Human Phosphomannomutase Research Articles

Insight on molecular pathogenesis and pharmacochaperoning potential in phosphomannomutase 2 deficiency, provided by novel human phosphomannomutase 2 structures.

Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (PMM2-CDG), is a severe condition, which has no cure. Due to the identification of destabilizing mutations, our group aims at increasing residual activity in PMM2-CDG patients, searching for pharmacochaperones. Detailed structural knowledge of hPMM2 might help identify variants amenable to pharmacochaperoning. hPMM2 structural information is limited to one incomplete structure deposited in the Protein Databank without associated publication, which lacked ligands and residues from a crucial loop. Here we report five complete crystal structures of hPMM2, three for wild-type and two for the p.Thr237Met variant frequently found among Spanish PMM2-CDG patients, free and bound to the essential activator glucose-1,6-bisphosphate (Glc-1,6-P2 ). In the hPMM2 homodimer, each subunit has a different conformation, reflecting movement of the distal core domain relative to the dimerization cap domain, supporting an opening/closing process during catalysis. Two Mg2+ ions bind to the core domain, one catalytic and one structural. In the cap domain, the site for Glc-1,6-P2 is well delineated, while a Cl- ion binding at the intersubunit interface is predicted to strengthen dimerization. Patient-found amino acid substitutions are nonhomogeneously distributed throughout hPMM2, reflecting differential functional or structural importance for various parts of the protein. We classify 93 of 101 patient-reported single amino acid variants according to five potential pathogenetic mechanism affecting folding of the core and cap domains, linker 2 flexibility, dimerization, activator binding, and catalysis. We propose that ~80% and ~50% of the respective core and cap domains substitutions are potential candidates for pharmacochaperoning treatment.

Structural Basis of the Molecular Switch between Phosphatase and Mutase Functions of Human Phosphomannomutase 1 under Ischemic Conditions.

The human phosphomannomutases PMM1 and PMM2 catalyze the interconversion of hexose 6-phosphates and hexose 1-phosphates. The two isoforms share 66% sequence identity and have kinetic properties similar to those of mutases in vitro but differ in their functional roles in vivo. Though the physiological role of PMM2 is catalysis of the mutase reaction that provides the mannose 1-phosphate (Man-1-P) essential for protein glycosylation, PMM1 is thought to provide a phosphohydrolase activity in the presence of inosine monophosphate (IMP), converting glucose 1,6-bisphosphate (Glu-1,6-P2) to glucose 6-phosphate (Glu-6-P), rescuing glycolysis during brain ischemia. To uncover the structural basis of how IMP binding converts PMM1 from a mutase to a phosphatase, the 1.93 Å resolution structure of PMM1 complexed with IMP was determined. The structure reveals IMP bound at the substrate recruitment site, thus inhibiting the mutase activity while simultaneously activating a phosphatase activity (IMP Kact = 1.5 μM) resulting from the hydrolysis of the phospho-enzyme. The bound structure and site-directed mutagenesis confirm that the long-range electrostatic interactions provided by Arg180 and Arg183 conserved in PMM1 are the major contributors to IMP binding, and their oblation removes phosphatase but not mutase activity. These residues are not present in the PMM2 isoform, which consequently lacks significant phosphatase activity in the presence of IMP. T2 relaxation nuclear magnetic resonance and small angle X-ray scattering together support the hypothesis that binding of IMP to PMM1 favors an enzyme conformation that is catalytically competent for water attack at the phosphoaspartyl intermediate. Such a mechanism may be generalizable to other enzymes that act through covalent intermediates.

Structure of Leishmania mexicana Phosphomannomutase Highlights Similarities with Human Isoforms

Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 Å of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 Å. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.

Evidence for phosphotransferases phosphorylated on aspartate residue in N-terminal DXDX(T/V) motif

The group of enzymes forming a phosphoaspartate intermediate comprises P-type ATPases, bacterial protein phosphatases acting in signal transduction, and a newly identified class of phosphotransferases that is characterized by a conserved DXDXT/V motif close to the N terminus. The chapter shows the formation of an acyl-phosphate for the following enzymes: spinach phosphoglycolate phosphatase, human phospho serine phosphatase, human phosphomannomutases 1 and 2, and Lactococcus lactis β- phosphoglucomutase.The position of the phosphorylated residue was identified as the first aspartate in the DXDXT/V motif in human phosphomannomutase and in human phosphoserine phosphatase. Results of site-directed mutagenesis of the two aspartates in phosphomannomutase and in phosphoserine phosphatase are compatible with this conclusion. In most of these enzymes, the DXDXT/V motif is located close to the N terminus. An exception—such as yeast trehalose-6-phosphate phosphatase —is because of the fact that this enzyme is bifunctional, with a trehalose-6-phosphate synthase domain N-terminal to the phosphatase domain. Other exceptions may correspond to proteins with several domains.

Identification of a IVS4-58delATG polymorphism in the human phosphomannomutase 2 (PMM2) gene.

Identification of a IVS4-58delATG polymorphism in the human phosphomannomutase 2 (PMM2) gene.

Identification of a IVS4 -58delATG polymorphism in the human phosphomannomutase 2 (PMM2) gene Communicated by: Mark H. Paalman Online Citation: Human Mutation, Mutation and Polymorphism Report #113 (2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/mpr113.pdf

Identification of a IVS4 -58delATG polymorphism in the human phosphomannomutase 2 (PMM2) gene Communicated by: Mark H. Paalman Online Citation: Human Mutation, Mutation and Polymorphism Report #113 (2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/mpr113.pdf

An AciI polymorphism in the 3' untranslated region of the human phosphomannomutase 2 (PMM2) gene.

We found an AciI polymorphism in the 3' untranslated region of the phosphomannomutase 2 (PMM2) gene located at 16p13. A G-to-C transition at nucleotide position 96 bp downstream from the PMM2 stop codon was detected in polymerase chain reaction (PCR) products after AciI digestion. The heterozygosity of the polymorphic alleles was 0.375 in a Japanese population. This polymorphism is useful for genetic analysis in patients with carbohydrate-deficient glycoprotein syndromes, of which there are four subtypes.

Effect of mutations found in carbohydrate-deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2

Seven mutant forms of human phosphomannomutase 2 were produced in Escherichia coli and purified. These mutants had a V max of 0.2–50% of the wild enzyme and were unstable. The least active protein (R141H) bears a very frequent mutation, which has never been found in the homozygous state whereas the second least active protein (D188G) corresponds to a mutation associated with a particularly severe phenotype. We conclude that total lack of phosphomannomutase 2 is incompatible with life. Another conclusion is that the elevated residual phosphomannomutase activity found in fibroblasts of some patients is contributed by their mutated phosphomannomutase 2.

A New Class of Phosphotransferases Phosphorylated on an Aspartate Residue in an Amino-terminal DXDX(T/V) Motif

When incubated with their substrates, human phosphomannomutase and L-3-phosphoserine phosphatase are known to form phosphoenzymes with chemical characteristics of an acyl-phosphate. The phosphorylated residue in phosphomannomutase has now been identified by mass spectrometry after reduction of the phosphoenzyme with tritiated borohydride and trypsin digestion. It is the first aspartate in a conserved DVDGT motif. Replacement of either aspartate of this motif by asparagine or glutamate resulted in complete inactivation of the enzyme. The same mutations performed in the DXDST motif of L-3-phosphoserine phosphatase also resulted in complete inactivation of the enzyme, except for the replacement of the second aspartate by glutamate, which reduced the activity by only about 40%. This suggests that the first aspartate of the motif is also the phosphorylated residue in L-3-phosphoserine phosphatase. Data banks contained seven other phosphomutases or phosphatases sharing a similar, totally conserved DXDX(T/V) motif at their amino terminus. One of these (beta-phosphoglucomutase) is shown to form a phosphoenzyme with the characteristics of an acyl-phosphate. In conclusion, phosphomannomutase and L-3-phosphoserine phosphatase belong to a new phosphotransferase family with an amino-terminal DXDX(T/V) motif that serves as an intermediate phosphoryl acceptor.

Comparison of PMM1 with the phosphomannomutases expressed in rat liver and in human cells

Carbohydrate-deficient glycoprotein syndrome type I (CDGI) is most often due to phosphomannomutase deficiency; paradoxically, the human phosphomannomutase gene PMM1 is located on chromosome 22, whereas the CDGI locus is on chromosome 16. We show that phosphomannomutases present in rat or human liver share with homogeneous recombinant PMM1 several kinetic properties and the ability to form an alkali- and NH 2OH-sensitive phosphoenzyme with a subunit mass of ≈30 000 M r. However, they have a higher affinity for the activator mannose-1,6-bisphosphate than PMM1 and are not recognized by anti-PMM1 antibodies, indicating that they represent a related but different isozyme. Phosphomannomutases belong to a novel mutase family in which the active residue is a phosphoaspartyl or a phosphoglutamyl.

Isolation of the Human Phosphomannomutase Gene (PMM1) and Assignment to Chromosome 22q13

Phosphomannomutase (PMM) catalyzes the interconversion between mannose-6-phosphate and mannoase-1-phosphate; the latter is the direct precursor of the sugar nucleotide GDP-mannose, serving as the mannose donor for glycosylation. To date, yeast and candida PMMs have been reported, but the vertebrate cDNA for this enzyme has not been isolated. Herein, we have cloned human PMM cDNA and determined its chromosomal localization. 10 refs., 1 fig.

Cloning and characterization of human phosphomannomutase, a mammalian homologue of yeast SEC53.

Phosphomannomutase (PMM) catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate, which is a substrate for the synthesis of GDP-mannose. This nucleotide sugar is then used in the synthesis of dolichol-phosphate-mannose, which is essential for N-linked glycosylation and thus the secretion of several glycoproteins as well as for the synthesis of glycosyl-phosphatidyl-inositol (GPI) anchored proteins. In the yeast Saccharomyces cerevisiae, SEC53, a gene required for viability, encodes PMM. Given the importance of PMM in glycoprotein synthesis, it is surprising that very little is known about the enzyme in higher eukaryotes. Recently, an autosomal recessive human disease, Carbohydrate-deficient glycoprotein syndrome type I (CDGS-I) has been correlated with severely reduced PMM activity. Here we report the isolation of a cDNA encoding human PMM, a protein of 29 kDa that is 55% identical and 66% similar to yeast Sec53p. Northern blot analysis shows a single 1.4 kb transcript that is ubiquitously expressed, although levels vary markedly among tissues. Expression of the human cDNA in a temperature-sensitive mutant sec53 yeast strain confers growth at the restrictive temperature, strongly suggesting that this gene encodes a functional PMM. Finally, when expressed in BHK cells, PMM is localized exclusively to the cytosol corresponding to its localization in yeast.