Glycobiology Advance Access originally published online on December 23, 2003
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Glycobiology vol 14 no 3 pp. 243-251, 2004
Glycobiology vol. 14 no. 3 © Oxford University Press 2004; all rights reserved.
Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha
2 Metabolic Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Oun-dong 52, Yusong-gu, Daejeon 305-600, Korea; 3 Department of Microbiology, Chungnam National University, Daejeon 305-764, Korea; and 4 Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba, Ibaraki, 305-8566, Japan
Received on August 27, 2003; revised on October 27, 2003; accepted on October 30, 2003
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Abstract |
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Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man8–12GlcNAc2). Analyses using anti-
1,3-mannose antibody and exoglycosidases specific for 1,2- or 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short 1,6 extensions and are mainly elongated in 1,2-linkages without a terminal 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha–derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal 1,3-linked mannoses. Key words: cell wall mannoproteins / glycan profiling / Hansenula polymorpha / N-linked oligosaccharides / recombinant glucose oxidase
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Introduction |
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As a eukaryotic microorganism, yeast can carry out N-linked glycosylation, a posttranslational modification of proteins that involves the attachment of oligosaccharides to newly synthesized polypeptides. Initial N-glycan processing in the endoplasmic reticulum (ER) is very well conserved among eukaryotes but subsequent processing of oligosaccharides in the Golgi apparatus is variable, even between yeast species. In the traditional yeast Saccharomyces cerevisiae, the core oligosaccharides assembled on secretory proteins are elongated by addition of mannoses, which often leads to the formation of hypermannose structures (Man50–200GlcNAc2) with outer chains that may contain up to 200 mannose units. The linear backbone of the outer chain, frequently composed of 50 or more
1,6-linked mannoses, is highly branched by the presence of 1,2-linked mannoses and is terminally capped with 1,3-linked mannoses (Dean, 1999
). Hypermannosylated glycoproteins, especially those with terminal 1,3-linked mannose residues, produced from S. cerevisiae have been reported to be highly antigenic in humans (Ballou, 1990). On the other hand, in the methylotrophic yeast Pichia pastoris, the mannose outer chains of N-linked oligosaccharides have been shown to be generally much shorter than those found in S. cerevisiae (Bretthauer and Castellino, 1999), although extensive hyperglycosylation has also been reported in a few cases (Grinna and Tschopp, 1989; Scorer et al., 1993). The major oligosaccharide species in P. pastoris are reported to be Man8–14 GlcNAc2 forms with short 1,6 extensions (Trimble et al., 1991). More significantly, P. pastoris oligosaccharides have been reported to have no hyperimmunogenic terminal 1,3 glycan linkages (Montesino et al., 1998; Trimble et al., 1991). However, phosphomannose has been detected in both elongated and core oligosaccharides on some recombinant proteins of P. pastoris (Miele et al., 1997; Montesino et al., 1998), as observed in S. cerevisiae oligosaccharides.
The thermotolerant methylotrophic yeast Hansenula polymorpha has recently emerged as a promising host system for the production of recombinant proteins, ranging from industrial enzymes to therapeutic proteins (Gellissen, 2000, 2002). As the numbers of the reports of foreign gene expression using H. polymorpha increases, interest in the structures of oligosaccharides attached to the polypeptide chains produced by this yeast have been raised. A few studies on the expression of heterologous glycoproteins in H. polymorpha have indicated that the recombinant glycoproteins obtained appear to be less hyperglycosylated than those from S. cerevisiae (Gellissen et al., 1995; Kang et al., 1998). However, currently almost no information is available on the structural characteristics of the N-linked oligosaccharides of H. polymorpha–derived glycoproteins. In this study, we analyze the structure of the N-linked oligosaccharides derived from recombinant Aspergillus niger glucose oxidase (GOD) secreted from H. polymorpha and compared it with that of the oligosaccharides from S. cerevisiae. To obtain more general information on the structure of N-glycans of the secretory pathway in H. polymorpha, we also analyze the N-linked oligosaccharides from H. polymorpha cell wall mannoproteins. This is the first report on the structure of N-linked glycans derived from H. polymorpha to show that most oligosaccharide species attached to glycoproteins secreted from H. polymorpha have core-type structures (Man8–12GlcNAc2) without terminal 1,3-linked mannose residues.
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Results |
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Glycosylation pattern of rGOD expressed by H. polymorpha
Our previous studies on the heterologous expression of A. niger GOD, a glycoprotein with eight potential N-linked glycosylation sites, have shown that the recombinant GOD (rGOD) was expressed in highly mannosylated forms by S. cerevisiae wild-type strains (Kim et al., 2000
; Ko et al., 2002). To obtain information on the N-linked glycosylation in H. polymorpha, we expressed A. niger GOD, as a model glycoprotein, in two H. polymorpha wild-type strains, DL1-L and A16, and compared the glycosylation patterns of rGOD expressed with those of S. cerevisiae strains. Western blot analysis of the purified rGOD with the antibody specific for GOD revealed that the electrophoretic mobility of H. polymorpha–derived rGOD was much faster than that of S. cerevisiae–derived rGOD but slower than that of authentic GOD from A. niger (Figure 1B). This result suggested that the rGOD secreted from H. polymorpha appeared to be hyperglycosylated compared to authentic GOD but less hyperglycosylated than the rGOD from S. cerevisiae. After digestion with peptide:N-glycanase F (PNGase F), rGOD from all yeast strains displayed the same mobility, appearing as a discrete band (data not shown), and thus demonstrating that the difference in electrophoretic mobility was due to a difference in the extent of the glycosylation of N-linked oligosaccharides attached to the rGOD.
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To determine whether the terminal
1,3-mannose epitope was present in H. polymorpha–expressed rGOD, the purified rGOD was further analyzed using an antibody specific for terminal 1,3-linked mannoses. As shown in Figure 1C, the anti-1,3-mannose antibody bound to the rGOD secreted from wild-type S. cerevisiae (lanes 5 and 6) but not to that of the S. cerevisiae mnn1
mutant (lanes 7 and 8), which is defective in adding the terminal 1,3-mannose residues on both N- and O-linked oligosaccharides due to the absence of 1,3-mannosyltransferase activity (Yip et al., 1994). It was notable that rGOD from the H. polymorpha strains DL1-L (lanes 1 and 2) and A16 (lanes 3 and 4) was not recognized by the anti-1,3-mannose antibody, implying that the N-linked oligosaccharides on H. polymorpha–derived rGOD are apparently devoid of terminal 1,3-linked mannoses.
Size analysis of N-glycans assembled on rGOD
To gain more detailed information on the size of N-glycans assembled on the rGOD secreted by H. polymorpha, oligosaccharides were enzymatically released from purified rGOD, labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS), and then analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-performance liquid chromatography (HPLC). For FACE analysis, the ANTS-labeled oligosaccharide pools from H. polymorpha–derived rGOD and from S. cerevisiae–derived rGOD were run in a parallel on a polyacrylamide gel with ANTS-labeled oligomannosides of known size and structure, used as standards for size estimation (Figure 2A). The electrophoretic migration pattern of the H. polymorpha oligosaccharide pool was found to be quite different from that of the S. cerevisiae oligosaccharide pool, especially in terms of the sizes of the major oligosaccharides. In the sample from S. cerevisiae, large oligosaccharide species containing nine or more mannose residues (Man9–14 GlcNAc2) were abundantly detected (Figure 2A, lane 1), whereas in the sample from H. polymorpha relatively short oligosaccharides containing eight or nine mannoses (Man8–9 GlcNAc2) were the major forms (Figure 2A, lane 2). This is in a good agreement with our immunoblot result, that is, that H. polymorpha–secreted rGOD had much lower molecular weight forms than S. cerevisiae–secreted rGOD, which strongly supports the notion that the overall length of the outer mannose chain attached to glycoproteins is generally shorter in H. polymorpha.
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The ANTS-labeled N-linked oligosaccharides were also fractionated by size on NH2P-50 HPLC column (Figures 2Ba and 2Ca). Oligosaccharide profiles of the samples from H. polymorpha and S. cerevisiae reflected the results of the FACE analysis, displaying evident differences in the relative proportions of oligosaccharide species generated by these two yeasts. In the profile from H. polymorpha, the core form oligosaccharide Man8GlcNAc2 was the predominant species. Nearly 80% of the H. polymorpha oligosaccharides were distributed among oligosaccharide species containing 8–10 mannoses (Man8–10GlcNAc2), and only minor fractions were shown to have structures larger than Man10GlcNAc2 (Figure 2Ba). In contrast, the profile from S. cerevisiae showed a more even distribution of oligosaccharide species, ranging from Man8GlcNAc2 to Man14GlcNAc2. Almost 50% of the S. cerevisiae oligosaccharides contained more than 10 mannose residues (Man11–14-GlcNAc2, Figure 2Ca). Interestingly, H. polymorpha oligosaccharides containing nine or more mannose residues exhibited slightly longer retention times on HPLC than the corresponding S. cerevisiae oligosaccharides, which is consistent with the relatively faster electrophoretic migration of the H. polymorpha oligosaccharides observed during FACE analysis. These results indicate that there might be significant differences between these two yeasts in generating the oligosaccharides larger than Man8GlcNAc2.
Structural analysis of rGOD-derived N-glycans by exoglycosidase digestion
The use of specific -mannosidases has proved useful in the examination of the structural configurations of oligosaccharides from glycoproteins (Ichishima et al., 1999; Wong-Madden and Landry, 1995). To confirm the immunoblot result indicating the absence of terminal 1,3-linked mannose residues on the N-glycans of the H. polymorpha–derived rGOD, the ANTS-labeled oligosaccharide pool was treated with 1,2-mannosidase from Aspergillus saitoi. This exoglycosidase is highly specific for nonreducing terminal 1,2-mannose linkages. The digested products obtained were subjected to FACE and HPLC. As shown in Figures 2A, 2Bb, and 2Cb, after digestion with 1,2-mannosidase, virtually all of the H. polymorpha–derived oligosaccharides were converted to two small oligosaccharide forms, Man5GlcNAc2 (M5) and Man6GlcNAc2 (M6). Some tiny peaks not shifted to M5 or M6, indicated by arrowheads in Figure 2Bb, were detected after 1,2-mannosidase digestion. However, these peaks were resistant to the subsequent digestion with 1-2,3-mannosidase, implying that they do not contain the terminal 1,3-linked mannose residues (data not shown). On the other hand, after the digestion of the oligosaccharide pool from S. cerevisiae–derived rGOD, which was expected to contain terminal 1,3-linked mannose residues, substantial fractions of the digested products still remained as large oligosaccharide species containing eight or more mannose residues and only minor fractions were converted to Man5GlcNAc2 and Man6GlcNAc2 (Figure 2Cb). Therefore, the susceptibility of H. polymorpha oligosaccharides to 1,2-mannosidase digestion strongly indicates the absence of terminal 1,3-mannose residues on these oligosaccharides. Moreover this result also suggests that the outer chains of the oligosaccharides on H. polymorpha–derived rGOD are elongated mostly by the addition of 1,2-linked mannoses.
We further analyzed the structure of the oligosaccharide species, Man5GlcNAc2 and Man6GlcNAc2, which were generated from the H. polymorpha oligosaccharide pool after 1,2-mannosidase treatment, by digestion with 1,6-mannosidase from Xanthomonas manihotis (Figure 3). This highly specific exoglycosidase removes the terminal 1,6-linked mannose residues that are linked to a nonbranched sugar. After digestion with 1,6-mannosidase, the peak corresponding to Man6GlcNAc2 was completely converted to a peak corresponding to Man5GlcNAc2, indicating the presence of an extra 1,6-linked mannose in the Man6GlcNAc2 species. Considering that the structure of the core oligosaccharide Man8GlcNAc2 synthesized in the ER is the same in all eukaryotes examined so far (Munro, 2001), one can speculate that the Man5GlcNAc2 species is the final product of specific 1,2-mannosidase digestion of Man8GlcNAc2 or of larger oligosaccharide species extended with only 1,2-mannose linkages. In contrast, Man6GlcNAc2 is presumed to be the final product of specific 1,2-mannosidase digestion of the oligosaccharides Man9–14GlcNAc2, in which the core oligosaccharide Man8 GlcNAc2 is elongated by a single 1,6-linked mannose addition and branched with a variable number of 1,2-linked mannose units (Figure 3C). Therefore the results shown in Figure 3 strongly suggest that the outer chains of the N-glycans on H. polymorpha–secreted rGOD are mainly elongated in 1,2-linkages without terminal 1,3-linked mannose addition and have only one 1,6-linked mannose extension.
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Oligosaccharide structure of H. polymorpha mannoproteins
To determine whether the glycosylation of the rGOD reflect the general pattern of N-glycosylation in H. polymorpha, we investigated the structure of oligosaccharides derived from cell wall mannoproteins. The oligosaccharides prepared from the cell wall mannoproteins of H. polymorpha DL-1 strain were labeled with 2-aminopyridine (PA) and fractionated by HPLC under the elution condition that can separate neutral oligosaccharides from negatively charged ones in a chromatogram. As a control sample to check the HPLC condition, the oligosaccharides of the cell wall mannoproteins from S. cerevisiae mnn1
van1 were analyzed. Due to its defects in extensive polymerization of 1,6-linked mannoses and in terminal addition of 1,3-linked mannoses on N-glycans, the S. cerevisiae mnn1 van1 strain generates much shorter oligosaccharides, ranging from Man8GlcNAc2 to Man13GlcNAc2, compared to the S. cerevisiae wild-type strain (Shimma, unpublished data). As shown in Figure 4A, the oligosaccharides from S. cerevisiae mnn1 van1 were separated into three distinctive pools, depending on the phosphorylation status. The oligosaccharides of pool I corresponded to the N-glycans free of net negative charge, whereas those of the pools II and III corresponded to mono- or dimannosylphosphorylated glycans, respectively (Wang et al., 1997). The same HPLC analysis of the oligosaccharides derived from H. polymorpha mannoproteins revealed that the majority of the H. polymorpha oligosaccharides were of Man8–12GlcNAc2 sizes with the retention time of neutral glycans (Figure 4B). Almost identical oligosaccharide profiles were also obtained from mannoproteins of the other H. polymorpha strains, CBS4732 and NCYC495 (data not shown). The relatively small Man8GlcNAc2 and Man9GlcNAc2 oligosaccharides were commonly present as major species in H. polymorpha mannoproteins, contrast to S. cerevisiae mannoproteins with much larger oligosaccharides as dominant species. The oligosaccharide profile of H. polymorpha mannoproteins was in fact quite similar to that of H. polymorpha–derived rGOD (Figure 4C), implying that small oligosaccharide species (Man8–12GlcNAc2) without mannosylphosphorylation might be the general features of N-glycans of the secretory pathway in H. polymorpha.
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Further analysis of the oligosaccharide structure of H. polymorpha mannoproteins was carried out by exoglycosidase digestion. After treatment with
1,2-linkage-specific mannosidase, all of the oligosaccharides were converted to Man5GlcNAc2 and Man6GlcNAc2 (Figure 4D). Sequential digestion with 1,6-mannosidase converted the peak corresponding to Man6GlcNAc2 into a peak corresponding to Man5GlcNAc2 (Figure 4E). The results strongly indicate that N-linked oligosaccharides of H. polymorpha mannoproteins were not terminally capped by 1,3-linked mannose residues and extended mainly by 1,2-linkages, as observed in the glycans assembled on the H. polymorpha–derived rGOD. During the HPLC analysis, we observed an unknown peak, designated x, in the H. polymorpha oligosaccharide samples. The elution pattern of peak x was not changed even after the treatment of jack bean -mannosidase, which cleaves all three (1,2, 1,3, and 1,6) types of nonreducing terminal -mannose linkages. The x peak was dominantly detected especially in the samples of H. polymorpha cell wall mannoproteins, implying that it may correspond to a contaminant generated during the preparation of cell wall materials (Figure 4F).
S. cerevisiae and P. pastoris are reported to add only mannose to their glycoprotein glycans, whereas Schizosaccharomyces pombe is shown to add mannose and galactose (Gemmill and Trimble, 1999). Monosaccharide contents of the N-linked oligosaccharides prepared from H. polymorpha mannoproteins were analyzed by using anion-exchange column. The peak corresponding to PA-Man was detected as one major peak, whereas the peaks to PA-Glc and PA-GlcNAc as minor peaks (Figure 5B). No peaks corresponding to PA-galactose or PA-fucose were detected. Although the possibility that the outer chains of H. polymorpha contain glucose as a minor structural component can not be excluded, it is more likely that glucose was generated from the contaminated cell wall glucan during monosaccharide preparation (Peat et al., 1961). The presence of glucose inhibits the digestion of oligosaccharides by -mannosidases. Judging from our results of 1,2 and 1,6-mannosidase digestion experiments, it appears that H. polymorpha also add only mannose to their glycoprotein glycans, as do the other yeasts S. cerevisiae and P. pastoris.
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Discussion |
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Yeasts have been considered for the production of glycoprotein biopharmaceuticals (Lehle and Tanner, 1995
). As an alternative to the conventional yeast S. cerevisiae, the methylotrophic yeast H. polymorpha is rapidly gaining favor for the production of recombinant proteins (Gellissen, 2000, 2002; van Dijk et al., 2000). However, little is known about the structure of the N-glycans assembled on glycoproteins secreted from this methylotrophic yeast. In this study, we determined the size distribution and the structures of the N-linked oligosaccharides attached on H. polymorpha–secreted rGOD and cell wall mannoproteins. The results of oligosaccharide profiling showed that the small oligosaccharides with core-type structures (Man8–12 GlcNAc2) were present as major species in the H. polymorpha oligosaccharide (Figures 2 and 4), demonstrating that the H. polymorpha N-glycans are much less hypermannosylated than the S. cerevisiae N-glycans. More significantly, the inability of the anti-1,3-mannose antibody to bind to H. polymorpha–secreted rGOD (Figure 1C), and the susceptibility of the H. polymorpha N-glycans from rGOD and mannoproteins to 1,2-mannosidase digestion (Figures 2Bb and 4D) strongly indicated that the H. polymorpha oligosaccharides were not terminally capped by 1,3-linked mannose residues and were extended mainly in 1,2-mannose linkages. These structural characteristics suggest that H. polymorpha has some advantages over S. cerevisiae for the production of recombinant glycoproteins. In particular, the highly immunogenic 1,3-terminal linkages are common on S. cerevisiae oligosaccharides and present a major problem for the use of S. cerevisiae as a host strain for the production of human pharmaceuticals (Romanos et al., 1992).
The results of the sequential digestion with 1,2- and 1,6-mannosidase (Figures 3B and 4E) further indicate that the outer chains of the H. polymorpha N-linked oligosaccharides from rGOD and mannoproteins had very short 1,6 extensions, mainly composed of a single 1,6-linked mannose. The outer chain of the budding yeast S. cerevisiae has a long 1,6-linked mannose backbone, which is often composed of more than 50 mannose residues and branched extensively by addition of 1,2- and 1,3-linked mannoses (Dean, 1999). In contrast, P. pastoris N-linked oligosaccharides have relatively short 1,6 extensions. The oligosaccharides assembled on the recombinant proteins secreted from P. pastoris were reported to contain one to four 1,6-linked mannose units in their outer chains (Kalidas et al., 2001; Miele et al., 1997). Therefore the N-linked glycosylation pathways in both methylotrophic yeasts H. polymorpha and P. pastoris appear to be quite similar but significantly different from that in S. cerevisiae, with the addition of much shorter 1,6 outer chain backbone to the core and no 1,3 outer extensions.
Further modification of N-linked oligosaccharides with the addition of mannosylphosphate often occurs both in S. cerevisiae (Jigami and Odani, 1999) and P. pastoris (Bretthauer and Castellino, 1999). It is known that the core oligosaccharide Man8GlcNAc2 also contains two potential phosphorylation sites (Hernandez et al., 1989). However, we could not yet observe intensively phosphorylated oligosaccharides in the samples prepared from the H. polymorpha–derived mannoproteins and rGOD (Figures 4B and 4C). The digestion of most fractions of H. polymorpha oligosaccharides to Man5GlcNAc2 and Man6GlcNAc2 neutral oligomannosides by 1,2-mannosidase treatment (Figures 2Bb and 4D) also supports the absence of phosphate in the major species of H. polymorpha oligosaccharides because phosphorylated mannose blocks 1,2-mannosidase treatment. Although we cannot exclude the possibility that some tiny peaks not shifted completely to the small oligosaccharide M5 and M6 species after 1,2-mannosidase digestion (Figure 2Bb) could be minor oligosaccharide species containing phosphate residues, the present results imply that the mannosylphosphorylation of the N-linked glycans may not occur actively in H. polymorpha. However, the extent of mannosylphosphorylation is known to depend on culture conditions, such as media and cultivation periods, and a more detailed study should be carried out to discuss the possibility of mannosylphosphorylation as another form of oligosaccharide modification in H. polymorpha.
Increasing evidence shows that oligosaccharides have profound effects on critical properties of glycoprotein products for human therapeutic use, such as plasma clearance rate, antigenicity, and specific activity. Therefore the way to get correct glycosylation has been important issues in the field of biotechnology industry (Jenkins et al., 1996; Koeller and Wong, 2000). Further studies on the structural characteristics of H. polymorpha N-linked oligosaccharides, especially those derived from mutant strains defective in glycosylation, should facilitate the delineation of the H. polymorpha–specific N-glycosylation pathway. This would provide valuable information for the development of glycoengineering strategies in H. polymorpha to achieve the optimal glycosylation of recombinant proteins.
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Materials and methods |
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Yeast strains and plasmids
The H. polymorpha strains, DL1-L (leu2) and A16 (leu2), used in this study were a derivative of DL-1 (ATCC26012) and CBS4732 (ATCC34438), respectively. The S. cerevisiae mnn1
strain (MATa ura3-52 leu2-3,112 his4-34 mnn1::LEU2) was derived from S. cerevisiae L3262a (Kim et al., 2000). The S. cerevisiae mnn1 van1 (MATa leu2 ura3 trp1 ade8 his3 mnn1::URA3 van1::LEU2) was derived from S. cerevisiae RA1-1B (Y. Shimma, unpublished data). To express A. niger GOD tagged with six residues of histidine (GOD-His) in H. polymorpha under the MOX promoter, the 1.9-kb EcoRI/SalI DNA fragment encoding GOD-His was obtained from the S. cerevisiae GOD expression vector pYGOD-His (Kim et al., 2000) and exchanged with the 0.7-kb EcoRI/XhoI fragment coding for hirudin in pDLMOX-HIR (Kang et al., 1998), generating pDLMOX-GOD(H).
Media and general techniques
Yeast strains were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or SD minimal medium (0.67% yeast nitrogen without amino acids, 2% glucose) containing appropriate nutritional supplements. Yeast transformation was performed by the modified dimethyl sulfoxide–lithium acetate method (Hill et al., 1991). General DNA manipulations were performed as described previously (Sambrook and Russell, 2001). To induce the expression of A. niger GOD in H. polymorpha, the transformants harboring pDLMOX-GOD(H) were precultured overnight in a synthetic complete medium lacking leucine and transferred to flasks containing YPM medium (1% yeast extract, 2% peptone, 2% methanol) for 36 h at 37°C. For the induction of GOD expression in the S. cerevisiae transformants harboring pYGOD-His, YPDG medium (1% yeast extract, 2% peptone, 1% glucose, 1% galactose) was used. Immunoblot analysis was carried out using the polyclonal antibodies raised against A. niger GOD (Accurate Chemical & Scientific, Westbury, NY) and against 1,3-linked mannose (provided by R. Schekman, University of California, Berkeley, CA) as described previously (Kim et al., 2000).
Purification of rGOD
Culture supernatants containing His6-tagged GOD were concentrated by ultrafiltration (YM30 membrane, Millipore, Bedford, MA). The 100-fold concentrated culture supernatants were dialyzed against 50 mM sodium phosphate (pH 6.0) and 300 mM NaCl, and His6-tagged GOD was purified using ÄKTAprime chromatography system (Amersham Pharmacia Biotech AB, Uppsala, Sweden).
Preparation of cell wall mannoproteins
Yeast cells were cultivated in YPD medium supplemented with 0.3 M KCl and harvested at early stationary phase (OD600 = 10). Total cell wall mannoproteins were extracted by hot citrate buffer (0.1 M citrate buffer, pH 7.0) followed by precipitation with ethanol (Peat et al., 1961). The precipitates were desalted by a PD-10 column containing Sephadex G-25 (Amersham Pharmacia Biotech). The eluent was further purified by a concanavalin A–agarose column (Amersham Pharmacia Biotech), which was equilibrated with conacanavalin A buffer (0.1 M Tris-HCl buffer [pH 7.2] containing 0.15 M NaCl, 1 mM MnCl2, and 1 mM CaCl2). The column was eluted by conacanavalin A buffer containing 1 M methyl -D-mannoside. The mannoprotein fraction was dialyzed against water and lyophilized.
Oligosaccharide preparation
N-linked oligosaccharides were released from the purified rGOD and cell wall mannoproteins by PNGase F (New England Biolabs, Beverly, MA) or Glycanase F (Takara Shuzo, Shiga, Japan) following the manufacturer's instructions. Oligosaccharides were labeled covalently with fluorogenic compounds ANTS (Glyko, Novato, CA) or PA (Takara Shuzo) at their reducing ends (Jackson, 1990; Kondo et al., 1990). Digestion of fluorophore-labeled oligosaccharides with 1,2-mannosidase from A. saitoi, Glyko), 1-2,3-mannosidase (from X. manihotis, New England Biolabs), 1,6-mannosidase (from X. manihotis, New England Biolabs), or -mannosidase (from jack bean, Sigma, St. Louis, MO) was carried out according to the manufacturer's instructions.
FACE and HPLC of oligosaccharides
The ANTS-labeled oligosaccharides were separated on high-resolution polyacrylamide gels using FACE N-linked oligosaccharide profiling kit. The electrophoretic migration of a band was compared to an ANTS oligomannoside standard (Man9GlcNAc2-ANTS) or ladder of maltooligosaccharides-ANTS (Glyko). Size-fractionation HPLC of ANTS-labeled or PA-labeled oligosaccharides was carried out with Shodex Asahipak NH2P-50 column (Showa Denko, Tokyo, 0.46 x 25 cm) using a Waters 247 chromatography system (Waters, Milford, MA, USA). Fluorescence was measured using a Waters 2475 fluorescence detector (Waters) for ANTS at 
ex 353 nm and em 535 nm and for PA at ex 320 nm and em 400 nm, respectively.
HPLC analysis of monosaccharides
Monosaccharides from N-linked oligosaccharides attached on H. polymorpha cell wall mannoproteins were prepared by acid hydrolysis as described previously (Takasaki et al., 1982). Twenty microliters of oligosaccharide samples (100 pmol) were hydrolyzed with 40 ml 6 M trifluoroacetic acid at 100°C for 3 h using a gas-phase hydrazinolysis apparatus (Hydraclub S-204, Honen Oil, Tokyo). After hydrolysis, the resulting monosaccharides were N-acetylated twice as described previously (Nakanishi-Shindo et al., 1993). The monosaccharides were labeled with PA and analyzed using anion-exchange column, PALPAK Type A (Takara Shuzo, 0.46 x 15 cm) at 65°C. The products were identified with the authentic PA-monosaccharides standard (Takara Shuzo).
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Acknowledgements |
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This study was supported by grants form the Korean Ministry of Science and Technology (Korea-Japan International Cooperation Program, 21st Century Frontier R&D program) and the Japan Society for the Promotion of Science. The authors deeply appreciated Drs. M. Kainuma and K. Nakayama for their helpful discussions.
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: hyunkang{at}mail.kribb.re.kr
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Abbreviations |
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ANTS, 8-aminonaphthalene-1,3,6-trisulphonic acid; ER, endoplasmic reticulum; FACE, fluorophore-assisted carbohydrate electrophoresis; GOD, glucose oxidase; HPLC, high-performance liquid chromatography; PA, 2-aminopyridine; PNGase F, peptide:N-glycanase F
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References |
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