Molecular insights on pathogenic effects of mutations causing phosphoglycerate kinase deficiency

Abstract

Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.

Figure 1. Ribbon representation of the human PGK1.

Three-dimensional structure of open (A) and closed (B) human PGK1. The figures were generated from the atomic coordinates of Protein Data Bank, entry 2XE7 and 2WZC, using the Swiss-Pdb viewer (http://expasy.org/spdbv/). The black spheres indicate the Cα atoms of the amino acid residues subjected to mutagenesis. The arrows point to the substrates binding sites.

Figure 2. Assessment of the oligomeric state of recombinant PGK1.

Elution profile of PGK1 from the analytical gel-filtration on a Superose 12HR 10/30 prepacked column. The position of the peak corresponds to a protein of approximately 45 kDa. The inset shows 12% SDS-PAGE of the purified PGK1 run in parallel with molecular mass standards, and stained with Coomassie Blue R-250.

Figure 3. Thermal stability of PGK1 enzymes.

Thermal stability of the PGK1 wild-type and variants at 45°C (panel A) and at 37°C (panel B). Each enzyme was incubated in buffer A and aliquots were collected at intervals for measuring residual activity. Plot of the residual activities at 10 minutes versus temperatures (panel C). Each enzyme was subjected to heat inactivation in a range of temperature from 25°C to 60°C. After 10 minutes of incubation at a given temperature, the enzyme sample was chilled and the residual activity measured. Residual activity was expressed as percentage of initial activity. •, wild-type; ▪, p.I47N; ▴, p.L89P; ▾, p.G158V; ⧫ p.D164V; , p.K191del; ○, p.R206P; □, p.E252A; ▵, p.I253T; ▿, p.V266M; ⋄, p.D268N; , p.D285V; , p.D315N; , p.C316R; , p.S320N; , p.A354P; , p.T378P.

Figure 4. Steady state kinetics of PGK1 enzymes.

Steady state kinetics of PGK1 wild-type and variants as a function of Mg-ATP at fixed 5 mM 3-PG (panel A) and as a function of 3-PG at fixed 5 mM MgATP (panel B). All experiments were performed at 37°C as reported in the "Material and Methods" section. •, wild-type; ▪, p.I47N; ▴, p.L89P; ▾, p.G158V; ⧫ p.D164V; , p.K191del; ○, p.R206P; □, p.E252A; ▵, p.I253T; ▿, p.V266M; ⋄, p.D268N; , p.D285V; , p.D315N; , p.C316R; , p.S320N; , p.A354P; , p.T378P.