Last September, we submitted Perlara’s second preprint to bioRxiv describing our yeast models of PMM2 deficiency. This work is currently under review at the Genetics Society of America’s peer-reviewed journal G3. Below is the description of the figures from the manuscript, detailing our work in modeling PMM2 patient alleles. We’ll start with Figures 1 – 4 on this post, and continue with Figures 5 – 7 in a second post. We hope this outreach encourages new researchers into the PMM2 community.

Modeling PMM2 patient alleles in yeast SEC53

As part of our original PerlQuest partnership with Maggie’s Cure, we set out to generate three PMM2 variants: R141H, F119L, and V231M. Since then, we’ve added two more variants that are associated with PMM2-CDG to our list: E93A and E139K. You can see where these residues lie in the gene and in the protein structure (Figure 1A and C). R141 is in the substrate-binding domain of PMM2 (Andreotti et al., 2015). R141H has no detectable enzymatic activity and never occurs in homozygosity in patients despite being the most commonly observed mutation (Kjaergaard et al., 1999). F119 is a component of the hydrophobic core within the dimer interface and is the second most commonly observed mutation (Silvaggi et al., 2006). F119L has 25% enzymatic activity in vitro and this deficiency is likely due to its diminished ability to dimerize and decreased dimer stability (Kjaergaard et al., 1999; Pirard et al., 1999; Andreotti et al., 2015). V231 is in the interior of the core domain and a mutation in this residue is detrimental to its native protein structure (Silvaggi et al., 2006; Citro et al., 2018). The folding and stability defect of the V231M allele contributes to its reported reduced in vitro enzymatic activity of 38.5% (Kjaergaard et al., 1999; Pirard et al., 1999). Existing data on E93A and E139K variants are limited. E93 directly interacts with R116 in trans within the PMM2 dimer and a mutation in this residue likely compromises dimerization (Andreotti et al., 2015). E139K is a result of a 415G>A transition mutation in the genomic sequence that interferes with RNA splicing, causing either skipping of exon 5 to form a partially deleted and nonfunctional protein or a full-length E139K mutant protein (Vuillaumier-Barrot et al., 1999).

Figure 1. Generating yeast models of PMM2 deficiency

  1. Sequence alignment of phosphomannomutase genes in human (PMM2) and yeast (SEC53). Asterisk (*) indicates an identical amino acid residue and colon (:) indicates similar amino acids. Red boxes show the conserved disease-causing amino acid residues that we’ve modeled.
  2. Table showing the PMM2 patient alleles and the equivalent variants we generated in yeast SEC53.
  3. Structure of PMM2 dimer highlighting the five modeled residues. Dimer structure was generated from 2AMY in the RCSB protein data bank and courtesy of Dr. Maria Vittoria Cubellis (University of Naples Federico II, Italy).
  4. Comparison of promoter strength. Different promoters are used to drive the expression of the gene of interest. GFP is placed under the REV1, SEC53, ACT1, or TEF1 promoter and the fluorescent intensity of GFP is measured by flow cytometry. The graph displays the fluorescence in arbitrary unit (a.u.) against the cell count. The raw numbers are shown in the table along with the expression level relative to the SEC53 promoter.

 

PMM2 F119L, R139K, R141H, and V231M correspond to SEC53 F126L, E146K, R148H, and V238M, respectively (Figure 1B). SEC53 E100K was unintentionally generated, but the residue is conserved in PMM2 and the patient allele is E93A. Some of these variants have low to no detectable enzymatic activity reported in the literature, so we placed the mutants under different promoters to determine if changes in protein abundance affect viability of each variant. The relative strength of the TEF1, ACT1, and REV1 promoters were compared to the native SEC53 promoter by driving expression of the green fluorescent protein (GFP) (Figure 1D). Based on fluorescence reading by flow cytometry, we found that relative to the native SEC53 promoter, the strength of TEF1, ACT1, and REV1 promoters are 10X, 2X, and 0.2X, respectively.

 

Growth of Sec53 variants correlates with enzymatic defects of the variant and promoter strength

To overcome the complication that SEC53 is an essential gene, we placed a wildtype SEC53 copy on a URA3 plasmid that we can conditionally remove by growing cells in 5-fluoroorotic acid (5-FOA). 5-FOA is an analog of uracil that is converted into a toxic intermediate in cells where the uracil biosynthetic pathway is active, which the URA3 marker enables. Each SEC53 variant is then individually integrated at the HO locus of sec53∆ cells. The phenotype of each variant is revealed when the wildtype URA3 containing plasmid is counter-selected in media containing 5-FOA.

Increasing the expression level of the hypomorphic alleles improves their growth in every case except the R148H variant and the sec53∆ negative control (Figure 2A, B). When expressed at native level, the V238M allele is sufficient for growth, but at a slower rate and achieving a lower final yield than wildtype cells (31.8%). Doubling the expression of the V238M allele with the ACT1 promoter restores growth of this mutant: 67% at 20 hours and near wildtype final yield at 24 hours. The F126L allele grows poorly under the endogenous promoter (26.9%), and also grows better when its expression is doubled (56.6%). The relative growth of F126L and V238M is consistent with their reported residual in vitro enzymatic activity (Pirard et al., 1999). Over-expression of F126L and V238M alleles under the TEF1 promoter completely rescues these cells. E100K is viable only under the ACT1 (16.2%) and TEF1 (66.5%) promoters, which indicates the severity of this mutation compared to the other variants. On the other hand, the R148H null allele is not compatible with growth at any promoter strength and phenocopies the genetic null sec53∆. Together, these data can be fully explained by mass action effects, where a reduction in enzymatic activity can be overcome by increasing the total amount of enzyme in the cell.

In contrast, reducing wildtype SEC53 mRNA expression to 20% of its native level with the REV1 promoter modestly, but consistently, compromises cell growth to 63.6% of the control (Figure 2A and B). This result shows that yeast cells are highly sensitive to the total amount of SEC53 protein. Interestingly, there was no toxicity associated with 10-fold over-expression of wildtype SEC53. Similar to wildtype, the E146K variant is defective only at the lowly expressing REV1 promoter (68.8%). This result suggests that the splicing defect of the 415G>A mutation in humans may reduce the abundance of still functional PMM2 E139K proteins to a sub-critical amount below the threshold for viability.

Comparing the strains relative to one another at a single time-point, we can see that the severity of growth defects of the SEC53 alleles correlates with the level of residual enzymatic activity of the PMM2 alleles reported using bacterial recombinant protein and PMM2-CDG patient fibroblasts (Figure 2B).

 

Figure 2. Comparison of growth of yeast SEC53 haploid alleles

  1. Graphs show growth of 10-2 dilution of OD 1.0 cells over time at 30oC in 50 µL SC+FOA media in a 384-well plate. 1X indicates the native SEC53 promoter. 2X indicates double the native promoter strength. 10X indicates 10X the native promoter strength. 0.2X indicates 20% of the native promoter strength.
  2. Comparison of growth relative to wild type SEC53 at a single time point (t=18).

 

SEC53 diploid variants recapitulate the growth of haploid variants

Most PMM2-CDG patients are compound heterozygotes with a recurring or novel mutation (usually missense) paired with the R141H null allele. The most common genotype is PMM2F119L/R141H. To further study genotype-phenotype relationships of the SEC53 variants, we generated homozygous and compound heterozygous diploids (Figure 3). As expected, a single copy of wildtype SEC53 is sufficient for normal diploid cell growth and there is no growth difference between homozygous wildtype and heterozygous SEC53+/R148H (99.7%) cells (Figure 3A).

Homozygous F126L diploids grow slower than their respective F126L/R148H heterozygous diploids (Figure 3A). In both 1X and 2X expression regimes, R148H heterozygosity improves growth. Normalizing to wildtype at 20 hours, the growth of pACT1-F126L/F126L is 51.8% compared to 65.7% in pACT1-F126L/R148H. pSEC53-F126L/F126L is 20.9% compared to 40.7% in pSEC53-F126L/R148H. These results could be explained by the diminished ability of the PMM2 F126L variant to homodimerize, and the presence of R148H monomers allows formation of hemi-functional F126L:R148H heterodimers (Andreotti et al., 2015). On the other hand, V238M causes protein misfolding but properly folded V238M monomers are competent to dimerize. The pSEC53-V238M/V238M homozygous diploid (43.2%) grows similarly to the pSEC53-V238M/R148H heterozygous diploid (46.4%) (Figure 3B). However, at the final 24 hour timepoint the pSEC53-V238M/V238M homozygous diploid was growing better than the pSEC53-V238M/R148H heterozygous diploid. This could be explained by the excess of R148H monomers relative to V238M monomers resulting in formation of nonfunctional R148H:R148H homodimers at a higher rate than hemi-functional V238M:R148H heterodimers.

 

Figure 3. Comparison of growth of yeast SEC53 diploid alleles

Graphs show growth of 10-2 dilution of OD 1.0 cells over time at 30oC in 50 µL SC+FOA media in a 384-well plate. 1X indicates the native SEC53 promoter. 2X indicates double the native promoter strength. 10X indicates 10X the native promoter strength. 0.2X indicates 20% of the native promoter strength. Bar graphs show comparison of growth relative to homozygous wild type SEC53 diploids at a single time point (t=20). Strains with the indicated promoter are listed in the legend: A) SEC53-F126L, B) SEC53-V238M, C) SEC53-E100K, and D) SEC53-E146K.

 

Just as in the case of the dimerization-defective F126L variant, E100K homozygous diploids grew more poorly than E100K/R148H heterozygous diploids, which in turn grew more slowly than E100K/WT diploids (Figure 3C). Under the TEF1 promoter, E100K/E100K is 53.4% compared to 98.8% in E100K/WT and 100% in E100K/R148H. Under the ACT1 promoter, E100K/E100K is 8.2% compared to 100% in E100K/WT and 77.9% in E100K/R148H. Like F126, E100 is expected to affect dimerization and R148H may facilitate the formation of partially functional heterodimers. E146K, which is indistinguishable from wildtype, grew as expected except with the REV1 promoter: pREV1-E146K/E146K homozygous diploids (96.2%) grow better compared to the E146K/R148H heterozygous diploids (65.6%) (Figure 3D). The presence of R148H monomers results in fewer E146K:E146K homodimers which is still compatible with viability but not maximal growth rate.

 

The phenotypes of human PMM2 alleles in yeast parallel SEC53 alleles

It was previously demonstrated that expression of human PMM2 rescues the lethality of a temperature-sensitive allele of SEC53, sec53-6 (Hansen et al., 1997). We initially expressed human PMM2 cDNA but this failed to rescue yeast sec53∆. For the first time we show that expression of human PMM2 cDNA that is codon-optimized for expression in yeast does in fact rescue sec53∆ (Figure 4). This suggests that the non-codon-optimized PMM2 was likely poorly expressed in yeast such that it was not sufficient for growth under the native SEC53 promoter. Subsequently, we expressed each of the human PMM2 variants in sec53∆ cells to determine whether PMM2 alleles behave the same as SEC53 alleles. Under the SEC53 promoter, PMM2 partially rescues sec53∆ to 71% of wildtype yeast SEC53 (Figure 4A). The degree to which each PMM2 variant rescues sec53∆ correlates with their reported residual enzymatic activity and supports the conclusion that the biochemical defect of each allele is conserved between yeast and humans. PMM2 E139K (68%) grows similarly to wildtype PMM2, in agreement with yeast SEC53 E146K. V231M compromises growth (55.6%) and F119L further compromises growth (44.8%). E93A, on the other hand, is not sufficient for growth under the SEC53 promoter and does not restore growth above sec53∆ cells. This is consistent with pSEC53-E100K and further supports the essentiality of E93 in PMM2 function.

Figure 4. Expression of human PMM2 rescues growth of yeast sec53∆ cells

  1. Expression of the indicated human PMM2 variant under the SEC53 promoter.
  2. Expression of the indicated human PMM2 variant under the ACT1 or TEF1 promoter. Left panels show growth over time and right panels show growth relative to wild type SEC53 at t = 20 hr.

 

Expressing PMM2 under the ACT1 promoter improves growth to 97.3% (Figure 4B). Expectedly, doubling the expression of F119L with the ACT1 promoter also improves its growth to 63.6%. Under the TEF1 promoter, PMM2 completely rescues sec53∆ cells. This suggests that PMM2 expressed in yeast may not be functioning optimally and requires higher expression levels. As is the case with SEC53, 10-fold over-expression of PMM2 is not cytotoxic. Based on these results, we can characterize the deficiency of a given PMM2 allele in yeast based on its effect on cell growth over a range of protein expression regimes and in different compound heterozygous combinations.

 

Check out part 2 of this post for figures 5 – 7, or read more about Perlara’s preprint

 

Feature image: modified from original by Macrovector