Yeast Models of Phosphomannomutase 2 Deficiency, a Congenital Disorder of Glycosylation

Abstract

Phosphomannomutase 2 Deficiency (PMM2-CDG) is the most common monogenic congenital disorder of glycosylation (CDG) affecting at least 800 patients globally. PMM2 orthologs are present in model organisms, including the budding yeast Saccharomyces cerevisiae gene SEC53 Here we describe conserved genotype-phenotype relationships across yeast and human patients between five PMM2 loss-of-function missense mutations and their orthologous SEC53 mutations. These alleles range in severity from folding defective (hypomorph) to dimerization defective (severe hypomorph) to catalytic dead (null). We included the first and second most common missense mutations - R141H, F119L respectively- and the most common compound heterozygote genotype - PMM2^R141H/F119L - observed in PMM2-CDG patients. Each mutation described is expressed in haploid as well as homozygous and heterozygous diploid yeast cells at varying protein expression levels as either SEC53 protein variants or PMM2 protein variants. We developed a 384-well-plate, growth-based assay for use in a screen of the 2,560-compound Microsource Spectrum library of approved drugs, experimental drugs, tool compounds and natural products. We identified three compounds that suppress growth defects of SEC53 variants, F126L and V238M, based on the biochemical defect of the allele, protein abundance or ploidy. The rare PMM2 E139K protein variant is fully functional in yeast cells, suggesting that its pathogenicity in humans is due to the underlying DNA mutation that results in skipping of exon 5 and a nonfunctional truncated protein. Together, these results demonstrate that yeast models can be used to characterize known and novel PMM2 patient alleles in quantitative growth and enzymatic activity assays, and used as patient avatars for PMM2-CDG drug screens yielding compounds that could be rapidly cross-validated in zebrafish, rodent and human organoid models.

Keywords: PMM2-CDG; Phosphomannomutase 2 Deficiency; congenital disorders of glycosylation; drug screens; yeast models of human disease.

Figure 1

Generating yeast models of PMM2 deficiency. A. 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. B. Table showing the PMM2 patient alleles and the equivalent variants we generated in yeast SEC53. C. 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). D. 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.

Figure 2

Comparison of growth of yeast SEC53 haploid alleles. A. Graphs show growth of 10⁻² dilution of OD 1.0 cells over time at 30° in 50 µL SC+FOA media in a 384-well plate. 1X indicates the native SEC53 promoter. 2X indicates double the native promoter strength (pACT1). 10X indicates 10X the native promoter strength (pTEF1). 0.2X indicates 20% of the native promoter strength (pREV1). B. Comparison of growth relative to wildtype SEC53 at a single time point (t = 20). Cell density of each strain at t = 20 was normalized against its corresponding promoter match SEC53 wildtype at t = 20. However, because pREV1-WT grows slower than wildtype cells, all pREV1 strains were normalized against pSEC53-WT at t = 20.

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 30°C in 50 µL SC+FOA media in a 384-well plate. 1X indicates the native SEC53 promoter. 2X indicates double the native promoter strength (pACT1). 10X indicates 10X the native promoter strength (pTEF1). 0.2X indicates 20% of the native promoter strength (pREV1). Bar graphs show comparison of growth relative to homozygous wildtype 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.

Figure 4

Expression of human PMM2 rescues growth of yeast sec53∆ cells. A. Expression of the indicated human PMM2 variant under the SEC53 promoter. B. 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 wildtype SEC53 at t = 20 hr.

Figure 5

Phosphomannomutase activity of SEC53 alleles. A. Graphs showing NADPH formation over time in 200 µL reaction with 0.1 µg/mL protein lysate from the strains indicated. B. Phosphomannomutase activity (at 40 min) of the indicated SEC53 variant relative to pSEC53-WT plotted against its cell growth (at 20 hr) relative to pSEC53-WT.

Figure 6

Summary of the 2,560 drug repurposing screen. A. Comparison of z-scores between the negative (blue) vs. positive (red) controls of representative datasets show separation of data. B. Comparison of z-scores between replicates show positive correlation between duplicate datasets. C. Pre-hit compounds and z-scores. We identified six compounds from the Microsource Spectrum library that showed a z-score of ≧2.0 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.

Figure 7

Three compounds show differential rescue of growth of SEC53 variants. A. Growth of cells in alpha-cyano-4-hydroxycinnamic acid at the indicated dose relative to growth in the absence of compound (0 µM). B. Growth of cells in 2’,2’-bisepigallocatechin digallate at the indicated dose relative to growth in the absence of compound (0 µM). C. Growth of cells in suramin hexasodium at the indicated dose relative to growth in the absence of compound (0 µM). D. Growth of cells in cysteamine hydrochloride (negative control) at the indicated dose relative to growth in the absence of compound (0 µM).