2 Glycofi, Inc., 21 Lafayette Street Suite 200, Lebanon, NH 03766 3 Velocity 11; 435 Acacia Ave., Palo Alto, CA 94306
Received on June 19, 2003; revised on October 21, 2003; accepted on October 22, 2003
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Abstract |
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Key words: ALG3 / endoplasmic reticulum / lipid-linked glycosylation / mannosyltransferase / N-glycosylation
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Introduction |
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Due to the high degree of conservation in the early stages of glycosylation, S. cerevisiae has been employed as a model system for the study of eukaryotic N-glycan processing, and a large body of literature exists (Aebi et al., 1996; Burda and Aebi, 1998
; Burda et al., 1996
; Herscovics and Orlean, 1993
; Huffaker and Robbins, 1982
, 1983
; Reiss et al., 1996
; Stagljar et al., 1994
; Zufferey et al., 1995
). Comparatively little is known about glycosylation in yeasts other than S. cerevisiae, such as Kluyveromyces lactis, Candida albicans, and Pichia pastoris.
Our laboratory's main interest lies in the reengineering of the glycosylation pathway to obtain human-like glycosylation in a variety of different yeasts and filamentous fungi. In particular, the methylotrophic yeast P. pastoris has gained importance as a host for the high-level expression of heterologous proteins (Cereghino and Cregg, 2000). Previous work on this yeast indicated that although it displays similarities in glycosylation with S. cerevisiae, distinct differences also exist, such as the lack of a terminal
-1,3-mannose linkage (Verostek and Trimble, 1995
).
To further explore glycosylation in this industrially important yeast, we recently created a P. pastoris strain lacking the -1,6-polymannose outer chain by deleting the P. pastoris OCH1 gene (Choi et al., 2003
). The och1 mutant strain is temperature sensitive, exhibits increased flocculation, and displays no hypermannosylation. Analysis of N-glycans released from a secreted reporter protein showed mostly Man812-GlcNAc2 mannans (Choi et al., 2003
), suggesting the presence of glycosyltransferase activities acting on the core oligosaccharide even in the absence of an outer chain. By gaining a better understanding of the role of individual glycosyltransferases that act in both the ER and Golgi of P. pastoris, we hope to be able to reduce the number of steps required to modify the yeast N-glycosylation pathway to produce completely "humanized" N-glycans in this yeast.
With this in mind, we have undertaken a reverse genetic approach to identify and characterize genes that play a role in N-glycosylation in industrially important yeasts, such as P. pastoris and K. lactis. One such gene, ALG3, has been shown in S. cerevisiae to encode the enzyme Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase, which is responsible for the first Dol-P-Man-dependent mannosylation step at the luminal side of the ER, converting Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol (Sharma et al., 2001). The glycosylation profile of a S. cerevisiae alg31 mutant has been well studied in several genetic backgrounds, and the ALG3 gene of S. cerevisiae has been cloned and knocked out (Aebi et al., 1996
; Nakanishi-Shindo et al., 1993
; Verostek et al., 1991
, 1993a
,b
). Given the conserved nature of the early steps of N-glycosylation, we sought to identify a P. pastoris ALG3 homolog. We reasoned that by employing an alg3 mutant strain, which transfers Glc3Man5GlcNAc2 instead of Glc3Man9GlcNAc2 (see Figure 1) to a nascent polypeptide, expression and proper targeting of active
-1,2 mannosidases would yield a Man3GlcNAc2 structure on secreted proteins, the common pentasaccharide core in mammalian-type complex N-glycans.
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Results |
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Analysis of N-glycans released from a secreted reporter protein in P. pastoris alg3
To monitor the effects of the alg3 deletion on N-glycosylation, the Kringle3 domain of human plasminogein (K3) was employed as a model protein (Choi et al., 2003). N-glycans from four strains were released from secreted K3 by treatment with protein N-glycanase (PNGase); a wild-type P. pastoris strain (BK64), the och1 mutant strain (BK3-1), the och1 alg3 mutant strains (RDP25 and RDP26), and the och1 alg3+OCH1 complemented strain (RDP30). MALDI-TOF MS analysis was performed on the released N-glycans and yielded expected results from the wild type (Hex10GlcNAc2 to Hex16GlcNAc2) and och1 mutant (Hex8GlcNAc2 to Hex12GlcNAc2) as seen previously (Choi et al., 2003
; Figures 3A and 3B). The N-glycans released from the och1 alg3 mutant strains revealed the expected Hex5GlcNAc2 peak (Figure 3C) as described previously in S. cerevisiae alg3 mutants (Verostek et al., 1991
), but also a large degree of higher-molecular-weight glycans, ranging from Hex6GlcNAc2 to Hex12GlcNAc2 (Figure 3C).
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Next, we individually purified the -1,2-mannosidase recalcitrant Hex6GlcNAc2, Hex7GlcNAc2, Hex8GlcNAc2, and Hex9GlcNAc2 (hexose 69) fractions on a P4 column, though we were not able to isolate enough material from the higher-molecular-weight (hexose 1012) structures. The four isolated fractions were subjected to individual jack bean (
-1,2-,
-1,3-,
-1,6-) mannosidase and ß-1,4-mannosidase digest. Surprisingly, each peak proved to be completely recalcitrant to the action of these enzymes (see Table I). Although the hexose 69 structures migrated as neutral glycans, we tried to determine whether mannosylphosphate transfer could partially explain the lack of digestability by
-1,2-mannosidase, jack bean (
-1,2-,
-1,3-,
-1,6-) mannosidase, and ß-1,4-mannosidase. Using high-performance liquid chromatography to separate the neutral and acidic glycans, we established that
80% of the total glycans were of neutral nature, whereas
20% were charged. However, the major hexose 712 peaks previously observed by MALDI-TOF MS separated completely with the neutral fraction (data not shown). The charged glycans in RDP25 consisted of a series of oligosaccharides possessing molecular masses that were consistent with Man512GlcNAc2, structures each containing a single mannosylphosphate moiety (data not shown). After purification of the charged fraction, mild hydrolysis and alkaline phosphatase digestions were carried out, and the glycans were treated with
-1,2-mannosidase. All of the glycans were reduced to a mass corresponding to Man3GlcNAc2 (data not shown). The presence of structures larger than Man5GlcNAc2 in both the charged and neutral fractions suggest the action of additional yet unidentified mannosyltransferases capable of transferring sugars to the alg3 Man5 glycan.
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Discussion |
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In S. cerevisiae, ALG3 encodes a Dol-P-Man:Man5GlcNAc2-PPDol mannosyltransferase that is responsible for performing the first step in N-glycan biosynthesis following flipping of the Dol-PP-linked Man5GlcNAc2 glycan to the luminal side of the ER (Aebi et al., 1996; Sharma et al., 2001
). Deletion of PpALG3 in an och1 mutant background resulted in a distinct decrease in the size of glycans on a secreted protein, as determined by MALDI-TOF MS. The smallest observable mass in the alg3 och1 double mutant strain was Hex5GlcNAc2 as compared to Hex8GlcNAc2 in the och1 parental strain, and this Hex5GlcNAc2 peak was converted to Hex3GlcNAc2 following in vitro
-1,2-mannosidase treatment, consistent with it being the core alg3 Man5GlcNAc2 isomer Man
1,2Man
1,2Man
1,3(Man
1,6)Manß1,4GlcNAcß1,4GlcNAc, previously described for S. cerevisiae (Verostek et al., 1991
). However, several larger structures corresponding to size Hex6GlcNAc2 to Hex12GlcNAc2 were also observed, which were recalcitrant to in vitro
-1,2-mannosidase treatment. Amongst these, the hexose 6 peak was shown to contain glucose by HPAEC PAD analysis in a ratio consistent with GlcMan5GlcNAc2. This is similar to experiments performed on an alg3 mutant in S. cerevisiae that revealed an increase in glucose-containing structures ranging from Hex6GlcNAc2 to Hex10GlcNAc2. These were attributed to a reduction in lipid-linked glucosyl transferase or glucosidase activity on the alg3 Man5GlcNAc2 structure (Verostek et al., 1993).
In an S. cerevisiae alg3 mutant a relatively large percentage of Hex8GlcNAc2 to Hex10GlcNAc2 (2540%) contains Glc (Verostek et al., 1993a), and similarly we expected to observe three peaks that contained Glc among the recalcitrant masses from the P. pastoris alg3 och1 mutant strain. However, because only the Hex6GlcNAc2 species contains Glc, the remaining non-Glc-containing Hex6GlcNAc2Hex12GlcNAc2 structures are therefore likely the result of Golgi-residing mannosyltransferase activity that acts downstream of the core ER modifications but independent of the Och1p-initiated outer chain. In S. cerevisiae, mutation of och1 and mnn1 virtually eliminates downstream Golgi addition of monosaccharide residues to the core Man8GlcNAc2 glycan, including the large outer chain that is dependent on Och1p (Nakanishi-Shindo et al., 1993
). Similarly, deletion of och1 in P. pastoris eliminates formation of the
-1,6-dependent outer chain on the lower arm of the core oligosaccharide (Choi et al., 2003
). However, as previously observed in the Ppoch1 mutant strain, glycans ranging from Hex9GlcNAc2 to Hex12GlcNAc2 are generated by Golgi modifications to the core oligosaccharide in the absence of Och1p and despite the lack of an Mnn1p
-1,3-mannosyltransferase activity in P. pastoris (Bretthauer and Castellino, 1999
; Choi et al., 2003
; Verostek and Trimble, 1995
). In an S. cerevisiae alg3 mutant strain, the only apparent additions to the Man5GlcNAc2 structure are
-1,3 and
-1,6-mannose, which are added by Och1p and Mnn1p. The generation of the larger structures extended from a P. pastoris mutant alg3 Man5 structure indicates that Golgi-residing P. pastoris glycosyltransferases are capable of acting on a significantly truncated substrate, and do not even require formation of the complete Man8GlcNAc2 core for activity. Interestingly, a majority of these glycans (Hex6GlcNAc2Hex12GlcNAc2) were resistant to
-1,2-, ß-1,4-, and jack bean
-mannosidase treatment, suggesting that they result from previously uncharacterized mannose transferase reactions. The ability of P. pastoris to generate these high-mannose glycans in the absence of Och1p stands in stark contrast to the minimal nature of the Golgi modifications in S. cerevisiae och1 mnn1 and alg3-1 mutant strains (Nakanishi-Shindo et al., 1993
; Verostek et al., 1993b
).
Complementation of the P. pastoris och1 alg3 deletion mutant with the full-length PpOCH1 gene resulted in virtually complete restoration of the wild-type phenotype, including abrogation of temperature sensitivity and of the clumping phenotype associated with och1 deletion and other mutants lacking outer-chain elongation in many yeast species. Furthermore, analysis of secreted glycans from this complemented och1 alg3 + OCH1 strain revealed that 100% of the core Man5GlcNAc2 structures observed in the parental och1 alg3 double mutant had received an -1,6-mannose, as determined by the complete loss of Man5GlcNAc2 structures in this strain and the failure of any glycans to convert to Man3GlcNAc2 following in vitro
-1,2-mannosidase treatment. The formation of no Man3GlcNAc2 following in vitro
-1,2-mannosidase digest indicates that in vivo, PpOch1p can act quantitatively on the alg3 Man5 structure that is found on glycoproteins entering the Golgi. Furthermore, the formation of no Man4GlcNAc2 as well as very little Man5GlcNAc2 probably indicates that the subsequent
-1,6-mannosyltransferases are capable of acting on the Och1p product with equal efficiency. This contrasts the greatly reduced in vitro and in vivo activity of the S. cerevisiae Och1p counterpart against N-glycans lacking any part of the 1,6 arm (Cipollo and Trimble, 2000
; Nakayama et al., 1997
; Verostek et al., 1993b
). Interestingly, in vitro experiments with Och1p from Schizosaccharomyces pombe show a similar divergence of substrate affinity compared to S. cerevisiae Och1p (Yoko-o et al., 2001
). Although a substrate lacking the 1,6 arm is not compared, these results combined with the in vivo data observed here in P. pastoris indicate that although the Och1p function in formation of an outer glycan chain is very conserved among yeast species, the substrate affinity of Och1p, the initiating
-1,6-mannosyltransferase, is quite divergent.
In conclusion, we have cloned and deleted the ALG3 gene from P. pastoris and demonstrated that this gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase enzyme in this industrially important fungus. An och1 alg3 double mutant strain was generated, and the predicted Man5GlcNAc2 processing intermediate was observed on secreted glycans from this strain, indicating a predicted block in lipid-linked glycan assembly. Further modification of this strain by addition of mammalian-specific glycosidases and glycosyltransferases to determine its viability as a production host for human therapeutic proteins is under way.
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Materials and methods |
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Deletion of PpALG3
The alg3::G418R allele used for deletion of the ORF predicted to encode the PpAlg3 -1,3-mannosyl transferase activity was generated by the PCR overlap method (Davidson et al., 2002
; Ho et al., 1989
; Horton et al., 1989
). Primers RCD142 (5'-CCACATCATCCGTGCTACATATAG-3') paired with RCD144 (5'-ACGAGGCAAGCTAAACAGATCTCGAAGTATCGAGGGTTATCCAG-3'), RCD145 (5'-CCATCCAGTGTCGAAAACGAGCCAATGGTTCATGTCTATAAATC-3') paired with RCD147 (5'-AGCCTCAGCGCCAACAAGCGATGG-3'), and RCD143 (5'-CTGGATAACCCTCGATACTTCGAGATCTGTTTAGCTTGCCTCGT-3') paired with RCD146 (5'-GATTTATAGACATGAACCATTGGCTCGTTTTCGACACTGGATGG-3') were used to amplify the 5' and 3' flanking regions of the ALG3 gene and the G418 resistance marker (G418R), respectively. Then, primers RCD142 and RCD147 were used in a second reaction with all three first-round templates to generate an overlap product that contained all three fragments as a single linear alg3::G418R allele. This PCR product was then directly employed for transformation with selection on medium containing 200 µg/ml G418. Primers MG1 (5'-TCTGGTAGATCCATTAGTTGCTGC-3') and PTEF (5'-AGCTGCGCACGTCAAGACTGTCAAGGA-3') and MG6 (5'-ATTCTCCATATGCTCAACCTAACG-3') and KAN10 were used to confirm proper integration of the alg3::G418R mutant allele at the 5' and 3' junctions, respectively, and primers RCD178 (5'-TTTATGGTTAGCTGATTCCATTGTTAT-3') and RCD179 (5'-ATCAAAACTAGAACAGGTGCCAAGGCT-3') were used to confirm the absence of the wild-type ALG3 allele.
Complementation of OCH1
A 2985-bp portion of the P. pastoris OCH1 locus was PCR-amplified using primers 5'-CCTTGCGGCCGCGGAATTATTGGCTTTATTTGTTTG-3' and 5'-CCTTTTAATTAATTATTCGGAAGCATGATTTCTTGG-3'. The resulting amplified fragment was cloned and sequenced. It was then subcloned into the P. pastoris/E. coli shuttle vector pRCD259, which contains the hph gene (obtained from plasmid pAG32; Goldstein and McCusker, 1999) encoding for hygromycin resistance and the P. pastoris URA3 gene. The resulting plasmid, pRCD267, was linearized with a unique XbaI site located in the URA3 gene and transformed into the P. pastoris och1 alg3 mutant strain RDP25 by electroporation. Transformants were selected on YPD medium containing 300 µg/ml hygromycin, and the resulting colonies were restreaked and two independent transformants (named RDP36-1 and RDP36-2) were subjected to further analysis.
MALDI-TOF MS
Molecular weights of glycans were determined using a Voyager DE PRO linear MALDI-TOF (Applied Biosciences, Foster City, CA) mass spectrometer as described previously (Choi et al., 2003).
Monosaccharide compositional analysis
N-glycan fractions separated by Biogel P4 gel permeation chromotography were hydrolyzed in 2.5 M trifluoracetic acid at 121°C for 70 min and then dried in a speed vac. Samples were resuspended in water and analyzed on a chromatography system (Dionex, Sunnyvale, CA) consisting of a GP50 pump and an ED50 electrochemical detector. The system was controlled by and data collected with Dionex PeakNet software. Sample injection was carried out with a Famos well plate microautosampler (LC packings, San Francisco, CA). CarboPAC PA10 column (4 x 250 mm) was used at a flow rate of 1 ml/min. The sample was eluted with 18 mM NaOH (eluent B) for 20 min; the column was washed with 200 mM NaOH (eluent A) for 15 min, then returned to initial condition to equilibrate for another 20 min before the next sample injection.
Characterization of acidic oligosaccharides
Acidic oligosaccharides were separated from the neutral glycans on a Glyco SepC column (4.6 x 100 mm, Glyko). The flow rate was 0.4 ml/min. After eluting isocratically (20% A:80% C) for 10 min, a linear solvent gradient (20% A:0% B:80% C to 20% A:50% B:30% C) was used over 35 min to elute the charged glycans. Solvent A was acetonitrile; solvent B was an aqueous solution of ammonium acetate, 500 mM (pH 4.5); and C was HPLC-grade water. The column was equilibrated with solvent (20% A:80% C) for 20 min between runs. Acidic oligosaccharides were eluted at 2030 min from the column, and these charged glycans were collected for further analysis.
Mild hydrolysis and alkaline phosphatase (Sigma) digest were based on Miele et al. (1997) with the following modification: samples were hydrolyzed with 50 mM trifluoracetic acid in a 0.3-ml glass V-Vial with screw cap (Wheaton, Millville, NJ) at 100°C for 60 min.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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