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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Key words: glycosylation / GNT1 / Golgi / N-acetylglucosaminyltransferase / yeast
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Introduction |
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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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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 GlcNAc2Man812 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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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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Discussion |
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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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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 Atagged 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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Footnotes |
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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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References |
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