Carbohydrate-deficient glycoprotein syndrome type 1A: expression and characterisation of wild type and mutant PMM2 in E-coli

We have identified the PMM2 genotypes of 22 unrelated Danish patients with carbohydrate-deficient glycoprotein syndrome type1A: R141H/F119L (18), R141 H/C192G (1), F119L/F119L (1), F119L/G117R (1) and D223E/T237R (1). The lack of patients homozygous for R141H is statistically highly significant, but unexplained. In order to investigate the effect of PMM2 mutations on phosph omannomutase (PMM2) activity, PMM2-cDNA was cloned into a pET3a vector. Fol lowing introduction of mutations into PMM2-cDNA by site-specific mutagenesi s, wild type and mutant PMM2-cDNA were expressed in E. coli B121(DE3) cells , and the activity of PMM2 was determined by an enzymatic assay using manno se l-phosphate as substrate. Recombinant R141H, G117R, and T237R PMM2 had n o detectable catalytic activity, and the F119L PMM2 had 25% of the activity of the wild type. The activity of the C192G and D223E PMM2 was in the norm al range, but the affinity for their substrate was lower, and the proteins were more sensitive to increased temperatures. Each patient has at least on e mutation which retains residual PMM2 activity. Our results support the hy potheses that a genotype conveying residual PMM2 catalytic activity is requ ired for survival, and that homozygosity for R141H impairs PMM2 to a degree incompatible with life.