title: The Effects of PMM2-CDG-Causing Mutations on the Folding, Activity, and Stability of the PMM2 Protein.
abstract: Congenital disorder of glycosylation type Ia (PMM2-CDG), the most common form of CDG, is caused by mutations in the PMM2 gene that reduce phosphomannomutase 2 (PMM2) activity. No curative treatment is available. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The effects of the mutations on PMM2/Pmm2 stability, oligomerization, and enzyme activity were explored in an optimized bacterial system. The mutant proteins were associated with an enzymatic activity of up to 47.3% as compared with wild type (WT). Stability analysis performed using differential scanning fluorimetry and a bacterial transcription-translation-coupled system allowed the identification of several destabilizing mutations (p.V44A, p.D65Y, p.R123Q, p.R141H, p.R162W, p.F207S, p.T237M, p.C241S). Exclusion chromatography identified one mutation, p.P113L, that affected dimer interaction. Expression analysis of the p.V44A, p.D65Y, p.R162W, and p.T237M mutations in a eukaryotic expression system under permissive folding conditions showed the possibility of recovering their associated PMM2 activity. Together, the results suggest that some loss-of-function mutations detected in PMM2-CDG patients could be destabilizing, and therefore PMM2 activity could be, in certain cases, rescuable via the use of synergetic proteostasis modulators and/or chaperones.
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Sanitaria IdiPaZ, Madrid, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Yuste-Checa
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Sanitaria IdiPaZ, Madrid, Spain.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Gámez
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Sanitaria IdiPaZ, Madrid, Spain.
authors.forename: Sandra
authors.initials: S
authors.lastname: Brasil
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Sanitaria IdiPaZ, Madrid, Spain.
authors.forename: Lourdes R
authors.initials: LR
authors.lastname: Desviat
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Sanitaria IdiPaZ, Madrid, Spain.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ugarte
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Sanitaria IdiPaZ, Madrid, Spain.
authors.forename: Celia
authors.initials: C
authors.lastname: Pérez-Cerdá
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Investigación Sanitaria IdiPaZ, Madrid, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Pérez
mesh_terms: D000818:Animals; D018981:Congenital Disorders of Glycosylation / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005347:Fibroblasts; D015870:Gene Expression; D006801:Humans; D051379:Mice; D009154:Mutation; D017875:Phosphotransferases (Phosphomutases) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017510:Protein Folding; D055503:Protein Multimerization; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D017875:Phosphotransferases (Phosphomutases); C519582:phosphomannomutase 2, human
keywords: PMM2; PMM2-CDG; congenital disorders of glycosylation; phosphomannomutase 2; protein stability