Alterations in protein N-glycosylation confer vanadate resistance in Ogataea polymorpha mutants defective in phosphomannosylation

Abstract

Different yeast species, including Ogataea polymorpha, are often used as hosts for recombinant protein production. One of the most important factors limiting such applications is yeast-specific modifications of glycoside chains attached to secretory proteins. This problem can potentially be solved by the identification and inactivation of genes responsible for these modifications. Previously we demonstrated that the exceptional resistance of O. polymorpha to vanadate depends on the ABV1 gene responsible for the mannosylphosphorylation of protein glycoside chain in the Golgi apparatus. Here we show that mutations altering protein glycosylation in the secretory pathway can be selected in the abv1Δ mutant by screening for vanadate resistance. For one such mutant, we identified the responsible gene, which encodes a putative α-1,2-mannosyltransferase. To ensure the absence of phosphomannosylation, both O. polymorpha genes, ABV1 and MNN4, which encode mannosylphosphate transferase homologs, were inactivated. Some vanadate resistant mutants generated in this strain showed defects in N-glycosylation of a recombinant glycoprotein. This demonstrates that the effects of N-glycosylation on vanadate resistance in O. polymorpha are not mediated by phosphomannosylation per se and that identification of certain genes responsible for N-glycosylation in this yeast can be performed via selection of vanadate resistant clones.

1 Introduction

Proteins that enter the eukaryotic secretory pathway may undergo different posttranslational modifications, one of which is N-glycosylation. It starts in the endoplasmic reticulum (ER) with the attachment of Glc₃Man₉GlcNAc₂ oligosaccharide to specific asparagine residues in the -N-X-S/T-motif, where X is any amino acid except proline. This oligosaccharide is processed to the Man₈GlcNAc₂ structure before the protein enters the Golgi apparatus, where the glycoside receives additional modifications that can be essentially different in different organisms. In Saccharomyces cerevisiae, this Man₈GlcNAc₂ core glycoside can be modified by the attachment of a branched chain of mannose residues, a process initiated by the Och1 α-1,6-mannosyltransferase. However, the core glycoside of some S. cerevisiae proteins, e.g., carboxypeptidase Y, receive only few additional mannose residues (for a review see (Munro, 2001)). These modifications are frequently undesirable when yeast cells are used as a host for production of recombinant glycoproteins. To solve this problem it is necessary to inactivate the host genes encoding specific mannosyltransferases and introduce some heterologous genes, which encode enzymes catalyzing the desired modifications [for review see (Madhavan et al., 2021; Li et al., 2022)].

Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris) and two species of the Ogataea genus, O. polymorpha and O. parapolymorpha, which were formerly classified as Hansenula polymorpha, are frequently used as hosts for highly efficient recombinant protein production (Manfrão-Netto et al., 2019; Barone et al., 2023). Proteins secreted by these yeasts are usually not hyperglycosylated and their N-linked glycoside chains possess 8–14 mannose residues (Bretthauer and Castellino, 1999; Kim et al., 2004). While the K. phaffii homolog of S. cerevisiae Och1 was shown to be responsible for the initiation of mannosylation of the core glycoside in the Golgi apparatus (Choi et al., 2003), the closest homolog of this protein in O. parapolymorpha may not play a key role in this process. Functional analysis of other genes encoding homologs of α-1,6-mannosyltransferase in the latter yeast species identified the OCR1 gene whose inactivation prevented the attachment of α-1,6-mannose to the core glycosides of a recombinant protein in the Golgi apparatus (Kim et al., 2006).

In yeast, in addition to mannose residues, N- and O-linked glycoside chains are also modified by attachment of phosphomannose (Jigami and Odani, 1999). This modification of cell-wall mannoproteins creates negative charge on the cell surface that can be revealed by the ability of cells to bind Alcian blue dye. In S. cerevisiae, phosphomannose attachment depends on the protein encoded by the MNN4 gene (Jigami and Odani, 1999), which belongs to the fukutin protein family and presumably catalyzes mannosylphosphate transfer from the GDP-mannose donor (Aravind and Koonin, 1999). There is an MNN4 paralog in the S. cerevisiae genome, designated MNN14, however the inactivation of MNN4 alone is sufficient to abolish the Alcian blue staining (Jigami and Odani, 1999).

It is well known that mutations conferring resistance to vanadate in S. cerevisiae frequently affect protein N-glycosylation (Kanik-Ennulat et al., 1995), however the mechanism of this effect is not clear. Vanadate is a potent inhibitor of a large variety of enzymes due to its similarity to phosphate (Stankiewicz et al., 1995). These multiple targets can be protected by restriction of vanadate uptake from the environment. Vanadate resistant mutants isolated in Candida albicans (Mahanty et al., 1991) and Neurospora crassa (Bowman, 1983; Bowman et al., 1983) were defective in phosphate transport. The yeast phosphate transport system includes low- and high-affinity transporters. S. cerevisiae possesses two highly homologous low-affinity transporters in the plasma membrane, namely Pho87 and Pho90, and two high-affinity transporters Pho84 and Pho89, which show no similarity. Additionally, there is the vacuolar membrane low-affinity phosphate transporter Pho91. Despite high similarity, Pho87 and Pho90 are not equally functional, since the former is more efficient as a phosphate sensor, while the latter is more efficient as a transporter. The high-affinity transporter Pho84 was also shown to have an external phosphate sensing function (Giots et al., 2003). Methylotrophic yeast Ogataea polymorpha and O. parapolymorpha possess only one gene for the plasma membrane low-affinity phosphate transporter also designated as Pho87. Its inactivation in O. parapolymorpha increases resistance to vanadate (Karginov et al., 2018), while inactivation of the Pho91 homolog does not affect this phenotype (Farofonova et al., 2024). The O. parapolymorpha low-affinity phosphate transporters are somehow related to the regulation of methanol oxidase activity since inactivation of Pho91 leads to its complete reduction (Farofonova et al., 2023) while additional inactivation of Pho87 restores it to the wild-type level (Farofonova et al., 2024).

In contrast to O. parapolymorpha, whose vanadate resistance is similar to that of S. cerevisiae (Kim et al., 2002), its closest relative O. polymorpha is highly resistant to this compound (Mannazzu et al., 1997). Screening for vanadate hypersensitive mutants in this yeast identified the gene designated ABV1 (Karginov et al., 2018), which encodes a protein homologous to the S. cerevisiae putative mannosylphosphate transferase Mnn4. Its inactivation in both O. polymorpha and O. parapolymorpha led to a drastic decrease in the Alcian blue staining indicating defect in mannosylphosphorylation of oligosaccharide chains of cell-wall glycoproteins (Karginov et al., 2018). These yeasts possess one more gene encoding protein, which is even more homologous to S. cerevisiae Mnn4, but is probably not generally involved in mannosylphosphorylation of oligosaccharides of cell wall proteins, since inactivation of ABV1 alone was sufficient to virtually abolish Alcian blue staining. This gene is annotated as MNN4 in the deposited in NCBI GenBank O. parapolymorpha genome sequence (Ravin et al., 2013) due to its high similarity to the S. cerevisiae MNN4.

The role of O. polymorpha Abv1 in vanadate resistance most probably is related to the regulation of expression of the gene for the high-affinity transporter Pho84, since deletion of the ABV1 gene activates the PHO84 promoter while increase in ABV1 dosage represses it (Karginov et al., 2018). This may explain why S. cerevisiae mutants defective in glycosylation frequently show increased vanadate resistance, if the same mechanism is relevant in this yeast. At the same time, it remains unclear whether mannosylphosphorylation per se is crucial for vanadate resistance, or other alterations of N-linked glycoside chains can also affect this trait even in the absence of Abv1 function. The aim of this study was to explore these possibilities as well as to identify O. polymorpha genes affecting vanadate resistance and altering the protein glycosylation in the secretory pathway.

2 Materials and methods

2.1 Yeast strains, transformation and culture conditions

The strains used in this study are listed in Table 1. The O. polymorpha strain M257 (leu2 zeo^RP_MOX-GOX) carrying an expression cassette of Aspergillus niger glucoseoxidase (GOX) and 1b27 (leu2 ade2 ura3::ADE2) were used as a wild-type strain to construct mutants defective in protein glycosylation. The 1B27 was described previously (Fokina et al., 2012). The M257 strain was obtained by transformation of the A16 strain (Veale et al., 1992) with the linearized pZAM518 plasmid, which carries the GOX expression cassette under the control of the MOX promoter and the zeocin resistance selectable marker.

Yeast strains were transformed as described previously (Karginov et al., 2021). To do this, cells from 300 μL of an exponentially grown culture were spun down in a bench-top microcentrifuge at 5,000 rpm for 30 s, washed, and resuspended in 42 μL of sterile water. Then, 2 μL of the DNA-carrier solution (10 mg/mL, sheared and denatured by boiling) and 6 drops (approx. 90 μL) of 70% PEG 4000 were added and mixed well. After that, the suspension was mixed with 9 μL of 1 M Li-acetate solution and dispensed by 20–23 μL to add 1 μL of transforming DNA to each portion. Suspensions were incubated first at 30 °C for 30 min then at 45 °C for 30 min. The cells were washed with YPD medium and spread on plates for the selection of transformants.

Synthetic complete medium with glucose as a carbon source (0.67% Yeast Nitrogen Base, 2% glucose, 2% agar) was used to select yeast transformants. YPD (1% Yeast Extract, 2% Peptone, 2% Glucose) and YPM (1% Yeast Extract, 2% Peptone, 1% Methanol) media were used to cultivate yeast strains. To induce GOX expression, yeast cells were grown in Y3PM (1% Yeast Extract, 3% Peptone, 1% Methanol) supplemented with 150 mM NaCl at 30 °C for 50–60 h.

2.2 Plasmids, oligonucleotides and gene disruption cassettes

Oligonucleotides used in this work are listed in Supplementary Table S1. The pZAM518 plasmid was based on the pDLMOX-GOD-H GOX expression vector (Kim et al., 2004), whose 2405 bp DraI-BamHI fragment bearing the ampicillin resistance and LEU2 selectable markers was replaced with the 923 bp BamHI-EcoRV fragment of the pGAPZα-A plasmid (Invitrogen) carrying the zeocin resistance marker. Construction of the pAM620 plasmid used to disrupt the ABV1 gene with cre/loxP-self excisable vector was described previously (Karginov et al., 2018). To obtain recombination arms for the ABV2 disruption cassette, O. polymorpha genomic DNA was digested with EcoRV, self-ligated and used as a template for PCR with primers ABV2U and ABV2L. The obtained fragment was inserted between the EcoRV and the SalI sites of the pAM619 vector (Agaphonov and Alexandrov, 2014). The resulting plasmid designated pAM759 was linearized with EcoRV and used for yeast transformation to disrupt the ABV2 gene. To recycle the leu2 auxotrophic marker, the disruptants obtained with plasmids pAM620 and pAM759 were grown overnight on liquid YPM and spread onto YPD plates to obtain separate colonies, which were then tested for the loss of leucine prototrophy.

In O. polymorpha, integration of gene disruption cassettes often occurs in random genomic sites via non-homologous recombination. This sometimes significantly hampers identification of clones with the “correct” integration of a disruption cassette if such clones do not display a selectable phenotype. Since there is the HIS2 gene adjacent to MNN4 in the O. polymorpha genome it could be used to select transformants with plasmid integrated into this locus. Thus, we have constructed two plasmids for the MNN4 disruption, one of which, pMP9V, was designed to replace MNN4 and portion of HIS2 with the LEU2 selectable marker, and another one pCMP7 designed to replace integrated sequence of pMP9V bearing LEU2 and to restore HIS2. To construct the pCMP7 plasmid, one recombination arm was obtained by PCR with primers OpoMNN4AU1 and OpoMNN4L1 and digested with HindIII, while the other was obtained by PCR with primers OpoMNN4AL1 and OpoMNN4U1 and digested with BamHI and HindIII. The obtained fragments were ligated with the BamHI-HincII-digested pBCKS + vector. To construct pMP9V plasmid, the 2963 bp ScaI Eco72I fragment of pCMP7 was inserted between the EcoRV and the BglII sites of the pAM773 vector (Agaphonov, 2017).

The OCH1 disruption cassette was constructed as follows. The DNA fragment representing the O. polymorpha genomic locus carrying OCH1 was obtained by PCR with primers OpoOCH1U1 and OpoOCH1L1, digested with HindIII and ligated with HindIII-Ecl136II-digested pUC18 vector. The NaeI-BglII 241 bp fragment within the OCH1 ORF in the resulting plasmid was replaced with the BamHI-EcoRV 1254 bp fragment of the pCLHX plasmid (Sohn et al., 1996) carrying the LEU2 selectable marker. The resulting plasmid designated, which was designated pMP6, was digested with HindIII and NcoI to excise the disruption cassette used to disrupt OCH1 in the O. polymorpha genome.

Sequences of the O. parapolymorpha OCR1 locus were used as recombination arms for the OCR1 disruption cassette. To construct it, the DNA fragment possessing this gene was obtained by PCR with primers OpaOCR1AL and OpaOCR1AU. The BamHI-PstI 1382 bp and XhoI-PstI 1316 bp were ligated with the BamH-XhoI-digested pBCKS + plasmid to obtain pAM1042. The recombination arms were excised from pAM1042 by BssHII-BamHI (971 bp fragment) and BssHII-PvuII (1346 bp fragment) and ligated with the EcoRV-BglII-cleaved pAM773 vector, which is capable of self-excision by cre/loxP recombination (Agaphonov, 2017). The resulting plasmid designated pAM1055 was cleaved with XhoI and BglII prior to yeast transformation.

2.3 UV mutagenesis

To obtain vanadate resistant mutants, yeast cells from logarithmic YPD culture were collected by centrifugation re-suspended in sterile water to OD₆₀₀ ∼0.5 and exposed to UV for 10 s. The cell suspension was mixed with an equal volume of YPD and incubated in 37 °C shaker incubator for 1 h. Cells were collected by centrifugation, re-suspended in YPD containing 17% glycerol dispensed by 0.5 mL in 1.5 mL Eppendorf tubes and stored at −70 °C. The frozen aliquots were thawed at room temperature and spread onto YPD plates containing 3.5 or 4 mM sodium orthovanadate.

2.4 Electrophoresis, immunoblotting and antibodies

Prior to electrophoresis, proteins from culture supernatants were precipitated with trichloroacetic acid (TCA) as follows. 1 mL of culture supernatant was mixed with 33 μL of 0.5% sodium deoxycholate, incubated 5 min at room temperature and then mixed with 72 μL of trichloroacetic acid 1 g/mL solution and incubated 1 h at room temperature. The precipitate was spun down by centrifugation in a table-top microcentrifuge at 15,000 g for 10 min. The pellet was washed 2 times by 1 h incubation and 1 time by overnight incubation with 1 mL of acetone. Then the pellet was air-dried 10 min at 37 °C and dissolved in either 50 μL of the 1^X electrophoresis sample buffer (1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetate, 5% glycerol, 0.5% β-mercaptoethanol, 25 mM tris-HCl pH 6.8), or 30 μL of solution containing 0.5% SDS and 40 mM dithiothreitol (DTT) if the treatment with the PNGase F was required and incubated 10 min at 98 °C. The samples destined for PNGase treatment were mixed with equal volume of 2% NP-40 solution in 100 mM sodium phosphate buffer pH 7.5. PNGase F was added to a final concentration of 5 μg/mL and the samples were incubated 1 h at 37 °C. The recombinant PNGase F (Boyko et al., 2023) was kindly provided by Dr. Nikolai Sluchanko (Research Center of Biotechnology RAS, Moscow, Russia). The PNGase F treated samples were mixed with 1/3 volume of 4^X electrophoresis sample buffer and incubated 3 min at 98 °C. Proteins were separated by SDS-PAGE and transferred to PVDF membrane according to standard protocols (Laemmli, 1970; Towbin et al., 1979). The membranes were blocked by 1 h incubation in 1% casein solution in TBS buffer (150 mM NaCl, 30 mM tris-HCl pH 7.4) containing 0.08% Tween 20. Then the membranes were placed in the same solution supplemented with a primary antibody and incubated overnight at room temperature. The unbound antibody was removed by incubation of membrane in TBS 3 times for 5 min. Then the membranes were incubated in the casein solution supplemented with the goat anti-rabbit IgG horseradish peroxidase conjugate (ThermoFisher Scientific, cat. #31460) at a dilution of 1:10,000. The unbound antibody was removed by incubation in TBS containing Tween-20 2 times for 5 min and 2 times for 5 min in TBS without Tween-20. The protein bands were detected by enhanced chemiluminescence technique using the SuperSignal™ West Dura kit (ThermoFisher Scientific, cat. #34076). Commercial antibody against Aspergillus niger glucose oxidase (Accurate Chemical and Scientific, Westbury, NY; cat. #YNNE0577S) was kindly provided by Prof. Hyun Ah Kang (Chung-Ang University, Seoul, Republic of Korea). This antibody was used at a dilution of 1:10,000. Immunoblotting of extracellular chitinase was performed using a rabbit antiserum, which was obtained as follows. The DNA fragment encoding chitinase was obtained by PCR using hpcts5′ and cbsCTS1_L2 primers and O. polymorpha genomic DNA as a template. The 775 bp BamHI-XhoI fragment of the obtained PCR product was inserted into the pET23a Escherichia coli expression vector (Novagen) between BamHI and XhoI sites that led to the in-frame fusion of the sequence encoding 17th-276th amino acid residues of O. polymorpha chitinase with the T7 tag- and the His tag-encoding sequences at the 5′and 3′ ends, respectively. The expression of the obtained recombinant gene in cells of the E. coli BL-21 (DE3) strain (Studier et al., 1990) and following purification of the protein product using Ni-NTA resin was performed according to the previously described protocol (Mierendorf et al., 1998). The purified protein was used for the rabbit immunization. The antiserum was used at a dilution of 1:3,000.

2.5 Alcian blue staining

Cell staining with Alcian blue was performed as described previously (Ballou, 1990) with minor modifications as follows. Cells from overnight cultures were precipitated by centrifugation at 2,500 g for 3 min, washed with 10 mM HCl and re-suspended in 0.1% Alcian blue solution in 10 mM HCl. Unbound dye was removed by washing cells with 10 mM HCl. Cells were transferred into a flat-bottom 96-well plate, which was centrifuged at 2000 g for 3 min and scanned.

3 Results

3.1 Mutations affecting N-linked glycosylation can increase vanadate resistance in O. polymorpha abv1Δ mutant

As it was mentioned in the Introduction, inactivation of the O. polymorpha ABV1 gene, which encodes mannosylphosphate transferase, decreases vanadate resistance (Karginov et al., 2018). To explore whether other mutations can increase vanadate resistance when this gene is deleted, the 1B27-620M1 strain possessing a deletion in ABV1 gene was UV-mutagenized and vanadate resistant clones were selected on YPD containing 4 mM sodium orthovanadate. Unlike the original strain, one of the obtained vanadate resistant clones, AV35, was unable to grow at 47 °C and in the presence of 0.005% SDS in culture medium (Supplementary Figure S1). As it was mentioned in the introduction, it was known that in S. cerevisiae some mutations affecting N-glycosylation also increase vanadate resistance (Kanik-Ennulat et al., 1995), while defects of glycosylation in the secretory pathway in Ogataea yeasts were shown to increase detergent sensitivity (Agaphonov et al., 2001; Agaphonov et al., 2005; Kim et al., 2006). Thus, the phenotypes observed in the AV35 mutant suggested possible defects in protein N-glycosylation. However the strain used for mutagenesis did not possess a convenient reporter for the analysis of N-glycosylation. To solve this problem, we constructed an abv1Δ mutant strain expressing A. niger extracellular glucose oxidase (GOX) as an N-glycosylation reporter since it has 8 potential N-glycosylation sites. This strain was also UV-mutagenized and vanadate resistant clones were selected on plates containing 3.5 mM sodium orthovanadate. Some of them grew very slowly even in the absence of vanadate, which hampered further analysis. Finally, 26 mutants with acceptable growth rates were chosen to test whether they were altered in protein N-glycosylation. To do this, electrophoretic mobility of secreted GOX was studied. As expected, GOX migrated on the SDS-PAGE as a smear due to the irregular size of N-glycoside chains. Electrophoretic mobility of this smear in some mutants differed from that in the original abv1Δ strain (Supplementary Figure S2, “AV” mutants) that indicated alterations in N-glycosylation. To confirm that this was due to the difference in the size of N-linked glycosides, electrophoretic mobility of glycosylated and deglycosylated GOX was studied in some mutants (Figures 1a,b). Unlike the untreated GOX migrating as a smear, which was different in different strains, the deglycosylated GOX migrated as two very close sharp bands (probably due to different proteolytic processing) with the same apparent molecular weight in all strains, which virtually corresponded to the calculated molecular weight of the mature polypeptide (64 kDa). This demonstrated that the difference in the mobility of the untreated GOX in different strains was due to alterations in N-linked glycosylation. Notably, there were fewer GOX species with lower electrophoretic mobility in some cases, particularly, in mutants AV13 and AV21 and this protein migrated as a more compact smear than in the original abv1Δ strain indicating the decrease in N-glycoside chain size, while a noticeable increase in N-glycoside chain size was observed in the AV20 mutant (Figure 1a). These results indicate that modifications of glycoside chains other than that catalyzed by the Abv1 protein can also affect vanadate resistance in O. polymorpha.

FIGURE 1

3.2 Inactivation of a O. polymorpha homolog of the Golgi apparatus α-1,2-mannosyltransferases increases vanadate resistance in the abv1Δ mutant

Since the AV35 mutant was hypersensitive to SDS and elevated temperature, we suggested that these phenotypes and vanadate resistance are caused by the same mutation. To identify the gene defined by this mutation, AV35 was transformed with an O. polymorpha genomic library and SDS resistant transformants were selected. The plasmids possessed by clones designated as “5” and “6” were recovered from yeast cells by the E. coli transformation. Their sequencing (Supplementary Figure S3) revealed that they contained overlapping fragments of the genomic locus with ORF encoding a presumable α-1,2-mannosyltransferase [GenBank gene ID: XM_018356748.1 (Riley et al., 2016)]. Notably, O. polymorpha genome possesses two more open reading frames (GenBank ID: XP_018209282.1 and ORF locating at 1396245-1398152 positions of the scaffold NW_017264699.1) encoding proteins homologous to S. cerevisiae and C. albicans α-1,2-mannosyltransferases (Supplementary Figure S4). The AV35 mutant was transformed with recovered plasmids to prove that they are able to complement the mutant phenotypes. The genomic library was based on the AMIpSL1 vector, which has a propensity for genome integration (Agaphonov et al., 1999). Taking this into account, the transformants were streaked on selective medium to obtain individual colonies, which were expected to be integrants. Indeed, the obtained clones were SDS-resistant and vanadate-sensitive, while the transformants obtained with the empty AMIpSL1 vector retained the mutant phenotypes (Supplementary Figure S5). This indicated that the plasmids acquired by the clones “5” and “6” possess the wild-type gene, whose mutation led to vanadate resistance in AV35.

Targeted disruption of the identified gene in the abv1Δ mutant using pAM759 disruption cassette (see Materials and Methods) led to the same phenotypes as those revealed in the AV35 mutant. Inactivation of the identified gene in the strain with wild-type ABV1 also increased sensitivities to SDS (Supplementary Figure S6) and high temperature (Figure 2), but surprisingly, it caused a severe growth defect, which was not observed in the double mutant (Figure 3). This indicated that the abv1Δ mutation may rescue the growth defect caused by inactivation of the identified gene.

FIGURE 2

FIGURE 3

This gene was designated ABV2 (Alcian blue staining, vanadate resistance), since its mutations improved vanadate resistance in the abv1Δ strain, while the cells of its knockout mutant obtained in the strain with wild-type ABV1 did not bind the Alcian blue dye that indicated a severe defect of phosphomannosylation of cell-wall proteins (Figure 4).

FIGURE 4

It was previously shown that deletion of the O. parapolymorpha OCR1 gene encoding α-1,6-mannosyltransferase, which is responsible for the N-glycoside outer chain elongation, also causes a detergent and high temperature sensitivity as well as retardation in growth rate (Kim et al., 2006). These phenotypes were also revealed when we inactivated the OCR1 gene in O. polymorpha, however the growth defect was less pronounced than in the abv2Δ strain (Figure 3). Interestingly, inactivation of OCR1 also led to a substantial decrease in Alcian blue staining, but it appeared to be not as severe as in the abv2Δ mutant (Figure 4). Inactivation of the OCH1 gene, which encodes another α-1,6-mannosyltransferase, also decreased Alcian blue staining, but less than the ocr1Δ mutation. This correlated with the observation that in O. parapolymorpha, Och1 has a smaller effect on N-glycoside outer chain formation than Ocr1 (Kim et al., 2006). In O. polymorpha, GOX secreted by ocr1Δ and abv2Δ mutants migrated on the SDS-PAGE not as a broad smear like in the wild-type strain, but as a more compact band (Figure 5) indicating more uniform glycosylation. In contrast, GOX secreted by the och1Δ mutant migrated as a smear, though this smear migrated faster than the smear of GOX secreted by the wild-type strain (Figure 5). Similar difference between the effects of inactivation of OCR1 and OCH1 was observed previously in O. parapolymorpha (Kim et al., 2006).

FIGURE 5

3.3 Inactivation of MNN4 in the abv1Δ mutant additionally decreases Alcian blue staining

Although some of the vanadate resistant mutants selected in the abv1Δ mutant were defective in protein glycosylation in the secretory pathway, we could not exclude that phosphomannosylation was still involved in the increase in vanadate resistance in these mutants, since they possessed the MNN4 gene, which encodes another homolog of the S. cerevisiae mannosylphosphate transferase Mnn4.

To ensure that the phosphomannosylation is completely abolished in O. polymorpha, inactivation of both ABV1 and MNN4 genes was required. However, we did not know whether inactivation of MNN4 was lethal or not, how this mutation affects Alcian blue staining and how it interacted with ABV1 inactivation. To explore this, the MNN4 gene was inactivated in the abv1Δ mutant and in the strain with wild-type ABV1. Both the single mnn4Δ and the double abv1Δ mnn4Δ mutants were found to be viable. Inactivation of MNN4 alone did not noticeably affect either vanadate resistance or Alcian blue staining (Figures 2, 4). At the same time, inactivation of this gene in the abv1Δ mutant additionally reduced Alcian blue staining (Figure 4), indicating that Mnn4 is involved in phosphomannosylation of a limited subset of the cell wall proteins or its mannosylphosphate transferase activity is much lower than that of Abv1.

Comparison of protein sequences of Mnn4 homologs from S. cerevisiae, Yarrowia lipolytica, K. phaffii and O. polymorpha (Figure 6) revealed that the common ancestor of these yeasts most probably possessed only one gene encoding a protein of this family, while the additional genes emerged after the lineages of S. cerevisiae and methylotrophic yeasts were separated. The O. polymorpha Mnn4 shows higher similarity to K. phaffii Pno1, while Abv1 falls into the same group with Y. lipolytica Mpo1 and K. phaffii Mnn4C and B. Alignment of protein sequences (Supplementary Figure S7) revealed conservative domains present in all these proteins, while some positions distinguish the group comprising O. polymorpha Mnn4 and K. phaffii Pno1 from the group of the O. polymorpha Abv1, K. phaffii Mnn4C and Y. lipolytica Mpo1.

FIGURE 6

3.4 Mutations affecting N-linked glycosylation can increase vanadate resistance in O. polymorpha abv1Δ mnn4Δ double mutant

To obtain vanadate resistant mutants, the cells of the abv1Δ mnn4Δ strain were irradiated with UV and spread on plates with 3.5 mM sodium orthovanadate. Some of the obtained clones grew on regular YPD medium much more slowly than the original strain that complicated further analysis. The clones with acceptable growth rates on YPD were tested for sensitivity to SDS and to elevated temperature since such phenotypes are often revealed in mutants defective in protein glycosylation (Agaphonov et al., 2005; Kim et al., 2006). Among 45 clones tested, 14 were unable to grow in presence of 0.004% SDS and at 46 °C (class A), 11 were hypersensitive to SDS but not to elevated temperature (class B), 6 were hypersensitive only to the elevated temperature (class C), growth of 7 was similar to that of the original strain (class D), and 7 clones grew even better than the original strain in these stress conditions (class E). The latter class could arise due to suppression of the negative effects caused by abv1Δ and mnn4Δ. Nine A clones, 6 B clones, 4 C clones, 2 D clones and 3 E clones were examined for the GOX electrophoretic mobility (Supplementary Figure S2, AMV mutants). GOX from some clones of class A and B, but not of other classes, demonstrated altered electrophoretic mobility indicating glycosylation defects. As described above for the mutants obtained in the abv1Δ single deletion strain, the involvement of N-glycosylation in the difference in electrophoretic mobility was confirmed in some mutants by immunoblotting of glycosylated and deglycosylated GOX (Figures 1a,b). Presence of SDS sensitivity in the mutants with altered glycosylation was in agreement with previous observations that defects in protein glycosylation in the secretory pathway can cause detergent sensitivity in yeast (Kanik-Ennulat et al., 1995; Agaphonov et al., 2001; Kim et al., 2006). This phenotype is usually explained in terms of compromising the cell wall integrity and propensity of cells to lyse in the presence of a detergent. However other explanations can also be valid since we have shown previously that SDS interferes with Ca²⁺ homeostasis by promoting its uptake from the environment (Fokina et al., 2012; Kulakova et al., 2024).

Similar to the mutants obtained in the abv1Δ strain, some of the mutants selected in the abv1Δ mnn4Δ strain secreted GOX with shorter N-glycoside chains than in the original strain (mutants AMV-5, AMV-18 and AMV-25), while in the mutant AMV-4 GOX glycosylation was significantly increased.

To study the effects of the obtained mutations on O-glycosylation, the electrophoretic mobility of extracellular chitinase was analyzed, since this protein possesses exclusively O-linked glycosides. Calculated molecular weight of its polypeptide chain is 59 kDa, but it migrates as an approximately 120 kDa protein due to O-glycosylation at multiple sites between the catalytic and binding domains (Kuranda and Robbins, 1991; Agaphonov et al., 2005). Five mutants obtained in the abv1Δ strain and 5 mutants obtained in the abv1Δ mnn4Δ strain were studied. None of the mutants obtained in the abv1-Δ strain showed alterations in the chitinase electrophoretic mobility despite at least four of them (AV13, AV20, AV21, and AV14) having altered N-glycosylation of GOX (Figure 1c). The chitinase content in culture medium of these mutants was similar to that in the wild-type, abv1Δ, and abv1Δ mnn4Δ strains. At the same time the mutants obtained in the abv1Δ mnn4Δ strain secreted noticeably less chitinase than the original strain and electrophoretic mobility of this protein was altered (Figure 1c). In particular, the AMV-4 mutant secreted more heavily glycosylated chitinase, while in AMV-5 it was less glycosylated. This correlated with alterations in GOX glycosylation in these two mutants (Figure 1a). This means that these mutations affect the length of both N- and O-linked glycosides attached to proteins in the secretory pathway and indicates that selection of vanadate resistant clones in the abv1Δ, and abv1Δ mnn4Δ strains may reveal different subsets of mutations affecting protein glycosylation. The latter suggestion was in agreement with the observation that inactivation of OCH1 in the abv1-Δ strain slightly increased vanadate resistance, but had almost no effect in the abv1Δ mnn4Δ strain (Figure 2).

4 Discussion

Previously, we have shown that vanadate resistance in the Ogataea yeasts depends on the ABV1 gene expression level that affects regulation of the promoter of the PHO84 gene encoding plasma membrane high-affinity phosphate transporter (Karginov et al., 2018). One could suggest that the presence of phosphomannose in the protein glycoside chains somehow downregulates the phosphate transport system and thus improves vanadate resistance. However here we show that even in the absence of the enzymes responsible for this modification other alterations of protein glycosylation can affect vanadate resistance in O. polymorpha. To do this, we first inactivated both O. polymorpha genes (ABV1 and MNN4) encoding homologs of the S. cerevisiae Mnn4 enzyme, which catalyzes phosphomannosylation of glycoside chains, and then obtained mutations improving vanadate resistance and altering glycosylation of a reporter protein.

Unlike ABV1, inactivation of MNN4, which encodes another homolog of S. cerevisiae mannosylphosphate transferase Mnn4, did not noticeably affect vanadate resistance in O. polymorpha. Inactivation of ABV1 alone conferred vanadate sensitivity sufficient to select genomic mutations improving vanadate resistance. One of these mutations defined a gene encoding a homologue of α-1,2-mannosyltransferases. Interestingly, inactivation of this gene in the strain with wild-type phosphomannosylation led to loss of the ability of cells to bind the Alcian blue dye. That was why this gene was designated ABV2 (Alcian blue staining, Vanadate resistance), though the effect of its inactivation on vanadate resistance in the abv1Δ mutant was opposite to the effect of ABV1 inactivation in the wild-type strain.

Inactivation of OCR1 encoding α-1,6-mannosyltransferase also led to the decrease in Alcian blue staining, however this effect was less pronounced than that in the abv1Δ mutant. A noticeably weaker decrease in the dye binding was observed in response to the inactivation of OCH1, which encodes another α-1,6-mannosyltransferase. This correlated with the effects of these mutations on growth rate. We suggest that mannosylphosphate is attached to residues, which are attached by Abv2 to the N-glycoside outer chains initiated by Ocr1 or Och1. In this case the absence of either Och1 or Ocr1 alone should not completely abolish the outer chain formation in all N-linked glycosides and thus the glycosides, which have received the outer chain, can still be modified by the attachment of α-1,2-mannose and then by phosphomannose. At the same time Abv2 presumably can modify the outer chains independently of whether they were initiated by Och1 or Ocr1. Probably, that was why ABV2 inactivation in the strain with the wild-type phosphomannosylation led to a more severe growth defect than inactivation of OCR1 or OCH1.

Although the O. polymorpha Mnn4 protein shows higher similarity to S. cerevisiae Mnn4 than Abv1 does, the effect of its inactivation on Alcian blue staining is much less pronounced and no effect on vanadate resistance was observed. Possibly, this protein phosphomannosylates a specific subset of proteins, which are less represented in the cell wall and are not involved in the phosphate transport control. Interestingly, Komagataella phaffii has four Mnn4 homologs, namely, Pno1 and Mnn4A-C (Miura et al., 2004; Bobrowicz et al., 2007). Inactivation of PNO1 significantly reduced phosphomannosylation of a model recombinant glycoprotein, but did not noticeably affect Alcian blue staining (Miura et al., 2004). Since O. polymorpha Mnn4 shows higher similarity to K. phaffii Pno1 and Mnn4A, while Abv1 is more homologous to Mnn4B and C, it is reasonable to expect that the similarity of sequences represent similarity of functions and that K. phaffii Mnn4B and C should be more involved in phosphomannosylation of cell wall proteins than Pno1 and Mnn4A are. Some biotechnological applications require complete ablation of phosphomannosylation of recombinant proteins produced by yeast. This seems to be more easily achieved in O. polymorpha and O. parapolymorpha than in K. phaffii, since the former two species have only two genes responsible for this modification and as we have shown here both these genes can be inactivated simultaneously.

Selection of vanadate resistant clones appears to be an efficient approach to inactivation and identification of yeast genes responsible for the Golgi apparatus modifications of N-glycosides. To do this in O. polymorpha, here we first used the vanadate sensitive abv1Δ mutant that allowed us to obtain mutants with altered glycosylation and identified the ABV2 gene. The use of the abv1Δ mnn4Δ strain may allow obtaining mutations in a different set of genes responsible for glycosylation if such mutants are selected by vanadate resistance. Indeed, the effect of OCH1 inactivation on vanadate sensitivity was different between the abv1Δ and abv1Δ mnn4Δ strains. The importance of MNN4 for the manifestations of mutations affecting glycosylation is also highlighted by the observation that none of the tested vanadate resistant mutants selected in the abv1Δ mutant showed alterations in O-glycosylation, while such alterations were revealed in mutants selected in the abv1Δ mnn4Δ strain.

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