Glycobiology Advance Access originally published online on March 19, 2004
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Glycobiology vol 14 no 5 pp. 399-407, 2004
Glycobiology vol. 14 no. 5 © Oxford University Press 2004; all rights reserved.
Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris
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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N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc3Man9GlcNAc2 structure that is subsequently transferred to a nascent protein in the endoplasmic reticulum. Once folded, glycoproteins are then shuttled to the Golgi, where additional but divergent processing occurs in mammals and fungi. We cloned the Pichia pastoris homolog of the ALG3 gene, which encodes the enzyme that converts Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP. Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man5GlcNAc2 structure that could be trimmed to Man3GlcNAc2 by in vitro
-1,2-mannosidase treatment. However, several larger glycans ranging from Hex6GlcNAc2 to Hex12GlcNAc2 were also observed that were recalcitrant to an array of mannosidase digests. These results contrast the far simpler glycan profile found in Saccharomyces cerevisiae alg3–1 och1, indicating diverging Golgi processing in these two closely related yeasts. Finally, analysis of the P. pastoris alg3 deletion mutant in the presence and absence of the outer chain initiating Och1p -1,6-mannosyltransferase activity suggests that the PpOch1p has a broader substrate specificity compared to its S. cerevisiae counterpart. Key words: ALG3 / endoplasmic reticulum / lipid-linked glycosylation / mannosyltransferase / N-glycosylation
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Introduction |
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The early stages of asparagine-linked glycosylation are strikingly conserved between fungal and mammalian cells. Across kingdoms, nearly every eukaryote studied to date synthesizes N-glycans in the same way, assembling an identical Glc3Man9GlcNAc2 glycan as a lipid-linked moiety with dolichyl pyrophosphate (Dol-PP) and transferring this to Asn residues on nascent proteins in the endoplasmic reticulum (ER) (Burda and Aebi, 1999
; Herscovics and Orlean, 1993; Kornfeld and Kornfeld, 1985; Lehle and Tanner, 1975). Once the core oligosaccharide has been transferred by the oligosaccharyltransferase complex, further processing yields the Man8GlcNAc2 core, which leaves the ER and enters the Golgi, where it is subjected to further processing. It is here where the pathways diverge significantly between animals and fungi. Mammalian cells trim the Man8GlcNAc2 to Man5GlcNAc2, which forms the basis for complex N-glycan synthesis through the ordered trimming by mannosidase II and additions of N-acetlyglucosamine, galactose, and sialic acid (Kornfeld and Kornfeld 1985; Kukuruzinska and Lennon, 1998; Moremen et al., 1994). In fungal cells, such as Saccharomyces cerevisiae, rather than trimming mannoses and adding back other sugars, additional mannose sugars are added by Golgi-residing mannosyltransferases to create large N-glycans consisting of up to 100 mannose residues (Dean, 1999; Gemmill and Trimble, 1999; Lussier et al., 1999).
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 Man8–12-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 alg3–1 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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Here we report the cloning and disruption of a functional homolog of the S. cerevisiae ALG3 gene in P. pastoris and the generation and analysis of an alg3 och1 double deletion mutant strain. N-glycans released from a reporter protein secreted by an alg3 och1 mutant strain yielded the predicted Man5GlcNAc2 structures, as described previously for S. cerevisiae alg3 mutants (Verostek et al., 1991
) but also a large degree of higher-order glycans. Glycosidase digests, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), high-performance anion exchange chromatography pulsed amperometric detection (HPAEC-PAD) analyses, as well as genetic complementation were employed to decipher the glycans observed in this double mutant strain. Together, these results reveal the presence of an important early glycosylation mannosyltransferase as well as detail the characterization of the N-glycan profile of a strain lacking this protein in the industrial yeast P. pastoris. |
Results |
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Cloning and deletion of PpALG3
Degenerate primers designed based on the ALG3 gene sequence from S. cerevisiae and homologs from human and Drosophila melanogaster were used to polymerase chain reaction (PCR)-amplify an 83-bp sequence from P. pastoris genomic DNA that exhibited homology with these other ALG3 genes. Gene-specific and universal primers were used to PCR from a P. pastoris genomic library and assemble a 4.6-kb genomic sequence spanning the entire 1395-bp open reading frame (ORF) of the PpALG3 gene. A 3.5-kb fragment encompassing the ORF was then cloned and sequenced from P. pastoris genomic DNA. The 465-amino-acid predicted protein encoded by this gene has 36% identity with S. cerevisiae Alg3 and 29% identity with the human homolog, Not56 (Figure 2).
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PCR fragments of the 5' and 3' flanking regions of the gene along with the amplified resistance gene for the drug G418 were combined in a PCR overlap amplification (Davidson et al., 2002
) to generate a linear alg3::G418R mutant allele. The amplified alg3::G418R allele was transformed into an och1 mutant strain (BK3-1) of P. pastoris. Out of 24 transformants screened by PCR, two were found (designated RDP25 and RDP26) in which a PCR pattern was consistent with replacement of the wild-type ALG3 gene with the alg3::G418 mutant allele (8%).
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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N-glycans from K3 were also subjected to in vitro
-1,2-mannosidase digest followed by MALDI-TOF MS. N-glycans derived from the wild-type and och1 mutant strains behaved as expected, yielding Hex8GlcNAc2 and Hex5GlcNAc2, respectively, with some recalcitrant structures (Figures 3E and 3F). When N-glycans from the alg3 och1 mutant were subjected to -1,2-mannosidase treatment, the Hex5GlcNAc2 peak shifted to Man3GlcNAc2 consistent with the canonical alg3 Man5 structure (Figure 3G). However, in contrast to the Man5GlcNAc2 species, the other glycans were mostly resistant to treatment with an -1,2-mannosidase (Figure 3G).
Next, we individually purified the -1,2-mannosidase recalcitrant Hex6GlcNAc2, Hex7GlcNAc2, Hex8GlcNAc2, and Hex9GlcNAc2 (hexose 6–9) fractions on a P4 column, though we were not able to isolate enough material from the higher-molecular-weight (hexose 10–12) 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 6–9 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 7–12 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 Man5–12GlcNAc2, 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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Beside the addition of hexoses to the alg3 Man5 structure, these data are also consistent with the secretion of glucose-containing N-glycans caused by reduced efficiency of ER-residing glucosidases I and II. This phenomenon has been observed in S. cerevisiae alg3 mutant strains, with the presence of mono-, di-, and triglucosylated Man5GlcNAc2 structures being secreted (hexose 6–8) (Verostek et al., 1993). To resolve this ambiguity, we performed HPAEC-PAD analysis to determine monosaccharide composition of certain peaks. Following digestion with
-1,2-mannosidase and purification by P4 gel permeation chromatography, the hexose 6–8 and hexose 3 peaks were hydrolyzed and subjected to HPAEC PAD (Figure 3F and Table II). Only the Hex6GlcNAc2 peak was shown to contain Glc by this analysis consistent with a sugar composition of two GlcNAcs, five Mans, and one Glc (Table II). In addition, in vitro digest with an endomannosidase, known to remove a terminal -1,2-linked Glc-Man disaccharide (Spiro and Spiro, 2000; Hamilton et al., unpublished data), yielded Hex4GlcNAc2, suggesting that the recalcitrant Hex6GlcNAc2 peak is GlcMan5GlcNAc2, or the alg3 Man5 structure with a terminal Glc (data not shown). However, the remaining peaks could not be explained by insufficient glucosidase I and II activity, because they were shown to be composed of ratios of GlcNAc and Man that are consistent with ManXGlcNAc2 structures (Table II).
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Complementation of OCH1 in the alg3 och1 mutant strain
To analyze the effects of an alg3 deletion in the presence of wild-type Och1p
-1,6 mannosyltransferase activity, the och1 mutation in the RDP25 alg3 och1 mutant strain was complemented. The OCH1 locus of P. pastoris (Choi et al., 2003) was PCR-amplified and cloned into a plasmid containing the hygromycin resistance gene hph and the P. pastoris URA3 gene to create pRCD267. After integration of this plasmid by transformation and selection on hygromycin, almost all transformants were shown to contain the complemented PpOCH1 locus by PCR and also displayed wild-type growth and lacked temperature sensitivity (Figure 4). Two of these strains were saved for further analysis and designated RDP36-1 and RDP36-2. Previously, it was reported that the initiating -1,6 mannosyltransferase Och1p of S. cerevisiae displays a very low activity toward the alg3 Man5 structure both in vivo and in vitro, as compared with its activity toward a Man8 or Man9 structure (Nakayama et al., 1997; Verostek et al., 1993). Surprisingly however, analysis of N-glycans released from K3 secreted by the OCH1-complemented alg3 och1 mutant revealed only Hex6GlcNAc2 and larger glycans, as opposed to the RDP25 strain where a significant amount of Man5GlcNAc2 is present (Figure 3D). This suggests complete conversion of the alg3 Man5 structure to larger structures consistent with addition of the Och1p -1,6-linked mannose and outer chain formation. Furthermore, in vitro digest of the released N-glycans with -1,2-mannosidase revealed no detectable Man3GlcNAc2 in the OCH1 transformants, in contrast to the complete conversion of Man5GlcNAc2 to Man3GlcNAc2 in the RDP25 parent strain (Figure 3H). This indicates that the PpOch1p has a broader substrate specificity with respect to the alg3 Man5 versus the wild-type Man8GlcNAc2 when compared to the ScOch1p.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Early N-glycan synthesis in eukaryotes proceeds through ordered assembly of a Glc3Man9GlcNAc2 core oligosaccharide that is linked to the lipid carrier Dol-PP. In this study, we used a reverse genetic approach to begin analyzing the early steps of N-glycan assembly in the methylotrophic yeast P. pastoris. Specifically, we cloned and deleted a P. pastoris homolog of the S. cerevisiae ALG3 gene and investigated the resulting effects on glycosylation using MALDI-TOF MS, glycosidase digests, and HPAEC PAD. Furthermore, we conducted analysis of glycans from secreted proteins from the same alg3 mutant strain in the presence and absence of the outer chain initiating
-1,6-mannosyl transferase OCH1. From this we conclude that there are differences in the substrate specificity of the PpOch1p enzyme compared to its ScOch1p counterpart, as well as differences in the profile of Golgi-residing glycosyltransferases responsible for the larger high-mannose glycans found in yeast.
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 Man1,2Man1,2Man1,3(Man1,6)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 (25–40%) 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 Hex6GlcNAc2–Hex12GlcNAc2 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 (Hex6GlcNAc2–Hex12GlcNAc2) 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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Strains, culture conditions, and reagents
Escherichia coli strains TOP10 or DH5
were used for recombinant DNA work. P. pastoris GS115 (his4; Invitrogen, San Diego, CA) were used for generation of yeast strains (see Table III). Transformation of yeast strains was performed by electroporation as previously reported (Cregg et al., 2000). Protein expression was carried out at room temperature in a 96-well plate format with buffered glycerol-complex medium consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4x10–5% biotin, and 1% glycerol as a growth medium; and buffered methanol-complex medium consisting of 1.5% methanol instead of glycerol in buffered glycerol-complex medium as an induction medium. YPD is 1% yeast extract, 2% peptone, 2% dextrose, and 2% agar. Restriction and modification enzymes were from New England BioLabs (Beverly, MA). Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). The enzymes N-glycosidase F, mannosidases, and oligosaccharides were obtained from Glyko (San Rafael, CA). DEAE ToyoPearl resin was from TosoHaas (Montgomeryville, PA). Metal-chelating HisBind resin was from Novagen (Madison, WI). Ninety-six-well lysate-clearing plates were from Promega (Madison, WI). Protein-binding 96-well plates were from Millipore (Bedford, MA). Salts and buffering agents were from Sigma (St. Louis, MO). MALDI matrices were from Aldrich (Milwaukee, WI).
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Cloning of ALG3
Degenerate primers were designed based on amino acid sequence homology of ALG3 homologs from S. cerevisiae, Candida albicans, Neurospora crassa, S. pombe, D. melanogaster, and Homo sapiens. Primers were designed following the CODEHOP (consensus degenerate hybrid oligonucleotide primer) strategy with a 3' variable region and a 5' "clamp" region (Rose et al., 1998
). One pair of primers, CO3 (5'-GGTGTTTTGTTTTCTAGATCTTTGCAYTAYCARTT-3') and CO6 (5'-AGAATTTGGTGGGTAAGAATTCCARCACCAYTCRTG-3'), amplified an 83-bp fragment by PCR using P. pastoris genomic DNA. This fragment was cloned and sequenced and shown to have homology to ALG3 genes, therefore sequence-specific primers ACT1 (5'-GCGGCATAAACAATAATAGATGCTATAAAG-3') and ACT2 (5'-CCTAAGCTGGTATGCGTTCTCTTTGCCATATC-3') were designed. These primers were paired with T3 and T7 primers, respectively, to amplify a 1929-bp and a 2738-bp fragment from a P. pastoris EcoRI genomic library (gift from Judah Folkman, Harvard Medical School, Boston, MA). These fragments were cloned and sequenced and shown to contain the entire ORF of a gene with homology to S. cerevisiae ALG3. Finally, genomic DNA from P. pastoris strain GS115 was digested with XbaI and SphI and ligated into a pUC19. One clone out of 138 screened by colony PCR was shown to harbor the 3.5-kb XbaI/Sph1 fragment containing the P. pastoris ALG3 gene, which was confirmed and sequenced.
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 20–30 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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We thank Piotr Bobrowicz for scientific discussions. We gratefully acknowledge the gift of reagents by Judah Folkman, Alan Goldstein, and John McCusker. We also thank Tillman U. Gerngross, Markus Aebi, Phil Robbins, and Roger Bretthauer for guidance and support.
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com
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Abbreviations |
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Dol-PP, dolichyl pyrophosphate; ER, endoplasmic reticulum; HPAEC PAD, high-performance anion exchange chromatography pulsed amperometric detection; K3, Kringle3 domain of human plasminogen; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; ORF, open reading frame; PCR, polymerase chain reaction
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