Selective induction of apoptosis in MCF7 cancer-cell by targeted liposomes functionalised with mannose-6-phosphate.

Liposomes are versatile platforms to carry anticancer drugs in targeted drug delivery; they can be surface modified by different strategies and, when coupled with targeting ligands, are able to increase cellular internalisation and organelle-specific drug delivery. An interesting strategy of antitumoral therapy could involve the use of lysosomotropic ligand-targeted liposomes loaded with molecules, which can induce lysosomal membrane permeabilization (LMP), leakage of cathepsins into the cytoplasm and subsequent apoptosis. We have previously demonstrated the ability of liposomes functionalised with a mannose-6-phosphate to reach lysosomes; in this research we compare the behaviour of M6P-modified and non-functionalised liposomes in MCF7 tumour cell and in HDF normal cells. With this aim, we first demonstrated by Western blotting the overexpression of mannose-6-phosphate/insulin-like growth factor (M6P/IGF-II) receptor in MCF7. Then, we prepared calcein-loaded liposomes and we revealed the increased uptake of M6P-functionalised liposomes in MCF7 cells respect to HDF cells by flow cytometry analysis. Finally, we loaded functionalised and not functionalised liposomes with N-hexanoyl-d-erythro-sphingosine (C6Cer), able to initiate LMP-induced apoptosis; after having studied the stability of both vesicles in the presence of serum by Dynamic Light Scattering and Spectrophotometric turbidity measurements, we showed that ceramide-loaded M6P-liposomes significantly increased apoptosis in MCF7 with respect to HDF cells.

Enzymatic phosphorylation of mannose by glucomannokinase from Mycobacterium phlei using inorganic polyphosphate.

Mannose-6-phosphate is an important phosphor-sugar, which is involved in many physiological functions and it is used to treat many diseases. Its production is however expensive since it requires costly substrate ATP as phosphorylation agent. This study has focused upon the direct synthesis of M6P by glucomannokinase using inorganic polyphosphate without involvement of ATP. The gene cloned for glucomannokinase has been sequenced from Mycobacterium phlei and it is transformed into Escherichia coli for expression. After purification involving affinity chromatography, a band of 30kDa corresponding to the enzyme has been isolated from induced crude supernatant. A total amount of 0.69mg/ml of enzyme has been successively obtained and the purity exceeds 90%. The kinetic assay studies show that this enzyme has more affinity towards polyphosphate and glucose than ATP and mannose respectively. The KM values of the enzyme for glucose, mannose, ATP and hexametaphosphate derived from experiments are 9.5, 203.7, 4.6, 1.7µM, respectively. The enzyme has shown a maximum production of mannose-6-phosphate at optimized conditions of pH 8.5, 25°C, poly(P)/mannose ratio 3:1 and in the presence of bivalent ion Mg2+. The results reveal that the glucomannokinase from Mycobacterium phlei suitable for further production of mannose-6-phosphate.

Excessive activity of cathepsin K is associated with cartilage defects in a zebrafish model of mucolipidosis II.

The severe pediatric disorder mucolipidosis II (ML-II; also known as I-cell disease) is caused by defects in mannose 6-phosphate (Man-6-P) biosynthesis. Patients with ML-II exhibit multiple developmental defects, including skeletal, craniofacial and joint abnormalities. To date, the molecular mechanisms that underlie these clinical manifestations are poorly understood. Taking advantage of a zebrafish model of ML-II, we previously showed that the cartilage morphogenesis defects in this model are associated with altered chondrocyte differentiation and excessive deposition of type II collagen, indicating that aspects of development that rely on proper extracellular matrix homeostasis are sensitive to decreases in Man-6-P biosynthesis. To further investigate the molecular bases for the cartilage phenotypes, we analyzed the transcript abundance of several genes in chondrocyte-enriched cell populations isolated from wild-type and ML-II zebrafish embryos. Increased levels of cathepsin and matrix metalloproteinase (MMP) transcripts were noted in ML-II cell populations. This increase in transcript abundance corresponded with elevated and sustained activity of several cathepsins (K, L and S) and MMP-13 during early development. Unlike MMP-13, for which higher levels of protein were detected, the sustained activity of cathepsin K at later stages seemed to result from its abnormal processing and activation. Inhibition of cathepsin K activity by pharmacological or genetic means not only reduced the activity of this enzyme but led to a broad reduction in additional protease activity, significant correction of the cartilage morphogenesis phenotype and reduced type II collagen staining in ML-II embryos. Our findings suggest a central role for excessive cathepsin K activity in the developmental aspects of ML-II cartilage pathogenesis and highlight the utility of the zebrafish system to address the biochemical underpinnings of metabolic disease.

[The effect of mannose-6-phosphate on adipocyte differentiation and its underlying molecular mechanism].

OBJECTIVE: Cathepsin K (CTSK) played an important role in adipocyte differentiation. The activation of CTSK needs to convey by mannose-6-phosphate receptors (M6PR) in osteoclasts. The aim of the present study was to identify the effects of mannose-6-phosphate (M6P) in adipocyte differentiation and its underlying molecular mechanism. METHODS: Oil red O staining, accumulation of cytoplasmic triglycerides and glycerine release were used to assess its effects on adipocyte differentiation in the 3T3-L1 cell line. The enzyme activity of CTSK was observed by laser confocal microscopy. The proliferation of 3T3-L1 preadipocytes was detected by MTT methods. mRNA expression of M6PR was determined by RT-PCR. RESULTS: M6P could prevent adipocyte differentiation in a dose-dependent manner as evidenced by absence of triglyceride accumulation and glycerol content. Statistical significance was showed when the concentrations of M6P were 5.0 mmol/L and 8.0 mmol/L respectively (P < 0.05). The mRNA expression of M6PR was detected during the whole process of adipocyte differentiation. With the increase of M6P concentration, enzyme activity of CTSK was inhibited in a concentration-dependent manner. MTT method showed that the absorbance at 570 nm of 3T3-L1 preadipocytes was 0.057 ± 0.091, increased about 62.9% at 10.0 mmol/L compared with the control group (P < 0.05). CONCLUSION: M6P inhibits the terminal differentiation of adipocyte, which may be associated with its effect of blocking CTSK activity by competitive binding with M6PR.

Altered chondrocyte differentiation and extracellular matrix homeostasis in a zebrafish model for mucolipidosis II.

Mucolipidosis II (ML-II) is a pediatric disorder caused by defects in the biosynthesis of mannose 6-phosphate, the carbohydrate recognition signal responsible for targeting certain acid hydrolases to lysosomes. The mechanisms underlying the developmental defects of ML-II are largely unknown due in part to the lack of suitable animal models. To overcome these limitations, we developed a model for ML-II in zebrafish by inhibiting the expression of N-acetylglucosamine-1-phosphotransferase, the enzyme that initiates mannose 6-phosphate biosynthesis. Morphant embryos manifest craniofacial defects, impaired motility, and abnormal otolith and pectoral fin development. Decreased mannose phosphorylation of several lysosomal glycosidases was observed in morphant lysates, consistent with the reduction in phosphotransferase activity. Investigation of the craniofacial defects in the morphants uncovered striking changes in the timing and localization of both type II collagen and Sox9 expression, suggestive of an accelerated chondrocyte differentiation program. Accumulation of type II collagen was also noted within misshapen cartilage elements at later stages of development. Furthermore, we observed abnormal matrix formation and calcium deposition in morphant otoliths. Collectively, these data provide new insight into the developmental pathology of ML-II and suggest that altered production and/or homeostasis of extracellular matrix proteins are integral to the disease process. These findings highlight the potential of the zebrafish system in studying lysosomal disease pathogenesis.

A Temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death.

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

Insulin-like growth factor 2 and its receptors (IGF 1R and IGF 2R/mannose 6-phosphate) in endometrial adenocarcinoma.

OBJECTIVE: To investigate the consequences of IGF proteins dysfunction in development of endometrial adenocarcinomas. METHODS: The expression of IGF 2 and IGF 1R was correlated with the expression of IGF 2R and apoptosis rate in 59 human endometrial adenocarcinomas, 10 endometrial hyperplasias and 7 normal tissues. The presence of mutations in the IGF 2R gene was followed in 46 adenocarcinomas. We also examined the effect of IGF 1 receptor blockage on cancer cell proliferation. In groups of either IGF 2-positive or IGF 2-negative tumors (stages III and IV) the expression of IGF 1 and IGF 1R was correlated with cell proliferation index and telomerase activity. RESULTS: The expression of IGF 2 and IGF 1R was much higher in malignant tissue of stages III and IV than in tumors of stages I and II and normal or hyperplastic endometrium. This correlated with a decreased apoptosis rate and IGF 2R expression. Eight adenocarcinomas expressed biallelic mutation of the IGF 2R gene. The specific inhibition of IGF 1R and IGF 2 decreased tumor cell proliferation in IGF 2/IGF 1R-positive tumors. Furthermore, the positive correlation between increased expression of IGF 1 and IGF 1R proteins and increased telomerase activity and cell proliferation index was found in both IGF 2-negative and IGF 2-positive tumors. CONCLUSION: Our data suggest that IGF 1, IGF 2 and their receptors are involved in the progression of endometrial adenocarcinomas. As cancer cell proliferation can be abrogated by blocking mRNA or protein products of these genes, tumors with extensive involvement of the IGF 2 pathway would be candidates for the therapeutics strategies aimed at interference with this pathway.

Identification of the minimal lysosomal enzyme recognition domain in cathepsin D.

Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265-293 of the beta loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the beta loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.

Bifunctional phosphomannose isomerase/GDP-D-mannose pyrophosphorylase is the point of control for GDP-D-mannose biosynthesis in Helicobacter pylori.

In this report a recombinant bifunctional phosphomannose isomerase/GDP-D-mannose pyrophosphorylase from Helicobacter pylori has been studied. The enzyme catalyzes the first and third steps of GDP-D-mannose biosynthesis from D-fructose-6-phosphate. The first step, isomerization from D-fructose-6-phosphate to D-mannose-6-phosphate, is found to be rate-limiting in GDP-D-mannose biosynthesis due to feedback inhibition. The inhibition is of non-competitive (mixed) type. As the enzyme is found only in bacteria probably participating in capsular polysaccharide biosynthesis, it could be a specific therapeutic target against bacterial infection.

Abnormal synthesis of mannose 1-phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency.

In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (

Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndromes are genetic multisystemic disorders characterized by defective N-glycosylation of serum and cellular proteins. The activity of phosphomannomutase was markedly deficient (< or = 10% of the control activity) in fibroblasts, liver and/or leucocytes of 6 patients with CDG syndrome type I. Other enzymes involved in the conversion of glucose to mannose 1-phosphate, as well as phosphoglucomutase, had normal activities. Phosphomannomutase activity was normal in fibroblasts of 2 patients with CDG syndrome type II. Since this enzyme provides the mannose 1-phosphate required for the initial steps of protein glycosylation, it is concluded that phosphomannomutase deficiency, which is first reported here for higher organisms, is a cause, and most likely the major one, of CDG syndrome type I.

Enzymes involved in the synthesis of mannose-6-phosphate from glucose are normal in carbohydrate deficient glycoprotein syndrome fibroblasts.

Fibroblasts of Carbohydrate Deficient Glycoprotein Syndrome (CDGS) patients synthesize smaller lipid-linked oligosaccharides and incorporate less [3H]-mannose into glycoproteins than controls. Adding mannose, but not glucose, to the culture medium simultaneously corrects both lesions, suggesting that CDGS cells lack sufficient mannose for normal glycosylation. Since mannose for glycoprotein synthesis is assumed to come from glucose, CDGS patients could have defective enzymes in this pathway. Here we show that these enzymes are normal in five CDGS cell lines. This suggests that much of the mannose for glycoprotein synthesis in fibroblasts may come from other, as yet unidentified, pathways.

Transport of hexoses in yeast. Re-examination of the sugar phosphorylation hypothesis with a new experimental approach.

The constitutive transport of hexoses in yeast has been re-examined with a new radioactive experimental approach devised to distinguish between association or independence of the transport step with phosphorylation of the sugar substrate. The approach takes advantage of the fact that the label of [2-3H]mannose disappears once it has been phosphorylated by the yeast, due to its conversion to fructose-6-phosphate. Our results with wild-type yeast and this fermentable sugar support the view that the transport of hexoses in yeast does not involve phosphorylation of the substrate. Other features of the transport process have been examined using this experimental procedure and are also reported.

An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.

A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]-mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.

Biosynthesis of methylphosphomannosyl residues in the oligosaccharides of Dictyostelium discoideum glycoproteins. Evidence that the methyl group is derived from methionine.

The phosphorylated oligosaccharides of Dictyostelium discoideum contain methylphosphomannosyl residues which are stable to mild-acid and base hydrolysis (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we present evidence that these methyl groups are derived from [methyl-3H]methionine, in vivo and [methyl-3H]S-adenosylmethionine in vitro. About 18% of the macromolecules secreted from vegetative cells labeled with [methyl-3H]methionine are released by digestion with preparations of endoglycosidase/peptide N-glycosidase F. The majority of the released molecules are sulfated, anionic high mannose-type oligosaccharides. Strong acid hydrolysis of the [3H]methyl-labeled molecules yields [3H]methanol with kinetics of release similar to those found for the generation of Man-6-P from chemically synthesized methylphosphomannose methylglycoside. Treatment of the [3H]methyl-labeled molecules with a phosphodiesterase from Aspergillus niger which is known to cleave this phosphodiester also releases [3H]methanol from a portion of the oligosaccharides. In vitro incorporation of [methyl-3H]S-adenosylmethionine into endogenous acceptors found in membrane preparations shows that the [3H]methyl group of the methylphosphomannose residues can be derived from this molecule.

The synthesis of mannose 1-phosphate in brain.

The interconversion of mannose-6-P and mannose-1-P in brain has been shown to be catalyzed by a distinct enzyme. The enzyme has been separated from most of the phosphoglucomutase activity of the brain. The residual phosphoglucomutase activity (less than 1%) may be associated with phosphomannomutase itself. Mannose-1,6-P2 or glucose-1,6-P2 is required for the reaction as well as a divalent cation (Mg2+ greater than Co2+ greater than Ni2+ greater than Mn2+). Glucose-1-P, glucose-6-P, and 2-deoxyglucose-6-P are also substrates or inhibitors. Other phosphorylated sugars tested, glucosamine-6-P, N-acetylglucosamine-6-P, galactose-6-P, fructose-6-P, ribose-5-P, and arabinose-5-P, do not affect the rate of the reaction when assayed in the presence of mannose-6-32P.

Recent studies on the involvement of retinyl phosphate as a carrier of mannose in biological membranes.

Rat liver microsomes synthesized [14C]mannosylretinylphosphate and dolichyl [14C]mannosylphosphate from guanosinedisphosphate [14C]mannose, retinylphosphate and dolichylphosphate. Two distinct enzyme activities were shown to be responsible for the biosynthesis of the two mannolipids. A higher affinity mannosyl transferase (EA I), responsible for dolichylmannosylphosphate synthesis, displayed a Km for GDP-mannose of 1.7 microM; while a lower affinity enzyme (EA II), responsible for mannosylretinylphosphate synthesis, displayed a Km for GDP-mannose of 12.5 microM. These Km values were unaffected by the addition of either dolichylphosphate for EA II, or retinylphosphate for EA I. The same Km values were found before and after solubilization of the enzyme activity with 1% Triton X-100. Differential solubilization of EA I and EA II was demonstrated, utilizing different concentrations of Triton X-100. Triple-labeled mannosylretinylphosphate was prepared from [3H]retinylphosphate, retinyl[32P]phosphate and GDP-[14C]mannose from incubations containing rat liver microsomes. This compound was shown to donate [14C]mannose to endogenous acceptors of rat liver microsomes.

Use of permeabilized yeast cells as a system of enzyme immobilization. Its use for the preparation of mannose 6-phosphate.

It was shown that hexokinase contained inside permeabilized yeast cells can be used successfully in the phosphorylation of mannose for the production of mannose 6-phosphate. We propose that enzymes entrapped within the semipermeable cell membrane be considered as an extension of the enzyme immobilization concept, since it shares many of its advantages and several of its properties with some unique characteristics.