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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Key words: cell wall mannoproteins / glycan profiling / Hansenula polymorpha / N-linked oligosaccharides / recombinant glucose oxidase
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Introduction |
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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. polymorphaderived 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 (Man812GlcNAc2) without terminal
1,3-linked mannose residues.
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Results |
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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. polymorphaderived rGOD and from S. cerevisiaederived 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 (Man914 GlcNAc2) were abundantly detected (Figure 2A, lane 1), whereas in the sample from H. polymorpha relatively short oligosaccharides containing eight or nine mannoses (Man89 GlcNAc2) were the major forms (Figure 2A, lane 2). This is in a good agreement with our immunoblot result, that is, that H. polymorphasecreted rGOD had much lower molecular weight forms than S. cerevisiaesecreted 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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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. polymorphaderived 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. polymorphaderived 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. cerevisiaederived 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. polymorphaderived 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 Man914GlcNAc2, 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. polymorphasecreted 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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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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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. polymorphaderived 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. polymorphaspecific 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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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 sulfoxidelithium 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 Aagarose 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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Footnotes |
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Abbreviations |
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References |
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