Mammalian phosphomannomutase PMM1 is the brain IMP-sensitive glucose-1,6-bisphosphatase

Abstract

Glucose 1,6-bisphosphate (Glc-1,6-P(2)) concentration in brain is much higher than what is required for the functioning of phosphoglucomutase, suggesting that this compound has a role other than as a cofactor of phosphomutases. In cell-free systems, Glc-1,6-P(2) is formed from 1,3-bisphosphoglycerate and Glc-6-P by two related enzymes: PGM2L1 (phosphoglucomutase 2-like 1) and, to a lesser extent, PGM2 (phosphoglucomutase 2). It is hydrolyzed by the IMP-stimulated brain Glc-1,6-bisphosphatase of still unknown identity. Our aim was to test whether Glc-1,6-bisphosphatase corresponds to the phosphomannomutase PMM1, an enzyme of mysterious physiological function sharing several properties with Glc-1,6-bisphosphatase. We show that IMP, but not other nucleotides, stimulated by >100-fold (K(a) approximately 20 mum) the intrinsic Glc-1,6-bisphosphatase activity of recombinant PMM1 while inhibiting its phosphoglucomutase activity. No such effects were observed with PMM2, an enzyme paralogous to PMM1 that physiologically acts as a phosphomannomutase in mammals. Transfection of HEK293T cells with PGM2L1, but not the related enzyme PGM2, caused an approximately 20-fold increase in the concentration of Glc-1,6-P(2). Transfection with PMM1 caused a profound decrease (>5-fold) in Glc-1,6-P(2) in cells that were or were not cotransfected with PGM2L1. Furthermore, the concentration of Glc-1,6-P(2) in wild-type mouse brain decreased with time after ischemia, whereas it did not change in PMM1-deficient mouse brain. Taken together, these data show that PMM1 corresponds to the IMP-stimulated Glc-1,6-bisphosphatase and that this enzyme is responsible for the degradation of Glc-1,6-P(2) in brain. In addition, the role of PGM2L1 as the enzyme responsible for the synthesis of the elevated concentrations of Glc-1,6-P(2) in brain is established.

FIGURE 1.

Stimulation of the glucose-1,6-bisphosphatase activity of PMM1 by IMP and GMP. The enzymatic activities (μmol/min/mg of protein) were assayed as described under “Experimental Procedures” with 150 μm Glc-1,6-P₂ and the indicated concentrations of IMP or GMP. The results shown are the means ± S.E. of three determinations with the same enzyme preparation.

FIGURE 2.

Inhibition by IMP of the phosphoglucomutase (a) and phosphomannomutase (b) activities of PMM1. Both enzymatic activities were assayed as described under “Experimental Procedures” with 1 μm Glc-1,6-P₂, increasing concentrations of IMP, and 25 μm glucose 1-phosphate (a) or mannose 1-phosphate (b). The results shown are the means ± S.E. of three determinations with the same enzyme preparation. PGM, phosphoglucomutase; PMM, phosphomannomutase; m, mouse.

FIGURE 3.

Effect of overexpression of human PGM2L1 or PGM2 and mouse PMM1 or PMM2 on the Glc-1,6-P₂ level in HEK293T cells. HEK293T cells were plated in 10-cm diameter dishes and transfected with the indicated quantities of plasmids encoding mouse (m) PMM1 or PMM2 or human (h) PGM2 or PGM2L1. Perchloric acid extracts were prepared 48 h after transfection for the assay of Glc-1,6-P₂. The results shown represent one example of at least four similar experiments. In the experiment shown, the concentrations of Glc-1,6-P₂ are the means ± S.E. of three independent transfections. Glc-1,6-P₂ values were compared using Student's t test, and the difference was considered non-significant (n.s.) at p > 0.05.

FIGURE 4.

Concentration of Glc-1,6-P₂ in tissues of control and PMM1-deficient mice. Mice were deeply anesthetized before organs were removed. Glc-1,6-P₂ was measured in neutralized HClO₄ extracts from brain hemispheres that were frozen as fast as possible after removal from the mouse skull (t = 0 min) or after 5 min at room temperature (t = 5 min) (a) or from other tissues that were frozen as soon as they were removed from the animal (b). The results shown are the means ± S.E. of determinations made in four to seven different mice. Glc-1,6-P₂ values were compared using Student's t test. When a p value is not given (p > 0.05), the difference was considered non-significant (n.s.). WT, wild-type; KO, knock-out; Sk. musc., skeletal muscle.