shown. UDP-galactose transporter is the limiting factor for galactosylation
From the above results on fluorescence staining and Western blot analyses using several strains which harbor a single copy of chromosomally integrated gma12+ gene, we hypothesized that the level of UDP-galactose transporter activity should be the limiting factor for the in vivo galactosylation rather than the GalT activity. To understand the relationship between the effect of in vivo galactosylation and UDP-galactose transporter activity, we performed the in vitro UDP-galactose transport assay using strains W303-1A, MKY8, MKY12, MKY13, and MKY14 (Figure
Figure 5. UDP-galactose transporter activities of various constructs. P3 microsomal fractions of W303-1A, MKY8, MKY12, MKY13, and MKY14 were prepared as described in Materials and methods. Each fraction (250 µg) was incubated with 5 µM UDP-[3H]Gal (456 cpm/pmol) at 30°C in a final volume of 300 µl. After incubation the transporter activity was measured as described in Materials and methods. Values are means ± SD from quadruplicate experiments. In vivo galactosylation of N-linked oligosaccharides in S.cerevisiae
By FITC conjugated lectin staining, we demonstrated incorporation of galactose to the cell surface of S.cerevisiae. However, it does not specify its transfer to N- or O-linked oligosaccharides of mannoproteins. Mannoproteins prepared from both wild type and MKY14 cells were subjected to Endo H digestion to release N-linked oligosaccharides. Monosaccharide contents of those oligosaccharides were analyzed as described in Materials and methods. As shown in Figure
Figure 6. Monosaccharides analysis of N-linked mannoproteins from strains W303-1A and MKY14. Total cell surface mannoproteins prepared either from W303-1A grown in galactose or MKY14 grown in either galactose or glucose were subjected to Endo H digestion and mild acid treatment followed by pyridylamination. Each sample was applied on anion exchange column PALPAK Type A to separate constituent monosaccharides. Separated PA-monosaccharide samples were compared with authentic PA-monosaccharides for the identification (data not shown). MKY14 grown in galactose (A) and glucose (B); W303-1A grown in galactose (C). PA-glucose (Glc), PA-mannose (Man) and PA-galactose (Gal) peaks are assigned after comparison of their retention times with those of authentic standards. The origin of glucose is unidentified. In vivo galactosylation of O-linked oligosaccharides in S.cerevisiae
We further examined the possibility of galactosylation of O-linked oligosaccharides. It was known that S.cerevisiae chitinase secreted into the culture medium is exclusively mannosylated with a series of short O-linked oligosaccharides (Kuranda and Robbins, 1991). Chitinase was purified from culture media by chitin binding after the wild type and MKY14 strains were grown in either YPD or YPG media. Its purity was confirmed by Coomassie staining (Figure
Figure 7. Lectin blot analysis of chitinase from strains W303-1A and MKY14. Extracellular chitinase was purified as described in Materials and methods from W303-1A (lanes 1 and 2) and MKY14 (lanes 3 and 4) cells grown in either galactose (lanes 1 and 3) or glucose (lanes 2 and 4). Equal amount of protein extracts was separated on SDS-PAGE (7.5%), subjected to Commassie staining (A), or electrotransferred, then followed by ConA (B) or GS I-B4 (C) lectin detection, as described in Materials and methods. Molecular mass is indicated in kilodaltons on the left of (A).
Further, to confirm which mannose is occupied by [alpha]1,2 linked galactose(s), HPLC analysis of O-linked oligosaccharides were conducted. O-Linked oligosaccharides of purified chitinase were prepared from MKY14 cultures grown in either galactose or glucose containing YP media. The PA labeled neutral O-linked oligosaccharides were analyzed by the amine column as described in Materials and methods. When the HPLC profiles of the samples prepared from strain MKY14 grown in either glucose or galactose were compared (Figure
Figure 8. HPLC analysis of O-linked oligosaccharides released from chitinase. O-Linked oligosaccharides were prepared from extracellular chitinase as described in Materials and methods. HPLC profiles using amine column are shown: MKY14 grown in YPD (A), MKY14 grown in YPG (B), total PA-oligosaccharides shown in panel B were digested with [alpha]-mannosidase (C), or with [alpha]-galactosidase (D), and the 20 min peak shown in (B) (asterisk) was pooled from four HPLC runs (E). The peak shown in (E) was digested with [alpha]-galactosidase resulting in a shift of the corresponding peak to 13 min (M1) (F). The peaks designated as M1 to M4 represent PA-labeled sugar chains containing one to four mannose residues after confirming their elution times with authentic PA-labeled M1-M4 oligosaccharides. Asterisk indicates the galactose containing peak. Generation of galactosylation system in S.cerevisiae
We have demonstrated the in vivo functional expression of a heterologous sugar nucleotide transporter, UDP-Gal transporter, in S.cerevisiae. Expression of this membrane protein directly contributed to the efficient in vivo galactosylation in S.cerevisiae. Our new strain which comprises integration of S.pombe [alpha]1,2 GalT and human UDP-Gal transporter genes in the [Delta]mnn1 background cell showed a highly efficient galactose incorporation into both N- and O-linked mannoproteins. By testing various combinations of factors involved in galactosylation, we confirmed that the following components are important for efficient in vivo glycosylation (1) expression of glycosyltransferase, (2) expression of sugar nucleotide transporter, (3) supply of donor substrates in cytosol, and (4) provision of acceptor oligosaccharides in the Golgi lumen. Our results on in vivo galactosylation and in vitro UDP-galactose transport assay strongly suggest that not the expression level of [alpha]1,2 GalT but the availability of UDP-Gal in the Golgi lumen plays a key role in de novo galactosylation in S.cerevisiae (Table I; MKY1 versus MKY5, MKY6 versus MKY8, and MKY12 versus MKY13 and 14; and Figure
In a previous report, the expression of S.pombe [alpha]1,2 GalT in the S.cerevisiae strain with mnn1 background displayed partial galactosylation in vivo (15-20% of total cells; Roy et al., 1998). In contrast, in this study the expression of gma12+ gene alone (MKY1) or the strain expressing gma12+ gene in the [Delta]mnn1 background (MKY12) did not evoke any in vivo galactosylation in S.cerevisiae cells, although the in vitro UDP-galactose transport activity was comparable between R16B expressing gma12+ gene and MKY12 (~10 pmol/mg protein/5 min). One possible reason is the difference in the strain backgrounds between R16B and W303-1A. Strain R16B used in the previous report (Roy et al., 1998) was constructed by various crossover with other non-isogenic strains which sometimes introduce unknown mutations. For example, different from W303-1A, aggregation of cells was observed in liquid medium when gma12+ was overexpressed in strain R16B (S. K. Roy, personal communication). In the work reported here, we performed stepwise the gene integration or gene disruption starting from well characterized haploid laboratory strain, W303-1A, without any cross. This enabled us to compare the effect of each gene function on galactosylation in parallel. The in vitro activity detected in MKY12 comparable to strain R16B may not be sufficient for in vivo galactosylation in this strain.
An aberrant cell morphology was observed when the BED1 gene was disrupted in S.cerevisiae (Mondésert and Reed, 1996). The BED1 gene was cloned based on its involvement in bud emergence and polarized growth. This gene is identical to the MNN10 gene which takes part in outer chain mannosylation in S.cerevisiae (Dean and Poster, 1996). Interestingly, the BED1/MNN10 gene has a significant sequence homology to the S.pombe gma12+ gene (Dean and Poster, 1996; Mondésert and Reed, 1996). Overexpression of the gma12+ gene under the TDH3 promoter in the multicopy plasmid also caused an abnormal cell morphology with an impaired growth phenotype. Elongated, larger and multibudded cells were observed (Figure
The growth rate of parental strain W303-1A and strain MKY14 was measured to see how galactosylation affects cell growth in S.cerevisiae. The wild type W303-1A and MKY14 strain grown in YPG media showed a doubling time of 2.5 h and 5.0 h, respectively, while both strains grown in glucose showed no significant differences in their growth rate. The galactosylation of mannoproteins may cause some inhibition of cell growth in S.cerevisiae.
The Gma12 p was originally purified based on its activity of galactose transfer from UDP-Gal to a variety of mannose-based acceptors to form an [alpha]1,2 galactosyl mannoside linkage (Chappell and Warren, 1989; Chappell et al., 1994). In in vitro studies [alpha]-methyl d-mannoside showed a fourfold increase in specific activity relative to d-mannose (Chappell and Warren, 1989). However, real in vivo acceptor substrates have not been identified and there is no direct evidence for involvement in galactosylation of N- and/or O-linked oligosaccharides. S.cerevisiae heterologously expressing GalT derived from S.pombe allowed us to study the in vivo acceptor specificity of the enzyme because no GalT has been identified in S.cerevisiae (Herscovics and Orlean, 1993). In this report, we have demonstrated that the [alpha]1,2 GalT encoded by the gma12+ gene acts on both N- and O-linked oligosaccharides in vivo (Figures
We can, however, postulate two reasons for detecting only Gal-Man-Ser/Thr structure but neither Gal-Gal-Man-Ser/Thr nor Gal-Man-Man-Ser/Thr structures which are constituents in S.pombe (Ballou et al., 1994). Since it has been reported that several galactosyltransferases are present in S.pombe (T. Yoko-o, personal communication; Chappell et al., 1994), it is likely that Gma12p is responsible for the first galactose addition and the other GalTs for the subsequent galactose addition in O-linked oligosaccharides of S.pombe. This is supported by the observation that Gma12p has no activity towards mannobiose in vitro (S. K. Roy, personal communication). Alternatively, since the galactosyltransferase is expressed in a heterologous system, the [alpha]1,2 galactosyltransferase is competing for the second sugar transfer with [alpha]1,2 mannosyltransferases (Ktr1p, Ktr3p, and Kre2p/Mnt1p), all of which are known to be involved in the second and third mannose additions in O-linked oligosaccharide of S.cerevisiae (Lussier et al., 1997). The [alpha]1,2 GalT was able to compete for the second sugar transfer but not for the third sugar transfer presumably due to the improper localization or less affinity towards disaccharides such as mannobiose or mannosylgalactose. This competition between Kre2 family proteins and Gma12p for the second sugar transfer also supports the idea that [alpha]1,2 GalT may be localized in the early Golgi compartment where Kre2 protein family is localized in S.cerevisiae (Chapman and Munro,1994; Lussier et al., 1995). Previously it was speculated that Gma12p resides in a cis compartment with other mannosyltransferase activity in S.pombe (Chappell et al., 1994). Further study on the specific localization and retention mechanism of Gma12p and other galactosyltransferases in S.pombe is awaited.
Our yeast strains constructed here producing sugar chains with [alpha]1,2 linked galactose are not directly applicable to produce glycoprotein therapeutics because normal human complex-type oligosaccharides contain only [beta]-linked galactose. Also the amount of actual galactose resides incorporated into the total oligosaccharides were not in a large quantity. However, the information obtained in this study will be useful for the construction of yeast strains which produces a desired glycoform of heterologous proteins in S.cerevisiae. In addition the use of such a heterologous system will contribute to further understanding the functional relationship of the expressed foreign proteins, such as glycosyltransferases and sugar nucleotide transporters, in the Golgi apparatus. Strains and genetic manipulations
The genotypes and sources of S.cerevisiae strains used or constructed in this work are summarized in Table I. YP medium (YP) and synthetic minimal medium (S), supplemented with the relevant amino acids, bases and 2% glucose (D) or 2% galactose (G), were prepared as described previously (Rose et al., 1990). Cultures were grown at 30°C and were monitored by measuring optical density at 600 nm (1 OD600 = 5 × 10 5 cells/ml). Yeast DNA transformation was carried out as follows: aliquots of 5 × 105 cells at early stationary phase were centrifuged and resuspended in 0.1 ml of 0.2 N lithium acetate, 40% polyethylene glycol 3350-4000, 10 mM DTT. Three micrograms of salmon sperm and 1 µg of the transforming DNA were mixed into the cell suspensions and incubated for 30 min at 45°C. The cell suspension was then directly plated on selective medium and incubated at 30°C. Plasmid and various strain constructions
YEpUGAPgma12+ constructed previously was used for [alpha]1,2 GalT expression in S.cerevisiae (Roy et al., 1998). The multicopy plasmid pUGT2 which contains human UDP-Gal transporter cDNA was constructed as follows: A 1.2 kb EcoRI fragment containing the coding region of hUGT2 from pGEM75zf(+)-UGT2 (Ishida et al., 1996) was excised and ligated into pKT10 (Tanaka et al., 1990; Roy et al., 1998) at the EcoRI site downstream of yeast TDH3 promoter. Then a 2.2 kb BamHI fragment which contained the TDH3 promoter, hUGT2, and TDH3 terminator was ligated into the BamHI site of YEp351 (Hill et al., 1986). The final plasmid was designated as pUGT2. The W303-1A strain which was transformed by YEpUGAPgma12+ was called MKY1. MKY3 was constructed by introducing pUGT2 into W303-1A. MKY1 was transformed with pUGT2 to obtain MKY5. To integrate gma12+gene in the S.cerevisiae chromosome, YEpUGAPgma12+ was linearized at the EcoRV site of the URA3 gene and introduced at the ura3 locus of W303-1A. Stable Ura+ transformants were chosen and the integration of single copy of gma12+ at the ura3 locus of W303-1A strain was confirmed by Southern blot analysis (data not shown). One of the resulting strains was named MKY6. MKY6 was transformed with pUGT2 to obtain MKY8.
To disrupt the MNN1 gene pHYH-[Delta]mnn1 was generated as follows. A 1.8 kb SphI fragment from pJJ244-[Delta]mnn1 (kindly provided by Y. Shimma) was ligated into the SphI site of pHYH (Schwelberger et al., 1993). pHYH-[Delta]mnn1 was digested at the NotI site and the linearized fragment containing the disrupted MNN1 gene was introduced into MKY6 by one-step gene disruption/deletion (Rothstein, 1983). Stable His+ transformants were selected, and the integrative disruption at the MNN1 locus was confirmed by PCR (data not shown). One of the transformants was designated as MKY12.
To integrate hUGT2 at the leu2 locus of chromosome, YIp352-UGT2 was constructed by excising the HpaI/AatII fragment to remove the 2 µm circle, followed by blunt-ending and religating the remaining fragment. The YIp352-UGT2 was linearized at the EcoRV site of the LEU2 gene and introduced into MKY12 to generate MKY14. The integration of single copy of hUGT2 at the leu2 locus was confirmed by Southern blot analysis (data not shown). Western immunoblotting
Yeast microsomal fraction was prepared by growing the strains in either YP or synthetic media containing galactose or glucose to the mid logarithmic phase. The cells were lysed by glass beads in the buffer containing 10 mM Tris-HCl, pH 7.5, 2 mM phenylmethanesulfonyl fluoride, 10 mM DTT, and 5 µg/ml protease inhibitor (leupeptin, pepstatin A and aprotinin) and centrifuged at 1000 × g for 5 min followed by 100,000 × g for 60 min. The pellet was resuspended in the same buffer. Microsomal fraction from Had-1 cells expressing hUGT2 gene was prepared as described previously (Yoshioka et al., 1997). The samples were fractionated on a 12% SDS-PAGE and Western immunoblotting was carried out using anti-hUGT2 polyclonal antibody as described previously (Yoshioka et al., 1997). FITC-conjugated lectin staining of various constructs
Each strain was grown in YP or synthetic media containing either glucose or galactose as a carbon source at 30°C to mid-logarithmic phase. Aliquots containing 1 × 105 cells were centrifuged and treated with fluorescein isothiocyanate (FITC)-conjugated Griffonia simplicifolia I-B4 (GS I-B4) lectin (Vector Laboratories, USA) as described previously (Roy et al., 1998). The cells were then subjected to the microscopic or FACS analysis. Fluorescent activated cell sorting (FACS) analysis
FITC-labeled cells were analyzed by FACSCalibur (Becton Dickinson) to detect fluorescence intensity. The instrument is equipped with argon-ion laser which is operated at 488 nm with 15 mW energy. Emission of FITC was measured at 560 nm. Cells were passed through flow cytometers, and 10,000 events were counted and analyzed with CELLQuest (Becton Dickinson). Subcellular fractionation and UDP-galactose transport assay
The microsomal fractionation was performed as described by Roy et al. (Roy et al., 1998). A P3 fraction (25 µg/µl) was used for the following UDP-galactose transport assay. The transport assay was performed as described previously (Roy et al., 1998) with some modifications. Briefly, a P3 fraction (250 µg) was incubated in a reaction mixture (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 5 mM MgCl2, 1 mM MnCl2, 10 mM 2-mercaptoethanol) with 5 µM UDP-[3H] galactose (specific activity, 456 cpm/pmol). After incubation at 30°C for 5 min, the reaction was terminated by 10-fold dilution with an ice-cold stop buffer (20 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl2) and placed on ice. Diluted samples were applied onto filtration apparatus containing HA filters (24 mm diameter, 0.45 µm pore size, Millipore). The filters were washed with the stop buffer, air-dried, and dissolved in 10 ml scintillation mixture (Clear-sol I, Nakarai Tesque). After 2 h at room temperature, the samples were counted in a liquid scintillation counter (Beckman LS1701). The amount of radioactivity that was bound nonspecifically to the outside of the vesicles was determined by zero time assay. HPLC analysis of N-linked monosaccharides
Total cell surface mannoproteins were prepared as described previously (Nakanishi-Shindo et al., 1993). Endoglycosidase H (Endo H) from Streptomyces griseus (Seikagaku Corp., Japan) was used to perform N-linked oligosaccharide digestion (Tarentino and Maley, 1974) according to the manufacturer's instructions. Monosaccharides were prepared from N-linked oligosaccharides by acid hydrolysis followed by N-acetylation as described previously (Suzuki et al., 1991). Each monosaccharide was labeled with 2-aminopyridine (PA) as described previously (Nakanishi-Shindo et al., 1993). Monosaccharide analysis was performed by using anion-exchange column, PALPAK Type A (Takara, Japan), and the products were identified with the PA-sugar standards (Takara, Japan). Isolation of extracellular chitinase by chitin binding
Extracellular chitinase was isolated as described previously with some modifications (Kuranda and Robbins, 1991). Briefly, intact chitinase (Cts1p) was isolated without any overexpression of the chitinase gene (CTS1). W303-1A and MKY14 cells were grown in 100 ml of either YPD or YPG medium until mid-logarithmic phase. The cells were pelleted and the supernatants were collected to which chitin pretreated with 1% SDS and 1% [beta]-mercaptoethanol was added to a final concentration of 1 µg/ml and then stirred at 4°C overnight. Chitinase bound to insoluble chitin was collected by filtration through Type HA membrane (0.45 mm, Millipore) and washed with Solution A (0.8% NaCl, 0.02% KCl, 0.12% Na2HPO4, 0.02% KH2PO4). Finally, the chitin-chitinase complex was suspended in 150 µl of the sample buffer (2% SDS, 5% [beta]-mercaptoethanol, 10% glycerol) and heated at 100°C for 10 min to release chitinase. Lectin blot analysis
Chitinase samples were fractionated by 7.5% SDS-PAGE (Laemmli, 1970) and subjected to electrotransfer to PVDF membranes (Amersham, Hybond-PVDF) using Towbin buffer (Towbin et al., 1979). Lectin blot analysis was carried out as described previously with some modifications (Cho et al., 1996). The blots were blocked with 5% BSA containing TBS (20 mM Tris-HCl, 500 mM NaCl, pH 7.5) with 2 µg/ml of streptavidin, then incubated with 3.5 µg/ml of biotinylated GS I-B4 (Vector Laboratories, USA) or biotinylated Con A (Vector Laboratories, USA) in a dilution buffer (TBS, 0.1% BSA, 0.3% Tween 20), followed by incubation with alkaline phosphatase-conjugated streptavidin (GIBCO-BRL, USA). The blots were developed as described previously (Wei et al., 1995). HPLC analysis of O-linked oligosaccharides
O-Linked oligosaccharides were prepared from chitinase bound chitin by hydrazinolysis (Hydrozinolysis Reagent C, Honen) using Hydraclub S-204 (Honen) according to the manufacturer's protocol. In brief, the sample was hydrolyzed by hydrazine at 65°C for 6 h, followed by N-acetylation by extracting the sample with 0.2 N ammonium acetate and acetic anhydride. The dried sample was pyridylaminated using pyridylamination kit (Takara Shuzo Co., Ltd.) according to the manufacturer's protocol. The PA-labeled sample was resuspended in H2O and applied to boronic acid column (Pierce Chemical Co.) and O-linked oligosaccharides were eluted by 0.2 M sorbitol. The boronic acid column binding fraction was further analyzed by Asahipak NH2P-50 (0.46 × 25 cm; Showa Denko Co., Tokyo) at the rate of 1.0 ml/min with solvent A (acetonitrile) and solvent B (200 mM acetic acid-triethylamine, pH 7.3). After the sample was injected, the proportion of B was increased linearly from 10% up to 60% for 60 min. Each peak was further collected and then subjected to either [alpha]-galactosidase (Sigma, EC 3.2.1.22) or [alpha]-mannosidase (Seikagaku Corp., EC 3.2.1.24) treatment according to manufacturers' protocols.
We are grateful to Y.Shimma and T.Odani for providing pJJ244-[Delta]mnn1; Y.Maeda and K.Nakayama for help with HPLC analysis; and S.K.Roy for suggestions on UDP-galactose transport assay, sharing unpublished data, and comments on the manuscript. M.K. is indebted to X.-D.Gao and Y.Maeda for helpful comments and discussion; and M.F.Cohen for English correction. This work was supported in part by a grant-in-aid of Research and Development Project of Basic Technologies for Future Industries from Ministry of International Trade and Industry, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be marked 'advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. M.K. was supported by a New Energy and Industrial Technology Development Organization (NEDO) postdoctoral fellowship.
4To whom correspondence should be addressed
Introduction
Results
Strain
Genotype
FITC-GSI-B4b
Reference
W303-1A
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100
-c
Wei et al.
MKY1
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [YEpUGAPgma12+]a
-
This study
MKY3
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [pUGT2]
-
This study
MKY5
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [YEpUGAPgma12+], [pUGT2]
50%d
This study
MKY6
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 URA3::gma12+
-
This study
MKY8
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 URA3::gma12+ [pUGT2]
50%~
This study
MKY12
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [Delta]mnn1::HIS3 URA3::gma12+
-
This study
MKY13
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [Delta]mnn1::HIS3 URA3::gma12+ [pUGT2]
60-70%
This study
MKY14
MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [Delta]mnn1::HIS3 URA3::gma12+ LEU2::UGT2
~80%
This study
Discussion
Materials and methods
Acknowledgments
References
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