Expression analysis revealing destabilizing mutations in phosphomannomutase 2 deficiency (PMM2‐CDG)

Abstract

Deficiency of phosphomannomutase (PMM2, MIM#601785) is the most common congenital disorder of glycosylation. Herein we report the genetic analysis of 22 Spanish PMM2 deficient patients and the functional analysis of 14 nucleotide changes in a prokaryotic expression system in order to elucidate their molecular pathogenesis. PMM2 activity assay revealed the presence of six protein changes with no enzymatic activities (p.R123Q, p.R141H, p.F157S, p.P184T, p.F207S and p.D209G) and seven mild protein changes with residual activities ranging from 16 to 54% (p.L32R, p.V44A p.D65Y, p.P113L p.T118S, p.T237M and p.C241S) and also one variant change with normal activity (p.E197A). The results obtained from Western blot analysis, degradation time courses of 11 protein changes and structural analysis of the PMM2 protein, suggest that the loss-of-function of most mutant proteins is based on their increased susceptibility to degradation or aggregation compared to the wild type protein, considering PMM2 deficiency as a conformational disease. We have identified exclusively catalytic protein change (p.D209G), catalytic protein changes affecting protein stability (p.R123Q and p.R141H), two protein changes disrupting the dimer interface (p.P113L and p.T118S) and several misfolding changes (p.L32R, p.V44A, p.D65Y, p.F157S, p.P184T, p.F207S, p.T237M and p.C241S). Our current work opens a promising therapeutic option using pharmacological chaperones to revert the effect of the characterized misfolding mutations identified in a wide range of PMM2 deficient patients.

Compromised Catalysis and Potential Folding Defects in in Vitro Studies of Missense Mutants Associated with Hereditary Phosphoglucomutase 1 Deficiency

“…1A). Similar correlations between mutants that misfold in vivo and also show problems in recombinant expression have been observed in many other inherited metabolic diseases, including deficiencies in cystathione ␤-synthase (23), medium-chain acyl-CoA dehydrogenase (24,25), phosphomannomutase 2 (26), and phenylalanine hydroxylase (27)(28)(29).…”

Simultaneous Sequencing of 24 Genes Associated with Steroid-Resistant Nephrotic Syndrome

“…The latter has previously been described as a polymorphism (22). Four different nsSNPs were predicted by Polyphen to be probably damaging: NPHS1 H1174Y (heterozygote in patient 21), MYO1E I531M (heterozygote in patient 34), MYH9 K910G (homozygous in patient 23; previously described in a patient with Fechtner syndrome but also in control) (23), and SMARCAL1 E377Q (heterozygous in patient 12; previously described; probable polymorphism) (24).…”

Phosphomannose Isomerase Inhibitors Improve N-Glycosylation in Selected Phosphomannomutase-deficient Fibroblasts

“…Structural studies show that the R141H mutant is impaired in substrate binding and has essentially no activity (25). If R141H allele is in combination with another allele that produces an unstable protein (V231M) or protein impaired in dimer interface interaction (F119L, P113L) (25,26), then no improvement is observed with MPI inhibitor. Even if Man-6-P pools were mobilized toward mutant PMM2, they could not be utilized for glycosylation.…”