Glycobiology Advance Access originally published online on March 19, 2003
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Glycobiology, 2003, Vol. 13, No. 8 581-589
© 2003 Oxford University Press
An N-acetylglucosaminyltransferase of the Golgi apparatus of the yeast Saccharomyces cerevisiae that can modify N-linked glycans
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Received on January 22, 2003; revised on February 26, 2003; accepted on February 26, 2003
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Abstract |
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The yeast Saccharomyces cerevisiae is widely regarded as being only capable of producing N-linked glycans with high-mannose structures. To investigate the glycan structures made in different mutant strains, we made use of a reporter protein consisting of a version of hen egg lysozyme that contains a single site for N-linked glycosylation. Mass spectrometry analysis of the attached glycans revealed that a large proportion contained an unexpected extra mass corresponding to a single N-acetylhexosamine residue. In addition, the glycosylated lysozyme was recognized by an N-acetylglucosamine specific lectin. The genome of S. cerevisiae contains an uncharacterized open reading frame, YOR320c, that is related to a known N-acetylglucosaminyltransferase. Deletion of this ORF resulted in the disappearance of the extra mass on the N-linked glycans and loss of lectin binding. We show that the protein encoded by YOR320c (which we term Gnt1p) is localized to the Golgi apparatus and has GlcNAc-transferase activity in vitro. The physiological role of Gnt1p is unclear because mutants lacking the protein show no obvious growth or cell wall defects. Nonetheless, these results indicate that heterologous glycoproteins expressed in yeast can receive N-glycans with structures other than high mannose. In addition, they indicate that the lumen of the yeast Golgi contains UDP-GlcNAc, which may facilitate reconstitution of higher eukaryotic N-glycan processing.
Key words: glycosylation / GNT1 / Golgi / N-acetylglucosaminyltransferase / yeast
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Introduction |
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N-linked glycans are based on a core structure that is attached to nascent glycoproteins as they are translocated into the endoplasmic reticulum (ER). This core is trimmed during protein folding to produce GlcNAc2Man8–9 structures that are then modified by enzymes in the Golgi apparatus in a manner that varies widely between species and even between individual cell types and proteins within a given species. In mammals several mannoses are removed before the generation of a diversity of complex structures containing such sugars as N-acetylglucosamine (GlcNAc), galactose, fucose, and sialic acid. In contrast, the yeast Saccharomyces cerevisiae does not trim the ER-derived N-glycan but extends it further to make one of two general structures (Dean, 1999
; Munro, 2001). These are a core-type structure, containing just a few extra residues, that is found on the glycoproteins of internal membranes and a mannan structure that consists of a long branched polymer of 
200 mannoses that is attached to many proteins of the cell wall and periplasmic space. Analysis of the core-type and mannan structures from both individual proteins and from bulk yeast cell wall protein has consistently found that they are made up entirely of mannose or phosphomannose in addition to the GlcNAc2Man8–9 core (Ballou et al., 1990; Hernandez et al., 1992; Nakanishi-Shindo et al., 1993; Olivero et al., 2000; Peat et al., 1961; Trimble and Atkinson, 1986). A large number of yeast mutants with defects in Golgi glycosylation have been isolated, which has allowed the identification of many (if not all) of the mannosyltransferases involved in Golgi processing. In addition, such mutants have revealed the transporters and other enzymes necessary to provide the Golgi lumen with nucleotide sugars and ion cofactors (Antebi and Fink, 1992; Dean et al., 1997).
Despite these differences from mammalian glycoprotein processing, yeast has attracted considerable interest as a system for the secretion of heterologous proteins. The folding environment of the yeast ER appears very similar to that of mammalian cells, and yeasts are genetically tractable and have low-cost growth requirements. The mannan structure represents a limitation because it is highly antigenic, but just as it is attached to only a subset of endogenous proteins, it is not attached to all exogenous proteins. The basis of this selectivity is not understood, but it has meant that both nonglycosylated and also glycosylated recombinant proteins with and without mannan have all been successfully secreted from yeasts. These include a hepatitis vaccine that receives no N-linked glycans in yeast (McAleer et al., 1984) and a recombinant granulocyte-macrophage stimulating factor that receives some O-linked sugars (but no mannan), which are in widespread clinical use. In addition, secretion of recombinant proteins has been investigated in mutants that lack mannan addition (Ip et al., 1992; Kang et al., 1998; Kniskern et al., 1994), or in other yeasts, such as Pichia pastoris, and filamentous fungi in which the mannan chain is shorter or more frequently absent (Bretthauer and Castellino, 1999; Maras et al., 1999; Murphy et al., 1998; Scorer et al., 1993; Zhu et al., 1997).
To understand more about the mechanism by which only some glycoproteins receive mannan we have examined the glycosylation of a simple reporter protein based on hen egg lysozyme. This protein is not normally glycosylated, but when a site for N-linked glycan is introduced by the mutation G49N, the resulting protein is glycosylated and then receives a mannan structure when expressed in yeast (Nakamura et al., 1993). The first modification step that is specific to the mannan pathway is the addition of an 
-1,6-linked polymer by mannan-polymerase I (M-Pol I), a complex of two mannosyltransferases Mnn9p and Van1p (Hernandez et al., 1989; Jungmann and Munro, 1998; Jungmann et al., 1999). Both of these proteins contain a DxD motif, a feature contained in many families of nucleotide-sugar using glycosyltransferases and shown to form part of the active site (Unligil and Rini, 2000; Wiggins and Munro, 1998). We have found that mutations in the DxD motif of either of Mnn9p or Van1p block mannan addition, even though the complex remains intact (Stolz and Munro, 2002). Lysozyme-G49N expressed from these two mutants had a slightly different mobility, suggesting that the two mutant complexes had retained differing residual activity. To investigate this further, the N-linked glycans on lysozyme-G49N were examined by mass spectrometry (MS). We report that the glycans from the two different mutants did differ in size, but in both cases most of the glycan structures contained an unexpected extra mass. We show that this is apparently a GlcNAc residue and that its attachment requires a previously uncharacterized and unanticipated GlcNAc-transferase that is present in the yeast Golgi apparatus.
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Results |
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MS analysis of the glycans attached to lysozyme-G49N
To follow the Golgi processing of N-linked glycans we previously used a reporter protein consisting of a glycosylated version of hen egg lysozyme (lysozyme-G49N) (Stolz and Munro, 2002
). This has the advantage that it is a small protein with just one N-glycan addition site, so any alteration in the gel mobility of the protein should reflect an alteration in glycan structure. When lysozyme-G49N is expressed in yeast it receives a mannan chain on the single N-linked glycan (Nakamura et al., 1993). However, in mutant strains in which either of the Golgi enzymes Van1p or Mnn9p are inactivated by mutation of their catalytic site DxD motifs (strains mnn9-AxD or van1-AxD) mannan synthesis is blocked as expected (Stolz and Munro, 2002). The mobility of the lysozyme-G49N produced by mnn9-AxD was slightly faster than that from van1-AxD, suggesting that Mnn9p might add the first residue of the mannan backbone. To examine this in more detail, the lysozyme-G49N from these strains was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the glycans removed by digestion with endoglycosidase F (endo F), and then examined by MS. To simplify analysis, the strains also lacked the Mnn1p -1,3-mannosyltransferase that adds terminal residues to both core-type and mannan structures (Alvarado et al., 1990).
Figure 1 shows the resulting spectra for the glycans from lysozyme-G49N secreted by the two mutant strains. As anticipated, the glycans from mnn9-AxD were smaller than those from the van1-AxD, but in both cases most of the glycans did not conform to the expected masses, that is, GlcNAc2Man8 with additional mannoses. Instead, the abundant species corresponded to GlcNAc2Man8–12 with an additional mass of 203 Da, which is that of a GlcNAc residue. To ensure that these unexpected masses were not a result of the isolation procedure, N-linked glycans from the well-characterized glycoprotein ribonuclease B were prepared and analyzed in the same manner. Figure 1C shows that these glycans showed the sizes and relative abundance expected from previous studies (Kuster et al., 1997), demonstrating that the unusual glycan masses were not a result of the methods used. This indicated that the glycan on lysozyme-G49N from these strains carries the addition of a single residue that does not appear to be mannose.
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The open reading frame YOR320c encodes a putative GlcNAc-transferase
Previous analyses of N-linked glycans from S. cerevisiae have not reported the addition to the N-linked core of residues other that mannose or phosphomannose (Ballou et al., 1990
; Olivero et al., 2000; Orlean, 1997; Peat et al., 1961). However, the S. cerevisiae genome contains an open reading frame (ORF) encoding a protein that is related to a known GlcNAc-transferase. This ORF, YOR320c, had not previously been characterized beyond being shown to be nonessential in the high-throughput analysis of the yeast genome (Winzeler et al., 1999). We have previously noted that the encoded protein contains a DxD motif, suggesting that it might be a glycosyltransferase (Wiggins and Munro, 1998), and it is also predicted to have a short transmembrane domain near its N-terminus characteristic of a Golgi localized enzyme (Levine et al., 2000) (Figure 2A). However, the sequence gave no further indication of its function until a related gene was cloned from the yeast Kluyveromyces lactis as corresponding to the mnn2-1 mutant that has defects in its mannan structure (Guillen et al., 1999). The mannan of K. lactis differs from that of S. cerevisiae in that it lacks phosphomannose but rather has terminal -1,2-linked GlcNAc residues on the side branches of the mannan outer chain. The mnn2-1 mutant lacks these residues and detectable GlcNAc-transferase activity (Smith et al., 1975). The K. lactis gene corresponding to mnn2-1 was termed Kl-GNT1 and encodes a protein that is 33% identical in its lumenal domain to that encoded by YOR320c. Related proteins sharing the DxD motif can also be found in the genomes of other yeasts and fungi including Candida and Aspergillus (Figure 2B). Although -1,2-linked GlcNAc has not been found in the mannan of S. cerevisiae, YOR320c seemed a plausible candidate to be a GlcNAc-transferase.
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Deletion of YOR320c affects the glycans attached to lysozyme-G49N
To determine whether the product of YOR320c was responsible for the unusual glycan structures we found on lysozyme-G49N, the YOR320c ORF was deleted from the mnn9-AxD and van1-AxD strains. Lysozyme-G49N was then expressed in these strains, and the protein was isolated from the media and the N-linked glycans released and analyzed by MS as before. Figure 3 shows that the unexpected peaks seen previously were now absent, and instead all the species observed were those with masses that can be accounted for by structures containing solely mannoses attached to the N-linked core structure. When the lysozyme-G49N secreted into the medium was analyzed by protein blotting, its mobility was apparently unaffected by deletion of YOR320c, although the protein from the mnn9-AxD strain still migrated slightly faster than that from van1-AxD, as we have previously reported (Stolz and Munro, 2002
) (Figure 3C). However, a lectin that is specific for GlcNAc residues, Griffonia simplicifolia lectin II (GS-II), showed greatly reduced binding to the lysozyme-G49N from the strains lacking YOR320c, whereas the binding to the mannose-specific lectin concanavalin A was unaltered. Taken together, these results indicate that the product of the YOR320c ORF is required for the unexpected structures seen on the lysozyme-G49N. In addition, the YOR320c-dependent binding by GS-II indicates that the extra residue present in these structures is GlcNAc. In light of these observations and the homology to K. lactis Kl-GNT1, and also data to be described shortly, we will refer to this S. cerevisiae YOR320c gene as GNT1 (GlcNAc-transferase).
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The pattern of N-glycan masses observed in the mnn9-AxD and van1-AxD strains in the absence of GNT1 activity shows an overall increase of one mannose in the latter case. This is consistent with the idea that Mnn9p in the M-Pol I complex adds the first mannose following the
-1,6-residue attached by Och1p and that further extension of the mannan backbone is dependent on the activity of Van1p (Stolz and Munro, 2002). The presence of a variable number of mannose residues is consistent with previous studies that have found that the ER-localized mannosidase Mns1p only acts on a fraction of GlcNAc2Man9 structures and that only a proportion of the truncated mannan backbone is branched with an -1,2-mannose (Ballou et al., 1991; Herscovics, 1999; Jakob et al., 1998; Trimble and Atkinson, 1992; Tsai et al., 1984).
GNT1 encodes a Golgi-localized membrane protein
To characterize the protein encoded by the GNT1/YOR320c gene, a triple hemagglutinin (HA) tag was inserted into the genome at the C-terminus of the ORF. Figure 4A shows that the resulting tagged Gnt1p migrated as a diffuse band of 70 kDa, which altered to a sharper band of 60 kDa following digestion with endo H to remove N-linked glycans. This is consistent with the amino acid sequence of Gnt1p, which predicts a size of 61 kDa and four sites for N-glycan attachment (Figure 2A). The presence of N-linked glycans on Gnt1p indicates that the portion of the protein C-terminal to the predicted transmembrane domain is in the Golgi lumen. To localize the protein within the secretory pathway, membranes from the strain expression Gnt1p-HA were separated on a velocity gradient, and fractions were blotted for organelle-specific markers and for the HA tag. Figure 4B shows that Gnt1p-HA comigrated with the Golgi and was clearly separate from the ER and vacuole. In addition, when the localization of Gnt1p-HA was examined by immunofluorescence, the protein was found to show substantial colocalization with the -1,3-mannosyltransferase Mnn1p, a resident of the medial Golgi (Lussier et al., 1995).
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Gnt1p has GlcNAc-transferase activity in vitro
To examine the enzymatic activity of Gnt1p in vitro, we used a protein A–tagged version of the protein isolated from cells on IgG Sepharose beads, an approach that we have been able to apply to a number of yeast Golgi enzymes (Jungmann et al., 1999
; Rayner and Munro, 1998; Stolz and Munro, 2002). Two copies of the protein A "Z" domain were inserted in the genome at the C-terminus of GNT1, and the resulting tagged protein (Gnt1p-ZZ) was isolated from detergent-solubilized cells. Previous studies on the K. lactis Kl-GNT1 gene had found that a mutation in the gene correlated with the loss of a transferase activity from cell lysates that could be measured using UDP-GlcNAc and the acceptor -1,3-mannobiose (Guillen et al., 1999). Thus, Gnt1p-ZZ immobilized on IgG Sepharose beads was incubated with UDP-[3H]GlcNAc and -1,3-mannobiose, and the products were separated by thin-layer chromatography (TLC). Figure 5A shows that there was some hydrolysis of the UDP-[3H]GlcNAc that was independent of substrate but was dependent on Gnt1p-ZZ because it was not seen with beads isolated from an untagged control strain. Such nucleotide sugar hydrolysis has been reported in a previous in vitro analysis of yeast glycosyltransferases (Doering, 1999) and may reflect the in vitro conditions not being a precise replica of the intra-Golgi milieu. Nonetheless, in the presence of the acceptor a labeled product was also produced demonstrating that Gnt1p has GlcNAc-transferase activity in vitro. As shown in Figure 5B, this activity required the divalent cation manganese, as has been observed for the activity of the K. lactis GlcNAc transferase and many other DxD-containing glycosyltransferases (Smith et al., 1975). Examination of different acceptors showed a preference for -1,3-mannobiose over other simple mannose-containing substrates (Figure 5C). However, the activity toward the larger GlcNAc2Man9 N-linked core structure showed a lower Km than that seen for -1,3-mannobiose (0.07 mM versus 6.0 mM; data not shown).
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Discussion |
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In this article we report that a heterologous glycoprotein expressed in S. cerevisiae receives an unexpected residue on its N-linked glycans that appears to be GlcNAc. This modification depends on the presence of a previously uncharacterized ORF, GNT1/YOR320c, which we show encodes a Golgi-localized glycoprotein that has GlcNAc-transferase activity in vitro. Presently the linkage formed by Gnt1p has not been defined, although the Gnt1p relative in K. lactis is required for the addition of an
-1,2-linked GlcNAc (Guillen et al., 1999; Smith et al., 1975). Indeed, we cannot at this stage exclude the formal possibility that in vivo Gnt1p carries out a GlcNAc-transferase reaction that is required for the activity of a second, unknown enzyme that is responsible for the addition of the GlcNAc observed on lysozyme-G49N. However, the simplest interpretation of our results is that Gnt1p is itself the GlcNAc-transferase that is responsible for directly modifying the N-linked glycan on lysozyme-G49N in vivo. In any case, these results have implications both for yeast cell biology and for the use of yeast as a system for the expression of recombinant glycoproteins.
Protein glycosylation in the yeast S. cerevisiae has been extensively studied for many decades, and this has revealed much of the enzymology of both Golgi and ER pathways of glycosylation, with the latter in particular being of direct relevance to mammalian systems (Aebi and Hennet, 2001; Dean, 1999; Orlean, 1997). The structure of N-linked glycans in yeast was initially addressed by examining total mannan released from cell walls (Peat et al., 1961). Further studies then examined the oligosaccharides attached to a number of endogenous proteins, including invertase, carboxypeptidase Y, and exoglucanase from both wild-type cells and those with mutants in mannan synthesis (Ballou et al., 1990; Hernandez et al., 1992; Lehle et al., 1979; Trimble and Atkinson, 1986). These studies have produced a consistent structure of yeast N-linked glycans that is based solely on mannose and phosphomannose, and we have not been able to find a single report suggesting the addition of further N-acetylhexosamine residues beyond the two GlcNAc residues found in the core structure. Although it is possible that minor species may have been missed or were not fully resolved by separation methods based on high-performance liquid chromatography (HPLC), it seems inconceivable that the Gnt1p-dependent modification is a universal feature of yeast N-linked glycans that has so far escaped detection. Indeed, we observed no difference in the binding of the GS-II lectin to total cellular proteins or to fixed cells when wild-type and 
gnt1 cells were compared (data not shown). This suggests that if Gnt1p does modify endogenous N-linked glycans, then it either acts on only a small percentage of proteins or only under special conditions.
The phenotype of yeast lacking GNT1 has provided few clues as to likely function. The gnt1 cells showed no change in sensitivity to caffeine, calcofluor white, or hygromycin, all of which have increased toxicity toward strains with cell wall defects (Dean, 1995; Ram et al., 1994), and there was no change in the mobility of invertase or increased secretion of the ER resident protein Kar2p (data not shown). It is possible that the normal substrate of the protein is not N-glycans, and it is perhaps noteworthy that GNT1 is located in the genome next to the PMT3 gene that encodes a protein O-mannosyltransferase (Immervoll et al., 1995). However, no GlcNAc has been found in the O-linked sugars from S. cerevisiae (Lussier et al., 1999). Nonetheless, the conservation of the gene in diverse yeasts and filamentous fungi, such as Candida, K. lactis, and Aspergillus, suggests that it must serve a function that is not highly species-specific. Of course, in K. lactis the protein appears to provide the GlcNAc in the mannan branches (Guillen et al., 1999; Smith et al., 1975). However, the other yeasts do not have this sugar in their mannan, so perhaps Kl-GNT1p in K. lactis was only recruited recently to mannan biogenesis. Mannan covers the outer surface of the yeast cell wall, and the structure of its branches varies greatly between yeast species, presumably reflecting an evolutionary pressure to evade hydrolytic enzymes and toxins, and in the case of pathogenic yeasts, neutralizing antibodies.
Irrespective of the in vivo role of this protein, the results described herein have possible implications for the use of S. cerevisiae as an expression system for recombinant glycoproteins. The Golgi-specific modification of N-linked glycans in yeast is clearly very different than that seen in mammals. However, the fact that yeast appear to have the capability to supply UDP-GlcNAc to the lumen of their Golgi means that converting yeast to make mammalian-type structures may require less engineering than previously anticipated. Yeast have already been found to have endogenous machinery capable of supplying UDP-GlcNAc and UDP-GalNAc to the lumen of the ER and Golgi, respectively (Roy et al., 1998, 2000). Indeed, the use of UDP-GlcNAc in the Golgi lumen by Gnt1p may provide an explanation for why S. cerevisiae has been found to have the capacity to degrade both GDP and UDP in the Golgi lumen when the only nucleotide sugar previously found to be required by endogenous Golgi glycosyltransferases was GDP-mannose (Abeijon et al., 1993; Gao et al., 1999; Lopez-Avalos et al., 2001).
Another implication of these findings is that not all heterologous glycoproteins expressed in yeast can be assumed to receive solely high-mannose structures on their N-linked glycans. S. cerevisiae has been tested as an expression system for a wide range of glycoproteins, including potential vaccines and therapeutic proteins. In many cases the recombinant glycoproteins receive mannan addition, and attempts have been made to avoid this by the use of mnn9 mutants or other yeasts. The N-glycans attached to some of these heterologous proteins have been examined in detail, including those from a glycosylated version of hepatitis surface antigen and from human trefoil factor expressed in S. cerevisiae (Ip et al., 1992; Kniskern et al., 1994; Kobayashi et al., 1992; Thim et al., 1993) and ß-lactoglobulin and tick antigens expressed in Pichia (Kalidas et al., 2001; Montesino et al., 1998). In these cases the glycans found conformed to the expected high-mannose structures, although in some cases this conclusion was based on the use of HPLC, which has a size resolution that is not as high as that of MS. However, the fact that Gnt1p appears to be able to efficiently modify lysozyme-G49N in vivo, and GlcNAc2 Man9 in vitro means that it seems possible that other heterologous glycoproteins could also be modified. It is not inconceivable that the presence of this extra residue could alter the circulation properties or the susceptibility to immunological responses of the resulting glycoprotein. Thus, it seems important to consider the Golgi addition of GlcNAc as a potential variable in the use of S. cerevisiae and other yeasts and fungi as expression systems for therapeutic glycoproteins. The apparent lack of effect on viability of deletion of the GNT1 gene at least provides a simple means to remove the modification if this is desired.
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Materials and methods |
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Yeast strains and plasmids
Yeast strains were based on the parental strain SEY6210 (MAT
ura3-52 leu2-3,112 his3-200 trp1-901 lys2-801 suc29) (Robinson et al., 1988). Strains lacking MNN1 and having AxD mutations in the genomic copies of MNN9 or VAN1 were as described (Stolz and Munro, 2002), and the YOR320c coding region was deleted in these by polymerase chain reaction (PCR)–based homologous recombination using Saccharomyces pombe his5+ (Wach et al., 1997). The GNT1 ORF was tagged with HA at the C-terminus using PCR-based homologous recombination and plasmid p3xHA-HIS5 (Jungmann et al., 1999). A similar approach was used for protein A–tagging Gnt1p for isolation for enzyme assays, except kanMX-based plasmid pFZ was used (Whyte and Munro, 2001), and the parental strain was the multiply protease deficient strain c13-ABYS 86 (MAT pra1-1 prb1-1 prc1-1 cps1-3 ura35 leu2-3,112 his3) (Heinemeyer et al., 1991). Lysozyme-G49N was expressed from the 2 µ plasmid pVT100-U-HELG49N (Stolz and Munro, 2002) and triple myc-tagged Mnn1p from its own promoter in a CEN plasmid (Wiggins and Munro, 1998).
Protein localization
Fractionation of yeast membranes on sucrose velocity gradients and localization of proteins by immunofluorescence were as described previously (Levine et al., 2000). Monoclonal antibodies against the HA epitope (3F10; Roche, Lewes, UK), Kar2p (2E7) (Napier et al., 1992), Vma1p (Molecular Probes, Eugene, OR), and rabbit polyclonal antibodies against Anp1p (Jungmann and Munro, 1998) and the myc-epitope (Santa Cruz Biotechnology, Santa Cruz, CA), were detected with species-specific secondary antisera labeled with fluorophores or peroxidase (Amersham Biosciences, Piscataway, NJ), and the latter was detected by chemiluminescence (Amersham Biosciences). For lectin blotting, biotinylated GS-II or concanavalin A (Vector Laboratories, Burlingame, CA) were used to probe blots at 0.25 µg/ml in phosphate buffered saline, 0.1% Tween-20, 200 µM CaCl2, and 200 µM MgCl2, followed by peroxidase-avidin (1 µg/ml; Vector Laboratories).
MS analysis of N-linked glycans
Lysozyme-G49N was isolated from the medium of strains harboring plasmid pVT100-U-HELG49N by ion exchange chromatography (Stolz and Munro, 2002). The N-glycans from typically 25 µg of protein were released by in gel digestion with endo F, followed by cleanup and MS as described previously (Kuster et al., 1997, 1998). Matrix-assisted laser desorption/ionization (MALDI) MS was performed on a PerSeptive Biosystems (Framingham, MA) Voyager-DE STR instrument.
In vitro assays of GlcNAc transferase activity
Protein A–tagged Gnt1p was precipitated from detergent lysates of spheroplasts using IgG Sepharose essentially as described previously (Rayner and Munro, 1998), except that 1% Triton X-100 was used as the detergent, and after binding and washing, the beads were washed into 50 mM 4-morpholine propane sulfonic acid (MOPS)–NaOH (pH 7.5). GlcNAc transferase activity was assayed in 50-µl reactions containing 20 µl beads (prepared from the lysate of 200 mg of cells) and 50 mM MOPS-NaOH (pH 7.5), 5 mM MnCl2, 0.24 µM (0.5 µCi) UDP-[3H]GlcNAc (41.6 Ci/mmol; New England Nuclear, Boston, MA), and acceptor. The mixture was shaken gently for 3 h at 30°C and, after addition of 200 µl water, applied to a 0.9-ml column of Dowex 1-X8 in the acetate form, the neutral reaction products eluted with 1.0 ml water, and the radioactivity quantified by scintillation counting. Analysis of products by TLC was as described previously (Doering, 1999).
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Acknowledgements |
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We are indebted to David Harvey for advice on the MS of N-glycans. Takehiko Yoko-o was supported by a 1-year fellowship from the Science and Technology Agency of Japan and Jürgen Stolz by an EMBO long-term fellowship (ALTF 495-1999).
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Footnotes |
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1 Present address: Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, AIST Central 6, Higashi, Tsukuba 305-8566, Japan
2 Present address: Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Universitätsstr. 31, D-93040 Regensburg, Germany
3 To whom correspondence should be addressed; e-mail: sean{at}mrc-lmb.cam.ac.uk
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Abbreviations |
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Endo F, endoglycosidase F; ER, endoplasmic reticulum; GS-II, Griffonia simplicifolia lectin II; HA, hemagglutinin; HPLC, high-performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; MOPS, 4-morpholine propane sulfonic acid; M-Pol I, mannan-polymerase I; MS, mass spectrometry; ORF, open reading frame; PCR, polymerase chain reaction; TLC, thin-layer chromatography.
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References |
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