This is a continuation of a previous post, which covered Figures 1 – 4 of Perlara’s preprint on bioRxiv, describing our work in modeling PMM2 patient alleles. This work is currently under review at the Genetics Society of America’s peer-reviewed journal G3. This post covers Figures 5 – 7 of the manuscript. We hope this outreach encourages new researchers into the PMM2 community.
Measuring phosphomannomutase activity levels in SEC53 variants
We performed a PMM2 enzymatic assay to determine whether the expression level of the variants correlates with the enzymatic activity measured in wildtype and mutant yeast cell lysates. We assayed phosphomannomutase (PMM) activity by measuring the reduction of NADP+ to NADPH spectrophotometrically at 340 nm with a plate reader (Van Schaftingen & Jaeken, 1995; Pirard et al., 1999; Kjaergaard et al., 1999; Sharma et al. 2011; and Citro et al., 2017). NADPH production is achieved by a series of four enzyme-coupled reactions (shown below). Mannose-1-phosphate and NADP+ are provided as substrates for the reactions along with phosphomannose isomerase, phosphoglucose isomerase, glucose-6-phosphoate dehydrogenase, and PMM2 from the protein lysate. Additionally, glucose 1,6-bisphosphate is used as an activator of PMM2 activity.
We determined that wildtype SEC53 expression level and growth correlates to the PMM activity detected by the assay (Figure 5A and B). Lysates from pTEF1 and pACT1 cells consistently show elevated activity compared to pSEC53 cells. Lysates from pREV1-SEC53 cells show less activity (70%), consistent with its growth relative (64%) to pSEC53-SEC53. For V238M, the expression level correlates with enzymatic assay and with growth, but not to the extent expected (Figure 5C). For example, pTEF1-V238M rescues growth completely (100%), but PMM activity in these cells are below half of wildtype cells (37.5%) (Figure 5C). pACT1-V238M also rescue growth to a higher degree than the enzymatic activity (29%) of these cells would indicate. This is also true for F126L (Figure 5C). However, pACT1-F126L (19.4%) and pSEC53-F126L (17.4%) does not show significant difference in PMM activity. This indicates that for SEC53 mutations, either mannose-1-phosphate levels or PMM enzymatic activity in cell lysates does not necessarily reflect the growth of these cells. In other words, SEC53 variants may only require a modest boost in PMM enzymatic activity and/or mannose-1-phosphate levels above their baselines to achieve maximal growth rate and presumably a fully glycosylated proteome.
Figure 5. Phosphomannomutase activity of SEC53 alleles
- Graphs show NADPH formation over time in 200 µL reaction with 0.1 µg/mL protein lysate from the strains indicated.
- Phosphomannomutase activity of the indicated SEC53 variant relative to pSEC53-WT plotted against cell growth relative to pSEC53-WT.
Drug repurposing screen in yeast models of PMM2-CDG identified three novel chemical modifiers
We advanced pACT1-F126L, pSEC53-V238M, and pSEC53-F126L haploids and pACT1-F126L/pACT1-R148H, pSEC53-V238M/pSEC53-R148H, and pSEC53-F126L/pSEC53-R148H heterozygous diploids to high-throughput drug screens. Screening was done with a 2,560 compound MicroSource Spectrum library consisting of FDA approved drugs, bioactive tool compounds, and natural products. Each strain was screened in duplicate in 384-well plates, with each plate containing 32 wells of the negative control (no drugs) and 24 wells of the positive controls (wildtype cells). As shown in Figure 6A, the positive and negative controls showed good separation of Z-scores, which allowed us to distinguish even a relatively modest rescue of growth in the screen. Additionally, there is good correlation between replicates (correlation >0.4 for all panels) (Figure 6B). With a Z-score cutoff of 2.0, we identified six “pre-hit” compounds that looked promising because their ability to rescue growth is conserved between the haploid and diploid strains (Figure 6C).
Figure 6. Summary of the 2,560 drug repurposing screen
- Comparison of z-scores between the negative vs positive controls of representative datasets show separation of data.
- Comparison of z-scores between replicates show positive correlation between duplicate datasets.
- Pre-hit compounds and z-scores. We identified six compounds from the Microsource Spectrum library that showed a z-score of ≧0 in growth in at least 2 replicates of the same allele (orange). Green indicates a z-score of ≧2.0, but did not replicate in the duplicate screen.
We reordered these compounds from the vendor as dry powder to retest them in multi-point dose response assays. We found that three of the six compounds showed consistent and dose-dependent rescues in haploid and heterozygous diploid cells (Figure 7). The negative control compound cysteamine hydrochloride did not rescue at any dose (Figure 7D). At the maximum dose tested, alpha-cyano-4-hydroxycinnamic acid rescues the F126L allele by 17.7% in haploid and 24.5% in diploid cells. Interestingly, this rescue is specific to the 2X pACT1 expression level (Figure 7A and Supplemental Figure 2).
2’-2’-bisepigallocatechin digallate rescues growth of pSEC53-F126L and pSEC53-V238M cells, and pACT1-F126L to a much lesser extent (Figure 7B). It also exerts a stronger response in heterozygous diploid cells than haploids. Growth of pSEC53-F126L improved by as much as 10.6% and pSEC53-F126L/R148H by 25%. pSEC53-V238M improved by 14.6% and pSEC53-V238M/R148H by 57.7%. pACT1-F126L cells only improved by 6.3% and 12.9% in pACT1-F126L/R148H (Figure 7B).
Similarly, suramin hexasodium also rescues growth of the pSEC53 variants more than the pACT1 variants, and diploids more strongly than haploids (Figure 7C). Growth of pSEC53-F126L improved by 24.5%, pSEC53-F126L/R148H by 47.4%, pSEC53-V238M by 16.1%, and pSEC53-V238M/R148H by 38.9%. In contrast, pACT1-F126L cells showed 6.7% improvement and pACT1-F126L/R148H showed 22.6%.
In summary, we found that alpha-cyano-4-hydroxycinnamic acid specifically rescues pACT1-F126L cells, while 2’-2’-bisepigallocatechin digallate and suramin hexasodium preferentially rescue pSEC53-F126L/R148H and pSEC53-V238M/R148H heterozygous diploid cells.
Figure 7. Three compounds show differential rescue of growth of SEC53 variants
- Growth of cells in alpha-cyano-4-hydroxycinnamic acid at the indicated dose relative to growth in the absence of compound (0 µM).
- Growth of cells in 2′,2′-bisepigallocatechin digallate at the indicated dose relative to growth in the absence of compound (0 µM).
- Growth of cells in suramin hexasodium at the indicated dose relative to growth in the absence of compound (0 µM).
- Growth of cells in cysteamine hydrochloride (negative control) at the indicated dose relative to growth in the absence of compound (0 µM).
Feature image: modified from original by Macrovector