Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2

Abstract

The most frequent disorder of glycosylation is due to mutations in the gene encoding phosphomannomutase2 (PMM2-CDG). For this disease, which is autosomal and recessive, there is no cure at present. Most patients are composite heterozygous and carry one allele encoding an inactive mutant, R141H, and one encoding a hypomorphic mutant. Phosphomannomutase2 is a dimer. We reproduced composite heterozygosity in vitro by mixing R141H either with the wild type protein or the most common hypomorphic mutant F119L and compared the quaternary structure, the activity and the stability of the heterodimeric enzymes. We demonstrated that the activity of R141H/F119L heterodimers in vitro, which reproduces the protein found in patients, has the same activity of wild type/R141H, which reproduces the protein found in healthy carriers. On the other hand the stability of R141H/F119L appears to be reduced both in vitro and in vivo. These findings suggest that a therapy designed to enhance protein stability such as those based on pharmacological chaperones or modulation of proteostasis could be beneficial for PMM2-CDG patients carrying R141H/F119L genotype as well as for other genotypes where protein stability rather than specific activity is affected by mutations.

Yeast Models Of Phosphomannomutase 2 Deficiency, A Congenital Disorder Of Glycosylation

“…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 positions of these residues are indicated in Figure 1C. 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).…”

Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG

“…The PMM2 enzyme forms an obligate homodimer in the cytoplasm that converts mannose-6-phosphate to mannose-1-phosphate, an initial essential step in the N-linked glycosylation of proteins. Glucose-1,6bisphosphate and mannose-1,6-bisphosphate are endogenous coactivators of PMM2 function, binding to and stabilizing PMM2 dimers (Andreotti et al, 2015). N-linked protein glycosylation is an evolutionarily In order to identify drug repurposing candidates that boost PMM2 enzyme function, we generated and characterized the first worm patient avatar of PMM2-CDG as an intermediate translational model situated between our previously published yeast models (Lao et al, 2019) and well-established PMM2-CDG patient fibroblasts.…”

“…Missense mutations fall into at least three biochemical classes: (i) protein destabilizing/misfolding mutations randomly distributed throughout the protein, (ii) dimerization defective mutations located in the monomer-monomer interface, and (iii) "catalytic dead" mutations in the active site (Yuste-Checa et al, 2015). Each PMM2 monomer forms a dimer with itself as a prerequisite for catalytic activity, though there need only be one functional active site per dimer (Andreotti et al, 2015). A plurality of PMM2-CDG patients across ethnic populations share the compound heterozygous genotype R141H/F119L, which pairs the aforementioned R141H null allele with the F119L dimerization-defective allele.…”