Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli.
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The Escherichia coli uhp T gene encodes an active transport system for sugar phosphates. When the uhp T gene was carried on a multicopy plasmid, amplified levels of transport activity occurred, and growth of these strains was inhibited upon the addition of various sugar phosphates. Two different mechanisms for this growth inhibition were distinguished. Exposure to glucose-6-phosphate, fructose-6-phosphate or mannose-6-phosphate, which enter directly into the glycolytic pathway, resulted in cessation of growth and substantial loss of viability. Cell killing was correlated with the production of the toxic metabolite, methylglyoxal. In contrast, addition of 2-deoxyglucose-6-phosphate, galactose-6-phosphate, glucosamine-6-phosphate or arabinose-5-phosphate, which do not directly enter the glycolytic pathway, resulted in growth inhibition without engendering methylglyoxal production or cell death. Inhibition of growth could result from excessive accumulation of organophosphates in the cell or depletion of inorganic phosphate pools as a result of the sugar-P/Pi exchange process catalysed by UhpT. The phosphate-dependent uptake of glycerol-3-phosphate by the GlpT antiporter was strongly inhibited under conditions of elevated sugar-phosphate transport. There are thus two separate toxic effects of elevated sugar-phosphate transport, one of which was lethal and related to increased flux through glycolysis. It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates. (+info)
The N-terminal carbohydrate recognition site of the cation-independent mannose 6-phosphate receptor.
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The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in the trafficking of newly synthesized mannose 6-phosphate-containing acid hydrolases to the lysosome. The receptor contains two high affinity carbohydrate recognition sites within its 15-domain extracytoplasmic region, with essential residues for carbohydrate recognition located in domain 3 and domain 9. Previous studies have shown that these two sites are distinct with respect to carbohydrate specificity. In addition, expression of truncated forms of the CI-MPR demonstrated that domain 9 can be expressed as an isolated domain, retaining high affinity (Kd approximately 1 nm) carbohydrate binding, whereas expression of domain 3 alone resulted in a protein capable of only low affinity binding (Kd approximately 1 microm) toward a lysosomal enzyme. In the current report the crystal structure of the N-terminal 432 residues of the CI-MPR, encompassing domains 1-3, was solved in the presence of bound mannose 6-phosphate. The structure reveals the unique architecture of this carbohydrate binding pocket and provides insight into the ability of this site to recognize a variety of mannose-containing sugars. (+info)
Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor.
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The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9. (+info)
Loss of cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition.
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The outer layer of the cell wall of the human pathogenic fungus Candida albicans is enriched with heavily mannosylated glycoproteins that are the immediate point of contact between the fungus and cells of the host, including phagocytes. Previous work had identified components of the acid-labile fraction of N-linked mannan, comprising beta-1,2-linked mannose residues attached via a phosphodiester bond, as potential ligands for macrophage receptors and modulators of macrophage function. We therefore isolated and disrupted the CaMNN4 gene, which is required for mannosyl phosphate transfer and hence the attachment of beta-1,2 mannose oligosaccharides to the acid-labile N-mannan side chains. With the mannosylphosphate eliminated, the mnn4Delta null mutant was unable to bind the charged cationic dye Alcian Blue and was devoid of acid-labile beta-1,2-linked oligomannosaccharides. The mnn4Delta mutant was unaffected in cell growth and morphogenesis in vitro and in virulence in a murine model of systemic C. albicans infection. The null mutant was also not affected in its interaction with macrophages. Mannosylphosphate is therefore not required for macrophage interactions or for virulence of C. albicans. (+info)
Among multiple phosphomannomutase gene orthologues, only one gene encodes a protein with phosphoglucomutase and phosphomannomutase activities in Thermococcus kodakaraensis.
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Four orthologous genes (TK1108, TK1404, TK1777, and TK2185) that can be annotated as phosphomannomutase (PMM) genes (COG1109) have been identified in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. We previously found that TK1777 actually encodes a phosphopentomutase. In order to determine which of the remaining three orthologues encodes a phosphoglucomutase (PGM), we examined the PGM activity in T. kodakaraensis cells and identified the gene responsible for this activity. Heterologous gene expression and purification and characterization of the recombinant protein indicated that TK1108 encoded a protein with high levels of PGM activity (690 U mg(-1)), along with high levels of PMM activity (401 U mg(-1)). Similar analyses of the remaining two orthologues revealed that their protein products exhibited neither PGM nor PMM activity. PGM activity and transcription of TK1108 in T. kodakaraensis were found to be higher in cells grown on starch than in cells grown on pyruvate. Our results clearly indicate that, among the four PMM gene orthologues in T. kodakaraensis, only one gene, TK1108, actually encodes a protein with PGM and PMM activities. (+info)
Conjugation of mannose 6-phosphate-containing oligosaccharides to acid alpha-glucosidase improves the clearance of glycogen in pompe mice.
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Clinical studies of enzyme replacement therapy for Pompe disease have indicated that relatively high doses of recombinant human acid alpha-glucosidase (rhGAA) may be required to reduce the abnormal glycogen storage in cardiac and skeletal muscles. This may be because of inefficient cation-independent mannose 6-phosphate receptor (CI-MPR)-mediated endocytosis of the enzyme by the affected target cells. To address this possibility, we examined whether the addition of a high affinity ligand to rhGAA would improve its delivery to these cells. Chemical conjugation of high mannose oligosaccharides harboring mono- and bisphosphorylated mannose 6-phosphates onto rhGAA (neo-rhGAA) significantly improved its uptake characteristics by muscle cells in vitro. Infusion of neo-rhGAA into Pompe mice also resulted in greater delivery of the enzyme to muscle tissues when compared with the unmodified enzyme. Importantly, this increase in enzyme levels was associated with significantly improved clearance of glycogen ( approximately 5-fold) from the affected tissues. These results suggest that CI-MPR-mediated endocytosis of rhGAA is an important pathway by which the enzyme is delivered to the affected lysosomes of Pompe muscle cells. Hence, the generation of rhGAA containing high affinity ligands for the CI-MPR represents a strategy by which the potency of rhGAA and therefore the clinical efficacy of enzyme replacement therapy for Pompe disease may be improved. (+info)
Mannose 6-phosphorylated proteins are required for tumor necrosis factor-induced apoptosis: defective response in I-cell disease fibroblasts.
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Whereas caspases are essential components in apoptosis, other proteases seem to be involved in programmed cell death. This study investigated the role of lysosomal mannose 6-phosphorylated proteins in tumor necrosis factor (TNF)-induced apoptosis. We report that fibroblasts isolated from patients affected with inclusion-cell disease (ICD), having a deficient activity of almost all lysosomal hydrolases, are resistant to the toxic effect of TNF. These mutant cells exhibited a defect in TNF-induced caspase activation, Bid cleavage, and release of cytochrome c. In contrast, TNF-induced p42/p44 MAPK activation and CD54 expression remained unaltered. Human ICD lymphoblasts and fibroblasts derived from mice nullizygous for Igf2 and the two mannose 6-phosphate (M6P) receptors, Mpr300 and Mpr46, which develop an ICD-like phenotype, were also resistant to CD95 ligand and TNF, respectively. Moreover, correction of the lysosomal enzyme defect of ICD fibroblasts, using a medium enriched in M6P-containing proteins, enabled restoration of sensitivity to TNF. This effect was blocked by exogenous M6P but not by cathepsin B or L inhibitors. Altogether, these findings suggest that some M6P-bearing glycoproteins modulate the susceptibility to TNF-induced apoptosis. As a matter of fact, exogenous tripeptidyl peptidase 1, a lysosomal carboxypeptidase, could sensitize ICD fibroblasts to TNF. These observations highlight the hitherto unrecognized role of some mannose 6-phosphorylated proteins such as tripeptidyl peptidase 1 in the apoptotic cascade triggered by TNF. (+info)
Human serum amyloid P is a multispecific adhesive protein whose ligands include 6-phosphorylated mannose and the 3-sulphated saccharides galactose, N-acetylgalactosamine and glucuronic acid.
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Carbohydrate recognition by amyloid P component from human serum has been investigated by binding experiments using several glycosaminoglycans, polysaccharides and a series of structurally defined neoglycolipids and natural glycolipids. Two novel classes of carbohydrate ligands have been identified. The first is 6-phosphorylated mannose as found on lysosomal hydrolases, and the second is the 3-sulphated saccharides galactose, N-acetyl-galactosamine and glucuronic acid as found on sulphatide and other acidic glycolipids that occur in neural or kidney tissues or on subpopulations of lymphocytes. Binding to mannose-6-phosphate containing molecules and inhibition of binding by free mannose-6-phosphate and fructose-1-phosphate are features shared with mannose-6-phosphate receptors involved in trafficking of lysosomal enzymes. However, only amyloid P binding is inhibited by galactose-6-phosphate, mannose-1-phosphate and glucose-6-phosphate. These findings strengthen the possibility that amyloid P protein has a central role in amyloidogenic processes: first in formation of focal concentrations of lysosomal enzymes including proteases that generate fibril-forming peptides from amyloidogenic proteins, and second in formation of multicomponent complexes that include sulphoglycolipids as well as glycosaminoglycans. The evidence that binding to all of the acidic ligands involves the same polypeptide domain on amyloid P protein, and inhibition data using diffusible, phosphorylated monosaccharides, is potentially important leads to novel drug designs aimed at preventing or even reversing amyloid deposition processes without interference with essential lysosomal trafficking pathways. (+info)