The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor substrate peptide

The retromer complex is involved in the retrograde transport of the CI-M6PR (cation-independent mannose 6-phosphate receptor) from endosomes to the Golgi. It is a hetero-trimeric complex composed of Vps26 (vacuolar sorting protein 26), Vps29 and Vps35 proteins, which are conserved in eukaryote evolution. Recently, elucidation of the crystal structure of Vps29 revealed that Vps29 contains a metallo-phosphoesterase fold [Wang, Guo, Liang, Fan, Zhu, Zang, Zhu, Li, Teng, Niu et al. (2005) J. Biol. Chem. 280, 22962-22967; Collins, Skinner, Watson, Seaman and Owen (2005) Nat. Struct. Mol. Biol. 12, 594-602]. We demonstrate that recombinant hVps29 (human Vps29) displays in vitro phosphatase activity towards a serine-phosphorylated peptide, containing the acidic-cluster dileucine motif of the cytoplasmatic tail of the CI-M6PR. Efficient dephosphorylation required the additional presence of recombinant hVps26 and hVps35 proteins, which interact with hVps29. Phosphatase activity of hVps29 was greatly decreased by alanine substitutions of active-site residues that are predicted to co-ordinate metal ions. Using inductively coupled plasma MS, we demonstrate that recombinant hVps29 binds zinc. Moreover, hVps29-dependent phosphatase activity is greatly reduced by non-specific and zinc-specific metal ion chelators, which can be completely restored by addition of excess ZnCl2. The binuclear Zn2+ centre and phosphate group were modelled into the hVps29 catalytic site and pKa calculations provided further insight into the molecular mechanisms of Vps29 phosphatase activity. We conclude that the retromer complex displays Vps29-dependent in vitro phosphatase activity towards a serinephosphorylated acidic-cluster dileucine motif that is involved in endosomal trafficking of the CI-M6PR. The potential significance of these findings with respect to regulation of transport of cycling trans-Golgi network proteins is discussed.     

Mannose 6-phosphate receptor-mediated uptake is defective in acid sphingomyelinase-deficient macrophages: implications for Niemann-Pick disease enzyme replacement therapy

Progressive accumulation of lipid-laden macrophages is a hallmark of the acid sphingomyelinase (ASM)-deficient forms of Niemann-Pick disease (i.e. Types A and B NPD). To investigate the mechanisms underlying enzyme replacement therapy for this disorder, we studied the uptake of recombinant, human ASM (rhASM) by alveolar macrophages from ASM knock-out (ASMKO) mice. The recombinant enzyme used for these studies was produced in Chinese hamster ovary cells and contained complex type, N-linked oligosaccharides. Binding of radiolabeled, rhASM to the ASMKO macrophages was enhanced as compared with normal macrophages, consistent with their larger size and increased surface area. However, internalization of the enzyme by the ASMKO cells was markedly reduced when compared with normal cells. Studies using receptor-specific ligands to inhibit enzyme uptake revealed that in normal cells rhASM was taken up by a combination of mannose and mannose 6-phosphate receptors (MR and M6PR, respectively), whereas in the ASMKO cells the M6PR had a minimal role in rhASM uptake. Expression of M6PR mRNA was normal in the ASMKO cells, although Western blotting revealed more receptors in these cells when compared with normal. We therefore hypothesized that lipid accumulation in ASMKO macrophages led to abnormalities in M6PR trafficking and/or degradation, resulting in reduced enzyme uptake. Consistent with this hypothesis, we also found that, when rhASM was modified to expose terminal mannose residues and target mannose receptors, the uptake of this modified enzyme form by ASMKO cells was approximately 10-fold greater when compared with the "complex" type rhASM. These findings have important implications for NPD enzyme replacement therapy, particularly in the lung.     

Vps26A and Vps26B subunits define distinct retromer complexes

The trimeric Vps29-Vps35-Vps26 sub-complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A-myc or Vps26B-myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian-specific Vps26 paralogues. Vps26B-retromer, like Vps26A-retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B-retromer and cation-independent mannose 6-phosphate receptor (CI-M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B-retromer with maturing endosomes relative to Vps26A-retromer. Interestingly, the cycling of CI-M6PR is restored upon deletion of the variable Vps26B C-terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C-terminal region can control cargo selection of the Vps26B-retromer.     

Chromosomal mapping of the gene for the type II insulin-like growth factor receptor/cation-independent mannose 6-phosphate receptor in man and mouse

The gene for insulin-like growth factor II (IGF-II) receptor (IGF2R) that has recently been found, by DNA sequencing, to be identical to the cation-independent mannose 6-phosphate receptor (CIM6PR) has been mapped in the human and murine species. Cloned cDNAs for human and rat IGF-II receptors were used to probe Southern blots of somatic cell hybrid DNA and for in situ chromosomal hybridization. The genes are located in a region of other conserved syntenic genes on the long arm of human chromosome 6, region 6q25----q27, and mouse chromosome 17, region A-C. The CIM6PR/IGF2R locus in man is asyntenic with the genes encoding IGF-II (IGF2), the IGF-I receptor (IGF1R), and the cation-dependent mannose 6-phosphate receptor (CDM6PR).     

Synthesis and receptor binding affinity of carboxylate analogues of the mannose 6-phosphate recognition marker

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is involved in multiple physiological pathways including targeting of lysosomal enzymes, degradation of IGF2, and cicatrization through TGFbeta activation. To target potential therapeutics to this membrane receptor, four carboxylate analogues of mannose 6-phosphate (M6P) were synthesized. Three of them, two isosteric carboxylate analogues and a malonate derivative, showed a binding affinity for the M6P/IGF2R equivalent to or higher than that of M6P. Contrary to M6P, all these analogues were particularly stable in human serum. Moreover, these derivatives did not present any cytotoxic activity against two human cell lines. These analogues represent a new potential for the lysosomal targeting of enzyme replacement therapy in lysosomal diseases or to prevent the membrane-associated activities of the M6P/IGF2R.     

In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.     

Cation-independent mannose 6-phosphate receptor inhibitor (PXS25) inhibits fibrosis in human proximal tubular cells by inhibiting conversion of latent to active TGF-beta1

Hyperglycemia and hypoxia have independent and convergent roles in the development of renal disease. Transforming growth factor-β(1) (TGF-β(1)) is a key cytokine promoting the production of extracellular matrix proteins. The cationic-independent mannose 6-phosphate receptor (CI-M6PR) is a membrane protein that binds M6P-containing proteins. A key role is to activate latent TGF-β(1). PXS25, a novel CI-MPR inhibitor, has antifibrotic properties in skin fibroblasts, but its role in renal fibrosis is unclear. The aim was to study the role of PXS25 in matrix protein production under high glucose ± hypoxic conditions in human proximal tubule (HK-2) cells. HK-2 cells were exposed to high glucose (30 mM) ± 100 μM PXS25 in both normoxic (20% O(2)) and hypoxic (1% O(2)) conditions for 72 h. Cellular fibronectin, collagen IV, and matrix metalloproteinase-2 (MMP-2) and MMP-9 were assessed. Total and active TGF-β(1) were measured by ELISA. High glucose and hypoxia independently induced TGF-β(1) production. Active TGF-β(1), but not total TGF-β(1) was reduced with concurrent PXS25 in the presence of high glucose, but not in hyperglycemia+hypoxia conditions. Hyperglycemia induced fibronectin and collagen IV production (P < 0.05), as did hypoxia, but only hyperglycemia-induced increases in matrix proteins were suppressed by concurrent PXS25 exposure. High glucose induced MMP-2 and -9 in normoxic and hypoxic conditions, which was not modified in the presence of PXS25. High glucose and hypoxia can independently induce endogenous active TGF-β(1) production in human proximal tubular cells. PXS25 inhibits conversion of high glucose-induced release of active TGF-β(1), only in the absence of hypoxia.     

Mannose 6-phosphate receptor and sortilin mediated endocytosis of α-galactosidase A in kidney endothelial cells

Prominent vasculopathy in Fabry disease patients is caused by excessive intracellular accumulation of globotriaosylceramide (GL-3) throughout the vascular endothelial cells causing progressive cerebrovascular, cardiac and renal impairments. The vascular lesions lead to myocardial ischemia, atherogenesis, stroke, aneurysm, thrombosis, and nephropathy. Hence, injury to the endothelial cells in the kidney is a key mechanism in human glomerular disease and endothelial cell repair is an important therapeutic target. We investigated the mechanism of uptake of α-galactosidase A (α-Gal A) in renal endothelial cells, in order to clarify if the recombinant enzyme is targeted to the lysosomes via the universal mannose 6-phosphate receptor (M6PR) and possibly other receptors. Immunohistochemical localization of infused recombinant α-Gal A in a renal biopsy from a classic Fabry disease patient showed that recombinant protein localize in the endothelial cells of the kidney. Affinity purification studies using α-Gal A resins identified M6PR and sortilin as α-Gal A receptors in cultured glomerular endothelial cells. Immunohistochemical analyses of normal human kidney with anti-sortilin and anti-M6PR showed that sortilin and M6PR were expressed in the endothelium of smaller and larger vessels. Uptake studies in cultured glomerular endothelial cells of α-Gal A labeled with fluorescence and (125)I showed by inhibition with RAP and M6P that sortilin and M6PR mediated uptake of α-Gal A. Biacore studies revealed that α-Gal A binds to human M6PR with very high affinity, but M6PR also binds to sortilin in a way that prevents α-Gal A binding to sortilin. Taken together, our data provide evidence that sortilin is a new α-Gal A receptor expressed in renal endothelial cells and that this receptor together with the M6PR is able to internalize circulating α-Gal A during enzyme replacement therapy in patients with Fabry disease.     

Synthesis of a targeting drug for antifibrosis of liver; A conjugate for delivering glycyrrhetin to hepatic stellate cells

The neoglycoproteins that consist of human serum albumin (HSA) modified with mannose 6-phosphate ([M6P]x-HSA) were synthesized, and they showed high binding property to hepatic stellate cells (HSC) by immunohistochemical analysis. In addition, an increased substitution (X) of 6-phosphated mannose (M6P) was associated with an increased accumulation in HSC. So the [M6P]x-HSA might be a carrier to deliver drugs to HSC. The antifibrotic drug, glycyrrhetin, was chosen to conjugate to M6P(26)-HSA. The result suggests there were 6 approximately 7 glycyrrhetin molecules having been conjugated to the carrier. Targeting glycyrrhetin to HSC might reduce its adverse affects and increase the efficacy.     

Synthesis and biological evaluation of new mannose 6-phosphate analogues

Three new analogues of mannose 6-phosphate (M6P)—a sulphate and two carboxylates—have been synthesized and their affinity toward the M6P/IGFII receptor evaluated by affinity column chromatography. These compounds display strong binding to the receptor and therefore are new M6P analogues which may find some dermatological applications, for example healing of post-surgical scars.     

A Cationic-Independent Mannose 6-Phosphate Receptor Inhibitor (PXS64) Ameliorates Kidney Fibrosis by Inhibiting Activation of Transforming Growth Factor-β1

The activity of transforming growth factor-β1 (TGF-β₁) is regulated by its conversion from the latent to the active form. We have previously shown that the conversion is at least in part mediated by the cationic-independent mannose 6-phosphate receptor (CI-M6PR), as the CI-M6PR inhibitor, PXS-25 has anti-fibrotic properties in human kidney tubular (HK-2) cells under high glucose conditions. However, its clinical use is limited by low bioavailability. Our aim was to determine the effects of PXS64, a pro-drug of PXS25, in in vitro and in vivo models of renal fibrosis. HK-2 cells were exposed to latent TGFβ₁+/- PXS64 for 48 hours. The mRNA and protein levels of pro-fibrotic and pro-inflammatory markers were determined. A 7 day unilateral ureteric obstruction (UUO) model was used and the following experimental groups were studied: (i) Sham operated, (ii) UUO, (iii) UUO + telmisartan (iv) UUO + PSX64. HK-2 cells exposed to PXS64 reduced TGFβ mediated effects on collagen IV, fibronectin, macrophage chemotactic protein-1 (MCP-1) and phospho-smad2 protein expression, consistent with inhibition of the conversion of latent to active TGF-β₁. PXS 64 treated UUO mice had a lower tubulointerstitial fibrosis index, collagen IV and fibronectin protein and mRNA expression when compared to untreated UUO mice. In addition, these animals had lower MCP-1 mRNA expression, reduced inflammarory cell infiltrate, as indicated by fewer CD45, F4/80 positive cells, and reduced phospho-Smad2 protein expression when compared to untreated UUO animals. Our data demonstrates that PSX64 is an effective anti-fibrotic agent by inhibiting the activation of latent TGF-β₁.     

Ligand interactions of the cation-dependent mannose 6-phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor

The interactions of the bovine cation-dependent mannose 6-phosphate receptor with monovalent and divalent ligands have been studied by equilibrium dialysis. This receptor appears to be a homodimer or a tetramer. Each mole of receptor monomer bound 1.2 mol of the monovalent ligands, mannose 6-phosphate and pentamannose phosphate with Kd values of 8 X 10(-6) M and 6 X 10(-6) M, respectively and 0.5 mol of the divalent ligand, a high mannose oligosaccharide with two phosphomonoesters, with a Kd of 2 X 10(-7) M. When Mn2+ was replaced by EDTA in the dialysis buffer, the Kd for pentamannose phosphate was 2.5 X 10(-5) M. By measuring the affinity of the cation-dependent and cation-independent mannose 6-phosphate receptors for a variety of mannose 6-phosphate analogs, we conclude that the 6-phosphate and the 2-hydroxyl of mannose 6-phosphate each contribute approximately 4-5 kcal/mol of Gibb's free energy to the binding reaction. Neither receptor appears to interact substantially with the anomeric oxygen of mannose 6-phosphate. The receptors differ in that the cation-dependent receptor displays no detectable affinity for N-acetylglucosamine 1'-(alpha-D-methylmannopyranose 6-monophosphate) whereas this ligand binds to the cation-independent receptor with a poor, but readily measurable Kd of about 0.1 mM. The spacing of the mannose 6-phosphate-binding sites relative to each other may also differ for the two receptors.     

Mannose 6-phosphate potentiates insulin-like growth factor II-stimulated inositol trisphosphate production in proximal tubular basolateral membranes

To ascertain whether mannose 6-phosphate affects insulin-like growth factor (IGF) II stimulation of phospholipase C activity in the basolateral membrane of the renal proximal tubular cell, we determined the effect of mannose 6-phosphate on IGF II-stimulated production of inositol trisphosphate (Ins-P3) in isolated basolateral membranes. Production of Ins-P3 measured in the presence of 10(-10), 10(-9), or 10(-8) M rat IGF II was potentiated approximately 2-fold by inclusion of 5 mM mannose 6-phosphate in incubations. Mannose 6-phosphate had no effect on Ins-P3 production in the absence of IGF II. Neither mannose 1-phosphate, mannose, glucose 6-phosphate, nor fructose 1-phosphate exerted similar potentiation. Enhancement of IGF II-stimulated Ins-P3 production required concentrations on the order of several millimolar mannose 6-phosphate. Total and specific binding of 10(-10) M 125I-IGF II to basolateral membranes was significantly increased by 5 mM mannose 6-phosphate. However, there was no significant effect on total or specific binding of 10(-9) or 10(-8) M 125I-IGF II. Our findings suggest that mannose 6-phosphate potentiates stimulation of phospholipase C by IGF II in the basolateral membrane of the renal proximal tubular cell and that potentiation is mediated via a mechanism in addition to enhanced binding of IGF II. Such potentiation could reflect a role for the mannose 6-phosphate moiety as a modulator of IGF II "signal" transmission in vivo.     

Mannitol Synthesis in Higher Plants : Evidence for the Role and Characterization of a NADPH-Dependent Mannose 6-Phosphate Reductase

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase ¹⁴C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K_m (M6P) was 15.8 millimolar, but the apparent K_m (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg²⁺, Ca²⁺, Zn²⁺, and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.     

Inhibition of pear fruit ripening by mannose

Softening of the flesh and the rise in ethylene evolution and respiration associated with ripening in pear (Pyrus communis L.) fruit was delayed when mannose was vacuum infiltrated into intact fruit. The extent of delay could be modified by altering the concentration or the volume of mannose applied to the fruit. Inhibition of ripening was associated with phosphorylation of mannose to mannose 6-phosphate (M6P), and accumulation of M6P was associated with lowered levels of inorganic phosphate (Pi), glucose 6-phosphate (G6P), and ATP in the fruit tissue. Subsequently, however, as the M6P was metabolized, the levels of Pi, G6P, and ATP increased and ripening processes were concomitantly released from inhibition. Hence, the degree of inhibition by mannose or the release from inhibition was related to the level of M6P in the fruit and its rate of metabolism. The data provide correlative evidence to support a view that one inhibitory effect of mannose is depletion of Pi in the cell as a result of phosphorylation of mannose to M6P. Inhibition of ripening by mannose was not alleviated by co-application of glucose as a competitive substrate for the hexokinase(s), or by Pi, presumably the depleted metabolite. Also, incubation of tissue disks with M6P resulted in inhibition of ethylene production and respiration. The structural analogs of mannose, glucosamine, and 2-deoxyglucose, which have been shown to mimic mannose action in several plant tissues, did not cause inhibition of ripening of pear fruit comparable with that associated with mannose. Both analogs stimulated respiration, and glucosamine caused only a small inhibition of softening and ethylene evolution. Another mannose analog, α-methylmannoside, did inhibit fruit ripening though to a lesser extent than mannose. Its influence was also associated with accumulation of M6P and a decrease of Pi levels. We conclude that the mannose effect may, in part, be due to M6P toxicity, as well as by depletion of Pi.     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

Quantitation of Mr 46000 and Mr 300000 mannose 6-phosphate receptors in human cells and tissues.

A highly specific enzyme-linked immunosorbent assay for quantitation of the Mr 46000 Da and Mr 300000 Da mannose 6-phosphate receptors was developed. The assay allows to detect ng amounts of human mannose 6-phosphate receptors. Analysis of human cells and tissues revealed significant differences in their contents of the two mannose 6-phosphate receptors, normalized for total cell protein. The ratio of the two mannose 6-phosphate receptors also differed among cells and tissues, suggesting that their steady state concentrations are regulated independently.     

Formation of a self-assembled phenylboronic acid monolayer and its application toward developing a surface plasmon resonance-based monosaccharide sensor

An assay has been developed to quantitate the amount of mannose 6-phosphate in glycoproteins using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The method was tested on a recombinant lysosomal enzyme, human alpha-galactosidase A, that contains mannose 6-phosphate. The assay includes two steps: hydrolysis of the glycoprotein in 6.75 M trifluoroacetic acid to release mannose 6-phosphate and quantitation of the released mannose 6-phosphate using HPAEC with PAD. There is a linear relationship between the amount of mannose 6-phosphate measured and the amount of alpha-galactosidase hydrolyzed. The assay is also sensitive for as little as 2.5 microg alpha-galactosidase, which contains 117 pmol mannose 6-phosphate. Further, the assay has been shown to have good day-to-day and operator-to-operator consistency. In order to evaluate the assay for glycoprotein in crude extract, the glycoprotein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The amount of mannose 6-phosphate in the electroblots following hydrolysis was determined using HPAEC-PAD. The assay was also linear when measuring mannose 6-phosphate on electroblots. Therefore, this assay has been shown to be specific, sensitive, and reproducible.     

Ligand interactions of the cation-independent mannose 6-phosphate receptor. The stoichiometry of mannose 6-phosphate binding

The interaction of the bovine cation-independent mannose 6-phosphate receptor with a variety of phosphorylated ligands has been studied using equilibrium dialysis and immobilized receptor to measure ligand binding. The dissociation constants for mannose 6-phosphate, pentamannose phosphate, bovine testes beta-galactosidase, and a high mannose oligosaccharide with two phosphomonoesters were 7 X 10(-6) M, 6 X 10(-6) M, 2 X 10(-8) M, and 2 X 10(-9) M, and the mol of ligand bound/mol of receptor monomer were 2.17, 1.85, 0.9, and 1.0, respectively. We conclude that the cation-independent mannose 6-phosphate receptor has two mannose 6-phosphate-binding sites/polypeptide chain.     

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.