Marina A Schwab

Sven W Sauer

Jürgen G Okun

Leo G J Nijtmans

Richard J T Rodenburg

Lambert P Van Den Heuvel

Stefan Dröse

Ullrich Brandt

Henk ter Laak Georg F Hoffmann

Ulrich Brandt

Henk Ter Laak

Stefan Kölker

Jan A M Smeitink

Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins

Keywords: propionic aciduria, propionyl-coenzyme A, mitochondrial dysfunction, mitochondrial DNA, oxidative phosphorylation, pyruvate dehydrogenase complex Biochemical

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INTRODUCTION

Propionyl-CoA is an intermediate metabolite in the final common catabolic pathways of the amino acids L-isoleucine, L-methionine, L-threonine and L-valine as well as odd-chain fatty acids and cholesterol. It is carboxylated by propionyl-CoA carboxylase (EC 6.4.1.3) to methylmalonyl-CoA and then is converted by methylmalonyl-CoA mutase (EC 5.4.99.2) to succinyl-CoA subsequently being fed as anaplerotic precursor into the tricarboxylic acid (TCA) cycle. Inherited deficiency of propionyl-CoA carboxylase, i.e. propionic aciduria (PA), is biochemically characterized by an accumulation of propionate, 3-hydroxypropionate, 2-methylcitrate, and propionylglycine, demonstrating alternative propionate oxidation in these patients. Furthermore, metabolic acidosis, ketosis, increased lactate concentrations, hypoglycemia, and hyperammonemia are found during metabolic derangement, highlighting secondarily compromised energy metabolism and ammonia detoxification [START_REF] Fenton | Disorders of propionate and methylmalonate metabolism[END_REF]. Clinically, PA is complicated by acute life-threatening metabolic crises which are precipitated by catabolic state and result in multiple organ failure or even death if untreated [START_REF] Fenton | Disorders of propionate and methylmalonate metabolism[END_REF]. Like other organic acidurias (e.g. methylmalonic aciduria), age at clinical onset and disease course may vary between individual patients presenting with neonatal metabolic encephalopathy (neonatal onset), recurrent episodes of ketoacidotic coma or Reye-like syndromes (chronic intermittent form), or psychomotor retardation and failure to thrive in the absence of acute crises (chronic progressive form). Similarly to neonatal onset of PA patients, PCCA -/-mice, a transgenic mouse model for PA, develop lethal ketoacidosis shortly after birth [START_REF] Miyazaki | Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene[END_REF].

Despite improvements in the diagnostic work-up and management of affected patients during the last decades, the neurologic outcome remains disappointing in PA patients [START_REF] Fenton | Disorders of propionate and methylmalonate metabolism[END_REF][START_REF] Brismar | CT and MR of the brain in disorders of the propionate and methylmalonate metabolism[END_REF]. In particular mental retardation and movement disorders secondary to cortical atrophy, leukoencephalopathy and basal ganglia injury are still frequently found even in early diagnosed children. The biochemical abnormalities found during acute metabolic crises have led to the suggestion that impairment of oxidative phosphorylation (OXPHOS) is crucial for the pathogenesis of this disease. Notably, accumulating metabolites of alternative propionate oxidation have been suggested to act as endogenous inhibitors of energy metabolism in different in vitro models [START_REF] Brock | On the mechanism of action of the antifungal agent propionate[END_REF][START_REF] Cheema-Dhadli | Effect of 2-methylcitrate on citrate metabolism: implications for the managment of patients with propionic acidemia and methylmalonic aciduria[END_REF][START_REF] Horswill | Studies of propionate toxicity in Salmonella enterica identify 2-methycitrate as a potent inhibitor of cell growth[END_REF][START_REF] Gregersen | The specific inhibition of the pyruvate dehydrogenase complex from pig kidney by propionyl-CoA and isovaleryl-CoA[END_REF], however, the pathophysiological impact of these findings on PA still remains unclear.

Here we report severe disturbance of mitochondrial energy metabolism in muscle tissues from two PA patients and demonstrate in vitro that propionyl-CoA-induced mitochondrial dysfunction plays a central role in this scenario. This girl was born at term as the second child of non-consanguineous Caucasian parents. The third day of life she was admitted because of progressive feeding refusal, lethargy and abnormal breathing. Laboratory investigations revealed a severe metabolic acidosis (pH 7.01, pCO 2 18 mm Hg, bicarbonate 5 mM, base excess -25 mM) and hyperammonemia (830 µM; normal <80 µM). The amino acids analysis in plasma revealed no abnormalities. Analysis of urine organic acids by gas chromatography/mass spectrometry showed biochemical abnormalities characteristic for PA with increased concentrations of betahydroxybutyric acid (6373 mmol/mol creatinine; control range: 0-45 mmol/mol creatinine), betahydroxypropionic acid (21461 mmol/mol creatinine; control range: 0-160 mmol/mol creatinine), betahydroxyisovaleric acid (176 mmol/mol creatinine; control range: 0-10 mmol/mol creatinine), lactic acid (776 mmol/mol creatinine; control range: 0-270 mmol/mol creatinine), propionylglycine, methylcitrate and tiglylglycine. The last three metabolites were not quantified. The concentration of free carnitine in plasma was decreased (10 µM; control range 20-200 µM), and the concentration of total carnitine was low-normal. A carnitine profiling was not done at the time of admission, but later on it repeatedly showed increased concentrations of propionylcarnitine varying between 40-80 µM (normal < 4 µM).

Biochemical

Extracorporal detoxification was performed. Besides a high caloric intake and transient stop of protein intake, metronidazol, lactulose, L-carnitine and biotin were added in the acute treatment phase. At age 10 days she was dismissed from the neonatal intensive care unit and then was nursed at the metabolic ward.

The only abnormal clinical sign at that time was a slight axial hypotonia. Maintenance treatment consisted of a natural protein intake (1.0 g/kg per day) supplemented with an amino acid mixture lacking precursor amino acids of propionyl-CoA (i.e., L-isoleucine, L-methionine, L-threonine, and L-valine), and application of L-carnitine and biotin. Analysis of propionyl-CoA carboxylase activity in leukocytes showed deficient activity of this enzyme (0.1 nmol/h/mg protein; control: 2.3 nmol/h/mg protein) and thus confirmed the diagnosis of PA. She was admitted to hospital numerous times due to metabolic derangements; the last one, at age five years, being fatal. The child had a severe myopathic appearance during life, which was, in combination with repeatedly increased blood lactate concentrations (2.0-7.2 mM; normal <2.1 mM), a reason to perform a muscle biopsy for mitochondrial studies.

Patient 2

This boy was born at term following an uncomplicated pregnancy and delivery. At the third day of life tachypnoea and metabolic acidosis led to a detailed metabolic investigation. The investigation of amino acids in plasma revealed an increased concentration of glycine (722 µM; 80-440 µM). As for patient 1 urine organic acid analysis showed an increased concentration of beta-hydroxybutyric acid (17818 mmol/mol creatinine), beta-hydroxypropionic acid (25096 mmol/mol creatinine), beta-hydroxyisovaleric Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221

Copyright 2006 Biochemical Society acid (34 mmol/mol creatinine), lactic acid (1322 mmol/mol creatinine), propionylglycine, methylcitrate and tiglylglycine. Propionyl-CoA carboxylase activity in cultured skin fibroblasts was strongly decreased (0.3 nmol/h/mg protein), confirming the diagnosis of PA that was treated according to standard protocols (see also patient 1).

The child always had a myopathic appearance and exercise intolerance. Standard exercise testing at age five years showed an increase of blood lactate from normal concentrations at rest to 20 mM lactate following a short run of about 50 meters. This was the reason for further bioenergetic analyses in muscle tissue (Table 1).

Muscle and Skin Biopsies

Muscle biopsies (Musculus vastus lateralis dextra) and forearm skin punch biopsies were performed in both patients. Furthermore, fibroblast cultures from patients with confirmed diagnosis of PDHc deficiency (n=3) and healthy volunteers (n=12) were used. Biopsies and subsequent experiments were performed after receipt of informed consent. The study was approved by the Institutional Review Board. Further investigations were performed in muscle tissue (Musculus quadriceps femoris) of wild-type C57Bl/6 mice. Animal care followed the official governmental guidelines and was approved by the government ethics committee.

Human Skin Fibroblast Cultures

Human skin fibroblasts from forearm skin biopsies were cultivated at standard conditions in DMEM at 37 °C supplemented with 10 % fetal calf serum (v/v), 100 µg/ml of penicillin, 100 µg/ml of streptomycin, 2.5 µg/ml of fungizone and 200 µM of uridine until confluency [START_REF] Schwab | Optimized spectrophotometric assay for the completely activated pyruvate dehydrogenase complex in fibroblasts[END_REF]. Medium was changed twice a week.

Each cell culture was tested for contamination with Mycoplasma spp. before enzyme analysis.

Preparation of Tissue Extracts

Fibroblast and muscle homogenates as well as submitochondrial particles from bovine heart (SMP) were prepared as previously described [START_REF] Schwab | Optimized spectrophotometric assay for the completely activated pyruvate dehydrogenase complex in fibroblasts[END_REF][START_REF] Okun | Three classes of inhibitors share a common binding domain in mitochondrial complex I (NADH:ubiqinone oxidoreductase)[END_REF][START_REF] Sauer | Bioenergetics in glutaryl-coenzyme A dehydrogenase deficiency, a role for glutaryl-coenzyme A[END_REF].

PDHc Activity

Spectrophotometric analysis of PDHc activity (E1, EC 1.2.4.1; E2, EC 2.3.1.12; E3, EC 1.8.1.4) was performed in purified porcine PDHc (Sigma-Aldrich, Schnelldorf, Germany), in SMP, and in homogenates from human skin fibroblasts and quadriceps muscle biopsy specimens using a piodonitrotetrazolium violet coupled system as previously described [START_REF] Schwab | Optimized spectrophotometric assay for the completely activated pyruvate dehydrogenase complex in fibroblasts[END_REF] Copyright 2006 Biochemical Society analyzed in temperature-controlled 96-well plates in a final volume of 300 µl. PDHc (purified enzyme, 160 milliunits/ml; SMP, 1.2 mg of protein/ml; fibroblast homogenate, 4 mg of protein/ml) was assayed in a buffer containing 0.05 M of potassium phosphate, 2.5 mM of NAD, 5 mM of L-carnitine, 0.2 mM of thiamine pyrophosphate, 0.1 mM of CoA, 0.1% Triton X-100 (v/v), 1 mM of MgCl 2 , 1 mg/ml of bovine serum albumin, 0.6 mM of p-iodonitrotetrazolium violet, and 6.5 µM of phenazine methosulfate, which was adjusted to pH 7.5 at 25 °C. PDHc activity was determined as p-iodonitrotetrazolium violet reduction at a wavelength of λ=500-750 nm. The specificity of this assay was confirmed by complete inhibition of PDHc activity by the specific E1 inhibitor 3-fluoropyruvate (5 mM).

To investigate the effect of acetyl-CoA and propionyl-CoA, we varied the concentrations of these CoA esters (0-1 mM) and pyruvate (0-0.1 mM). Additional experiments were performed using short-chain

(acetyl-CoA [C 2 ], propionyl-CoA [C 3 ], butyryl-CoA [C 4 ]), medium-chain (hexanoyl-CoA [C 6 ], octanoyl- CoA [C 8 ], decanoyl-CoA [C 10 ]) and long-chain (myristoyl-CoA [C 14 ], palmitoyl-CoA [C 16 ]
) acyl-CoA esters as well as corresponding short-and medium-chain fatty acids (up to 1 mM; all adjusted to pH 7.5).

Spectrophotometric Analysis of α-Ketoglutarate Dehydrogenase Complex (KGDHc) Activity

Spectrophotometric analyses of KGDHc (E1k, EC 1.2.4.2; E2k, EC 2.3.1.61; E3, EC 1.8.1.4) was performed in SMP and muscle homogenates as previously described [START_REF] Sauer | Bioenergetics in glutaryl-coenzyme A dehydrogenase deficiency, a role for glutaryl-coenzyme A[END_REF]. In brief, KGDHc (650 milliunits/ml) was assayed in a buffer containing 35 mM of potassium phosphate, 5 mM of MgCl 2 , 0.5 mM of EDTA, 0.5 mM of NAD, 0.2 mM of thiamine pyrophosphate, 0.04 mM of CoA-SH, and 2 mM of α-ketoglutarate, which was adjusted to pH 7.4 at 30 °C. KGDHc activity was determined as NAD reduction at a wavelength of λ=340-400 nm. The effect of propionyl-CoA on KGDHc activity was tested in analogy to PDHc (n=8).

Spectrophotometric Analysis of OXPHOS Complexes I-V

The catalytic activities of respiratory chain complexes I-V in SMP and muscle homogenates were investigated as previously described [START_REF] Okun | Three classes of inhibitors share a common binding domain in mitochondrial complex I (NADH:ubiqinone oxidoreductase)[END_REF][START_REF] Sauer | Bioenergetics in glutaryl-coenzyme A dehydrogenase deficiency, a role for glutaryl-coenzyme A[END_REF][START_REF] Schägger | Isolation of the eleven protein subunits of the bc 1 complex from beef heart[END_REF][START_REF] Brandt | Role of deprotonation events in ubihydroquinone:cytochrome c oxidoreductase from bovine heart and yeast mitochondria[END_REF][START_REF] Okun | Neurodegeneration in methylmalonic aciduria involves Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221 Copyright 2006 Biochemical Society inhibition of complex II and the tricarboxylic acid cycle, and synergistically acting excitotoxicity[END_REF][START_REF] Kölker | Methylmalonic acid, a biochemical hallmark of methylmalonic acidurias but no inhibitor of mitochondrial respiratory chain[END_REF]. The addition of standard respiratory chain inhibitors

(complex I, 2-n-decylquinazolin-4-yl-amine [1 µM]; complex II, thenoyltrifluoroacetone [8 mM]; complex III, antimycin A [1 µM]; complex IV, NaCN [2 mM]; complex V, oligomycin [80 µM])
revealed good inhibitory responses (93-100% of control activity, p<0.001 versus controls), confirming a high specific enzyme activity in our assay system. Acyl-CoA esters (C 2 , C 3 , C 4 , C 6 ) and corresponding fatty acids (all adjusted to pH 7.4) were added and effects on single respiratory chain complexes I-V were determined at concentrations up to 1 mM each (complex I, 0.5 mg of protein/ml; complex II, 0.3 mg/ml of protein; complex III, 0.1 mg/ml of protein, complex IV, 0.01 mg/ml of protein; complex V, 1.1 mg/ml of protein). In analogy, complex III activity was also investigated in isolated complex III from bovine heart (0.1 mg/ml of protein).

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Radiometric Analysis of Mitochondrial OXPHOS

Radiometric analysis was performed in freshly prepared quadriceps muscle biopsy specimens from PA patients. 600×g supernatants were prepared and oxidation rates of [1-14 C]pyruvate, [U- 14 C]malate and [1,4-14 C]succinate were determined as previously described [START_REF] Kölker | Methylmalonic acid, a biochemical hallmark of methylmalonic acidurias but no inhibitor of mitochondrial respiratory chain[END_REF][START_REF] Trijbels | Disorders of the mitochondrial respiratory chain: clinical manifestations and diagnostic approach[END_REF][START_REF] Sperl | Measurement of totally activated pyruvate dehydrogenase complex activity in human muscle: evaluation of a useful assay[END_REF].

ATP Production

Spectrophotometric analysis of ATP production was determined in muscle biopsies of PA patients using unlabeled pyruvate, malate, and succinate as described previously [START_REF] Kölker | Methylmalonic acid, a biochemical hallmark of methylmalonic acidurias but no inhibitor of mitochondrial respiratory chain[END_REF]. In brief, spectrophotometric analysis of ATP production is coupled to the formation of NADPH (λ=320-400 nm, 25 °C). The test principle consists of two enzyme reactions. In the first step, glucose (30.3 mM) and ATP are catalysed to glucose 6-phosphate and ADP by hexokinase (EC 2.7.1.1). Subsequently, glucose 6-phosphate dehydrogenase (EC 1.1.1.49) catalyses glucose 6-phosphate and NADP + to 6-phosphoglucolacton and NADPH.

Protein Electrophoresis of Muscle Tissue

One and two dimensional blue native polyacrylamide-gelelectrophoresis (BN-PAGE) and subsequent ingel activity assays and Western blotting were performed in muscle biopsies of PA patients as described previously [START_REF] Nijtmans | Blue native electrophoresis to study mitochondrial and other protein complexes[END_REF]. Antibodies against OXPHOS complex I (subunits 39 kDa and ND6), complex II (subunit 70 kDa), complex III (subunit Core 2) were obtained from Molecular Probes (Eugene, Oregon, USA), and against cyclophilin b from Affinity BioReagents (Golden, Colorado, USA).

Electron Microscopy of Muscle Tissue

Electron microscopy in muscle biopsies of PA patients was done according to standard protocols.

Data Analysis

Enzyme activities were normalized to citrate synthase (EC 2.3.3.1) activity and protein concentration of the same sample [START_REF] Srere | Citrate synthase, EC 4.1.3.7., Citrate oxaloacetate-lyase (CoA-acetylating)[END_REF][START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF]. Data were expressed as mean±SD of at least three independent experiments.

One-way analysis of variance (ANOVA) followed by posthoc Bonferroni's multiple comparison test (for three or more groups), or Student's t test (for two groups) were used to calculate statistical differences between groups. Results are presented as the mean±SD if not indicated differently. Statistics were calculated using SPSS for Windows 12.0 software. p<0.05 was considered significant.

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RESULTS

Propionyl-CoA Inhibits PDHc

In purified porcine PDHc, propionyl-CoA showed an inhibition of PDHc activity being uncompetitive with respect to pyruvate (Figure 1). Notably, this inhibitory effect was in the same range as for acetyl-CoA, the physiologic product and known inhibitor of PDHc (Figure 2). For propionyl-CoA (0.125-1 mM) the inhibition constant (K is ) ranged from 101-422 µM (3-12 times K m ), and for acetyl-CoA (0.125-1 mM) from 90-256 µM (2-7 times K m ). The K m was 35±0.5 µM for pyruvate. Similar results were found in SMP from bovine heart (data not shown).

Figure 1 and 2

Next, we determined PDHc activity in fibroblast homogenates from two PA patients. Assay conditions allowed enzyme analysis in the absence of intracellularly formed propionyl-CoA. Notably, fibroblasts from both patients revealed PDHc activities within the control range confirming our hypothesis of secondary PDHc inhibition induced by propionyl-CoA (Figure 3). As expected, patients with confirmed PDHc deficiency showed a reduced PDHc activity in fibroblasts (Figure 3).

Figure 3

Acyl-CoA Esters Inhibit PDHc in a Chain-length Specific Manner

To investigate whether PDHc inhibition was dependent on the chain length of acyl-CoA esters, we investigated the inhibitory effect of homologous monocarboxylic CoA esters ranging from C 4 to C 16 in purified porcine PDHc. In fact, PDHc was also effectively inhibited by C 4 , C 10 , C 14 , and C 16 but less effectively by C 6 and C 8 (Figure 4A). In contrast, short-chain fatty acids (up to 1 mM) did not exert an inhibition of PDHc (Figure 4B). These results were confirmed in SMP from bovine heart (data not shown).

Figure 4 Alternative Effects of Propionyl-CoA on Energy Metabolism?

Bioenergetic investigations in muscle tissue of both PA patients showed strongly reduced oxidation of pyruvate and PDHc activity as well as a diminished ATP plus phosphocreatine production (Table 1). In addition, decreased oxidation of succinate and malate as well as of almost all measured enzymes and enzyme complexes of mitochondrial respiratory chain and TCA cycle were found in these patients (Table 1).

Copyright 2006 Biochemical Society

Consistent with the enzymatic measurements is the decrease of the OXPHOS complexes I, II, III and IV in patient 2 observed with one and two dimensional BN-PAGE electrophoresis in combination with western blotting and a complex I in-gel activity assay (Figure 5).

Figure 5

The general decrease in the amount of these OXPHOS complexes suggests a destabilisation and increased breakdown of mitochondria. The observed reduction in mitochondrial DNA (mtDNA) content of about 50% in patient 1 and 2 is in line with this suggestion. In analogy with these biochemical results, histopathology of muscle biopsies showed numerous signs of mitochondrial alteration indicating disturbance of mitochondrial energy metabolism, such as inclusion of many lipid droplets in muscle fibers of patient 1 (Figure 6A), cristalline inclusions (Figure 6B) and large swollen mitochondria with dark globular inclusions (Figure 6C).

Figure 6

Since bioenergetic analyses in both PA patients suggested additional mechanisms acting in concert with propionyl-CoA-induced PDHc inhibition, we investigated whether propionyl-CoA and other shortchain acyl-CoA esters revealed inhibitory effects on respiratory chain and TCA cycle enzymes. We found no effect of short-chain acyl-CoA esters and fatty acids on respiratory complexes I, II, IV, and V in SMP (data not shown). However, in analogy to muscle tissue of PA patients, propionyl-CoA (1 mM) induced a mild inhibition of complex III activity in SMP (72±5% of controls) and in isolated complex III from bovine heart (81±8% of controls). In addition, the activity of KGDHc, a rate-limiting TCA cycle enzyme complex, was also decreased by propionyl-CoA (1 mM propionyl-CoA: 38±3% of controls).

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DISCUSSION

The accumulation of metabolites from the alternative oxidative pathways of propionyl-CoA is the biochemical hallmark of PA. During metabolic crisis additional biochemical abnormalities such as lactic acidosis, hyperketosis and hypoglycemia indicate the development of severe mitochondrial dysfunction resulting in impairment of energy metabolism [START_REF] Fenton | Disorders of propionate and methylmalonate metabolism[END_REF]. The present study significantly adds to the understanding of these mechanisms demonstrating 1) a severe bioenergetic disturbance and ultrastructural changes of mitochondria in muscle tissue of PA patients and 2) propionyl-CoA-induced synergistic inhibition of PDHc, KGDHc, and complex III in vitro. These results support the hypothesis that secondary mitochondrial dysfunction induced by accumulating toxic metabolites plays a major role in the pathomechanisms of propionic aciduria [START_REF] Gregersen | The specific inhibition of the pyruvate dehydrogenase complex from pig kidney by propionyl-CoA and isovaleryl-CoA[END_REF]. We clearly demonstrated that propionyl-CoA but not propioniate is the major toxic metabolite involved in this scenario.

PDHc is a bioenergetic bottle neck coupling cytosolic anaerobic glycolysis with the mitochondrial TCA cycle and OXPHOS. Not surprisingly, inherited PDHc deficiency results in a severe disturbance of energy metabolism and frequently in a fatal disease course [START_REF] Demeirleir | Defects of pyruvate metabolism and the Krebs cycle[END_REF]. In analogy, secondary inhibition of PDHc by propionyl-CoA can be suggested as an important mechanism inducing energy failure in PA which is confirmed by severely reduced ATP and phosphocreatine concentrations in muscle tissue of PA patients and increased serum lactate concentrations. In line with this, multiple organ failure in PA patients predominantly manifests in tissues with a high energy demand, such as central nervous system (encephalopathy, movement disorders, developmental retardation), skeletal muscle (myopathy), heart muscle (cardiomyopathy), and bone marrow (pancytopenia) [START_REF] Fenton | Disorders of propionate and methylmalonate metabolism[END_REF][START_REF] Brismar | CT and MR of the brain in disorders of the propionate and methylmalonate metabolism[END_REF]. Apart from mammalian species, propionyl-CoA-induced inhibition of PDHc has recently been suggested as major mechanism underlying the antibacterial and antifungal properties of propionate [START_REF] Brock | On the mechanism of action of the antifungal agent propionate[END_REF]. Thus propionyl-CoA-induced mitochondrial dysfunction can be regarded as common final pathway involved in different conditions, i.e. inherited disorders of propioniate metabolism and propionate toxicity. Future studies should focus on concentration-dependent effects of propionyl-CoA in tissues of affected children with PA. However, since the amount of a muscle biopsy specimen in children is always very limited because of ethical and functional considerations, this important aspect could not be investigated additionally in the present study.

In addition to PDHc inhibition, alternative mechanisms for propionyl-CoA and metabolites of alternative propionate oxidation should be considered as relevant. In particular inhibition of respiratory chain complex III (this study) and the TCA cycle enzymes KGDHc (this study), citrate synthase [START_REF] Cheema-Dhadli | Effect of 2-methylcitrate on citrate metabolism: implications for the managment of patients with propionic acidemia and methylmalonic aciduria[END_REF] and succinyl-CoA synthetase [START_REF] Brock | On the mechanism of action of the antifungal agent propionate[END_REF] may also contribute to impaired energy metabolism in this disease.

Interestingly, inherited deficiency of succinyl-CoA synthetase caused by deleterious mutations in the SUCLA2 gene has recently been associated with encephalomyopathy and mtDNA depletion [START_REF] Elpeleg | Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion[END_REF], linking the TCA cycle with mtDNA homeostasis [START_REF] Elpeleg | Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion[END_REF][START_REF] Saada | Deoxyribonucleotides and disorders of mitochondrial DNA integrity[END_REF]. In addition, increased oxidative stress, which has been Copyright 2006 Biochemical Society [START_REF] Fontella | Propionic and L-methylmalonic acids induce oxidative stress in brain of young rats[END_REF][START_REF] Wallace | The mitochondrial genome in human adaptive radiation and disease: on the road to therapeutics and performance enhancement[END_REF]. Interestingly, the amount of mtDNA and the activities of OXPHOS complexes I, III, and IV, which are partially encoded by mtDNA, were significantly decreased in muscle tissue of both PA patients, however, it remains unclear whether this result reflects a causal link. Besides mtDNA homeostasis, other secondary or tertiary targets might be involved but have not yet been identified.

PA shares a variety of biochemical and clinical similarities with methylmalonic aciduria which is caused by inherited deficiency of methylmalonyl-CoA mutase or the synthesis or transport of its cofactor, 5'-adenosylcobalamin [START_REF] Fenton | Disorders of propionate and methylmalonate metabolism[END_REF]. We have recently hypothesized that propionyl-CoA and metabolites deriving from propionyl-CoA, such as 2-methylcitrate, might act as endogenous neurotoxins also in this disease, whereas methylmalonate most likely plays a minor role [START_REF] Okun | Neurodegeneration in methylmalonic aciduria involves Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221 Copyright 2006 Biochemical Society inhibition of complex II and the tricarboxylic acid cycle, and synergistically acting excitotoxicity[END_REF][START_REF] Kölker | Methylmalonic acid, a biochemical hallmark of methylmalonic acidurias but no inhibitor of mitochondrial respiratory chain[END_REF][START_REF] Kölker | Methylmalonic acid -an endogenous toxin? Cell[END_REF]. Since the manifestation of secondary metabolic blocks are pathophysiologically relevant in PA and methylmalonic aciduria, it is of interest to investigate whether alternative energy substrates such as succinate and citrate might be beneficial for metabolic maintenance treatment and intensified emergency treatment of these patients helping to restore mitochondrial energy metabolism and to prevent multiple organ failure.

  demonstrated in an in vitro model for disorders of propionate metabolism, induces mtDNA damage Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221

FIGURE LEGEND FIGURE 1 .

 LEGEND1 FIGURE LEGEND

FIGURE 2 .

 2 FIGURE 2. Inhibition of PDHc by Acetyl-CoA. Spectrophotometric analysis in purified PDHc from porcine heart demonstrates uncompetitive inhibition of PDHc activity by acetyl-CoA with respect to pyruvate (Pyr) as demonstrated by a Lineweaver Burk plot. Values are given as the mean±SD. Experiments were performed at least in triplicates.

FIGURE 3 .

 3 FIGURE 3. Spectrophotometric Analysis of PDHc Activity in Human Skin Fibroblasts.Spectrophotometric analysis of PDHc activity was performed in human skin fibroblasts from healthy volunteers (n=12), patients with propionic aciduria (n=2) and primary PDHc deficiency (n=3). The horizontal bar represents the mean of the control group and the dotted line indicates the range of the control group.

FIGURE 4 .

 4 FIGURE 4. Chain-length specific effects of Acyl-CoA Esters and fatty acids on PDHc activity. (A) Acyl-CoA Esters: Spectrophotometric analysis of PDHc activity was performed in purified enzyme from porcine heart using short-, medium-and long-chain monocarboxylic acyl-CoA esters. The figure summarizes the I 50 values of acyl-CoA esters. Values are given as mean±SD. Experiments were performed at least in triplicates. *p<0.01; **p<0.001 versus control (ANOVA followed by posthoc Bonferroni's multiple comparison test, n=8). (B) Fatty Acids: In contrast to acyl-CoA esters, corresponding short-and medium-chain fatty acids (up to 1 mM) did not inhibit PDHc activity in purified enzyme from porcine heart. PDHc activity was determined at standard conditions. Values were normalized to 100% (dotted line) and values were given as the mean±SD. Experiments were performed at least in triplicates.

FIGURE 5 .

 5 FIGURE 5. BN-PAGE of OXPHOS Complexes in Muscle Homogenates. Protein (40 µg) of solubilised muscle homogenates from patient 2 (P) and control (C) were analyzed on a 5-15% BN-polyacrylamide gel for the separation of multi-subunit complexes. (A) In-gel activity assay of mitochondrial complex I confirming a decrease in activities of patient 2 compared to control samples. (B)A second gel was run in duplicate and western blot analysis was performed using antibodies against complex I subunit 39 kDA, complex II subunit 70 kDa and complex III subunit Core 2 (C). A second

Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221

  . In brief, steady state activities of PDHc were recorded using a computer tunable spectrophotometer (SPECTRAmax Plus 384 Microplate Reader, Molecular Devices, Sunnyvale, CA) operating in the dual wavelength mode. Samples were

Table 1 Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221

 1

Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221 Copyright 2006 Biochemical Society FOOTNOTES

  To whom correspondence should be addressed: Stefan Kölker, Department of General Pediatrics, Division of Inborn Metabolic Diseases, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany. Tel.: +49-6221-561714; Fax: +49-6221-565565; E-mail:Stefan_Koelker@med.uni-heidelberg.de § Both authors contributed equally to the study.

	Abbreviations used: Coenzyme A, CoA; PDHc, pyruvate dehydrogenase complex; TCA, tricarboxylic
	acid; PA, propionic aciduria; OXPHOS, oxidative phosphorylation; SMP, submitochondrial particles
	from bovine heart; E1, pyruvate decarboxylase; E2, dihydrolipoyl transacetylase; E3, dihydrolipoyl
	dehydrogenase; C 2 , acetyl-CoA; C 3 , propionyl-CoA; C 4 , butyryl-CoA; C 6 , hexanoyl-CoA; C 8 , octanoyl-
	CoA; C 10 , decanoyl-CoA; C 14 , myristoyl-CoA; C 16 , palmitoyl-CoA; KGDHc, α-ketoglutarate
	dehydrogenase complex; E1k, α-ketoglutarate dehydrogenase; E2k, dihydrolipoyl succinyltransferase;
	BN-PAGE, blue native polyacrylamide-gelelectrophoresis; ANOVA, one-way analysis of variance; K is ,
	inhibition constant; mtDNA, mitochondrial DNA; K m , Michaelis-Menten constant; Pyr, pyruvate; n.m.,
	not measured
	1

ACKNOWLEDGEMENTS

This study was supported by a research grant from the University of Heidelberg to Marina A. Schwab (# 19/2003) and a grant from the Deutsche Forschungsgemeinschaft (SCHW1367/1-1). We are grateful to Roel Smeets and Sonja Exner-Camps for excellent technical support. Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060221 Copyright 2006 Biochemical Society

dimension was run and western blot analysis was performed using antibodies against OXPHOS complexes I (ND6), III (Core 2) and IV (COX2) and cyclophilin B as a loading control. Arrows indicate the first and second dimension. Radiochemical analysis of coupled mitochondria (A) using [1-14 C]pyruvate, [U- 14 C]malate and [1,4-14 C]succinate. Spectrophotometric analysis of single enzymes of the respiratory chain, TCA cycle, PDHc (B), ATP and phosphocreatine production rate (C), and citrate synthase (D). Analysis were performed in muscle biopsy specimens of both patients. Activities are expressed as nmol/h×mg protein (A), milliunits/unit citrate synthase (B), nmol/h×milliunits citrate synthase (C) and milliunits/mg protein (D).

The production of ATP and phosphocreatine was determined to establish the total mitochondrial energy generating capacity of the muscle mitochondria of both patients. n. m., not measured. Copyright 2006 Biochemical Society

Experiment


Elena Odintsova
email: odintsoe@bham.ac.uk

Terry D Butters

Eugenio Monti

Hein Sprong

Gerrit Van Meer

Fedor Berditchevski

GANGLIOSIDES PLAY AN IMPORTANT ROLE IN THE ORGANISATION OF CD82-ENRICHED MICRODOMAINS

Keywords: tetraspanins, CD82, microdomains, EGFR, GD1a, Neu3 sialidase. Abbrevations used

Four transmembrane domain proteins of the tetraspanin superfamily are the organisers of specific microdomains at the membrane (tetraspanin-enriched microdomains or TERM) that incorporate various transmembrane receptors and modulate their activities. The structural aspects of the organisation of TERM are poorly understood. Here we investigated the role of gangliosides in the assembly and stability of TERM. We demonstrated that inhibition of the glycosphingolipid biosynthetic pathway with specific inhibitors of glucosylceramide synthase (NB-DGJ and PPMP) resulted in specific weakening of the interactions involving tetraspanin CD82. Furthermore, ectopic expression of the plasma membrane-bound sialidase Neu3 in mammary epithelial cells also affected stability of the complexes containing CD82: its association with tetraspanin CD151 was decreased but the association with EGFR was enhanced. The destabilization of the CD82-containing complexes upon ganglioside depletion correlated with the re-distribution of the proteins within plasma membrane. Importantly, depletion of gangliosides affected EGF-induced signalling only in the presence of CD82. Taken together our results provide strong evidence that gangliosides play an important role in supporting the integrity of CD82-enriched microdomains. Furthermore, these data demonstrate that the association between different tetraspanins in TERM is controlled by distinct mechanisms and identify Neu3 as a first physiological regulator of the integrity of these microdomains.

Introduction

The plasma membrane is a dynamic highly compartmentalised structure.

Compartmentalisation is important for the induction of signalling, for the assembly of adhesion complexes and for the generation of immune responses. Currently there are a few models of membrane organisation and they all agree on the fact that certain lipids and proteins within the plasma membrane are arranged in small aggregates (microdomains) that carry out signalling functions [1;2]. These microdomains enlarge or dissolve over time depending on the cell's activities. A number of different types of microdomains have been described including lipid rafts and caveolae [START_REF] Razani | Caveolae: from cell biology to animal physiology[END_REF], detergent-resistant membranes (DRM), glycosphingolipid-enriched microdomains (GEM) and glycosynapses [START_REF] Hakomori | Glycosylation defining cancer malignancy: new wine in an old bottle[END_REF]. With the exception of glycosynapses they are all based on a classic model of "lipid rafts". The structure of lipid rafts in the intact plasma membrane is unknown but an operational definition has been adopted according to which they are cholesterol-dependent and contain components that are isolated as detergent-insoluble membranes [5;6]. Tetraspanin-enriched microdomains (or TERM) represent a novel type of molecular aggregates that are distinct from all the mentioned above. They could be operationally defined as the membrane complexes maintained after solubilisation with mild detergents such as Brij 96 or Brij 98 [7;8]. Indeed, in contrast to classical lipid rafts TERM are sensitive to Triton X-100 and do not contain GPI-linked proteins [START_REF] Hemler | Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain[END_REF]. The principal components of TERM are tetraspanins, which constitute a large family of four transmembrane domain proteins. Although the exact biochemical function of tetraspanins is not well defined, it has been established that these proteins play an important role in membrane compartmentalisation and dynamics [START_REF] Hemler | Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain[END_REF][START_REF] Yunta | Tetraspanin proteins as organisers of membrane microdomains and signalling complexes[END_REF][START_REF] Delaguillaumie | Tetraspanin CD82 controls the association of cholesterol-dependent microdomains with the actin cytoskeleton in T lymphocytes: relevance to co-stimulation[END_REF][START_REF] Levy | The tetraspanin web modulates immune-signalling complexes[END_REF]. Tetraspanins interact with each other thereby providing a structural platform for the recruitment of other transmembrane and cytoplasmic proteins into TERM [8;11;12]. A number of transmembrane receptors are known to associate with tetraspanin microdomains, including integrins [START_REF] Berditchevski | Complexes of tetraspanins with integrins:more than meets the eye[END_REF], receptor tyrosine kinases and G-protein coupled receptors [11;12]. Importantly, association of a particular receptor with TERM may lead either to the enhancement or to the attenuation of its activity [10;14]. The contribution of individual tetraspanins to the generation of co-stimulatory or inhibitory effects has not been studied in detail.

Gangliosides are essential structural components of the membranes. These glycosphingolipids are involved in the regulation of signalling through the growth factor and adhesion receptors [START_REF] Wang | Ganglioside modulates ligand binding to the epidermal growth factor receptor[END_REF][START_REF] Miljan | Regulation of growth factor receptors by gangliosides[END_REF][START_REF] Kawakami | Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility[END_REF]. Previous reports established various links between tetraspanins and gangliosides. It was shown that CD9 (and, possibly, CD82) and GM3 cooperatively down-regulate motility of tumour cells by attenuating signalling induced by laminin/integrin interactions [START_REF] Kawakami | Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility[END_REF][START_REF] Ono | GM3 Ganglioside Inhibits CD9-Facilitated Haptotactic Cell Motility: Coexpression of GM3 and CD9 Is Essential in the Downregulation of Tumor Cell Motility and Malignancy[END_REF][START_REF] Ono | Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation[END_REF] The cross-talk between integrins and FGFR has also been influenced by the changes in gangliosides and/or tetraspanin expression levels [START_REF] Toledo | Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor[END_REF]. Moreover, our previous data suggested that the activity of EGFR is attenuated by the tetraspanin CD82 through the modification of ganglioside composition at the membrane [START_REF] Odintsova | Tetraspanin CD82 regulates compartmentalisation and ligand-induced dimerization of EGFR[END_REF]. Specifically, we found that there was a correlation in the expression levels of CD82 and gangliosides in mammary epithelial cells [START_REF] Odintsova | Tetraspanin CD82 regulates compartmentalisation and ligand-induced dimerization of EGFR[END_REF]. However, the underlying mechanism of the interactions of gangliosides and tetraspanins has not been identified. Most likely gangliosides provide stability and structural support for TERM. Gangliosides may also control localisation of the transmembrane proteins to the segregated microdomains.

In this report we examined the importance of gangliosides for the assembly of TERM. By manipulating ganglioside content pharmacologically and enzymatically we demonstrated that removal of gangliosides affects associations of CD82 with its partners -components of TERM. We also found that there is a clear specificity in the effect that ganglioside depletion had on the interactions involving different tetraspanins. Particularly, only associations of CD82 (but not CD9) with EGFR, α3β1 integrin and other tetraspanins have been affected. Thus, our data indicate that structural integrity of CD82-containing microdomains relies on the presence of gangliosides.

Materials and Methods.

Cell lines, antibodies and reagents. Human mammary epithelial cells HB2/ZEO and HB2/CD82 [START_REF] Odintsova | Tetraspanin CD82 regulates compartmentalisation and ligand-induced dimerization of EGFR[END_REF] were maintained in DME medium (Gibco) supplemented with 10% foetal calf serum (FCS), 10 µg/ml of hydrocortisone and 10 µg/ml of insulin.

Mouse melanoma cells GM95 and GM95/CGlcT-ER were grown in DME medium (Gibco) supplemented with 10% FCS. The anti-CD82 mAbs γC11 and ΙΑ4 were kindly provided by Dr.H.Conjeaud. The anti-CD82 mAb M104 was kindly provided by Dr.O.Yoshie. The anti-CD82 mAb TS82b and anti-CD9 mAb TS9 were kindly provided by Dr.E.Rubinstein. 5C11, anti-CD151 mAb, and C9BB, anti-CD9, mAb were described elsewhere [22;23]. Dr.L.Ashman generously provided polyclonal anti-CD151 antibody and 11G1B4 anti-CD151 mAb. Dr.F.Watt kindly provided polyclonal antibody to α3-integrin subunit. Anti-EGFR mAb Ab-15 was purchased from Neomarkers; phosphospecific anti-EGFR polyclonal antibody was from Cell Signalling. Anti-phosphotyrosine monoclonal antibody (4G10) was purchased from the Upstate Biotechnology. Monoclonal (H7) and polyclonal (Y-11) antibodies against HA-tag sequence were purchased from Autogen Bioclear. Dr.R.Schnaar kindly provided mouse mAbs to gangliosides GD1a and GM1. FITC-conjugated cholera toxin B subunit (CT) was purchased from Sigma. NB-DGJ was prepared as described [START_REF] Platt | Nbutyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis[END_REF]. D-threo-1-Phenyl-2-hexadecanoylamino-3-morpholino-1propanol•HCl (PPMP) was purchased from Matreya, Inc. Other reagents were from Sigma or Fishers unless stated otherwise.

Transfections. GM95 and GM95/CGlcT-ER transfected with CD82 and CD151 tetraspanins were selected in Zeocin-containing medium, and pools of drug-resistant clones were sorted with anti-CD82 mAb IA4 and anti-CD151 mAb 11G1B4. HB2 cells were transfected with sialidase Neu3 cDNA by using FuGene6 (Roche) and selected (when necessary) in Zeocin-containing medium. Exogenous administration of gangliosides. For exogenous administration of gangliosides GD1a or GM3 cells were plated 2 days before the experiment.

Inhibition

Subsequently, GD1a (Calbiochem), or GM3 (Matreya) were added to serum-free medium at final concentration 50 µg/ml. DMSO (25µl/ml) was used in the control experiments. After the incubation (2-24 hours), cells were washed with PBS and used in various assays. Ganglioside extraction and thin-layer chromatography. The gangliosides were extracted according to the small-scale isolation of gangliosides protocol [START_REF] Schnaar | Thin-layer chromatography of glycosphingolipids[END_REF] which is described briefly further. Cells were collected, washed and pelleted. A small cell pellet was adjusted to 0.6 ml with water, homogenized in Dounce homogeniser (15 strokes) on ice, 1.6 ml of methanol was added. After vigorous agitation chloroform (0.8 ml) was added, and the capped mixture was vigorously agitated again at ambient temperature. The tube was centrifuged in a table-top centrifuge (3000g, 10 min) at ambient temperature, the supernatant transferred to a fresh tube, and the pellet reextracted with 2 ml of chloroform-methanol-water (4:8:3, v/v). After centrifugation, the second supernatant was added to the first, the volume of the combined supernatants was measured, and water was added to bring the final chloroformmethanol-water ratio to 4:8:5.6 (v/v). After vigorous agitation, the two phases were separated by centrifugation (1500g, 10 min, ambient temperature) and the upper phase was transferred to a fresh tube. The lower phase was re-extracted with theoretical upper phase, and the combined upper phases were applied to a pre-washed experiment was tested by measuring the surface expression level of gangliosides by flow cytometry. Interestingly, depletion of gangliosides led to the increase in the amount of CD82 (Fig. 1B). We have previously demonstrated that there is a correlation between the expression levels of CD82 and gangliosides: increase in CD82 amounts led to the rise in GD1a concentration [START_REF] Odintsova | Tetraspanin CD82 regulates compartmentalisation and ligand-induced dimerization of EGFR[END_REF].

Immunoprecipitation with the anti-CD82 antibody revealed significant changes in the associations of CD82 with other proteins under the conditions of ganglioside depletion (Fig. 1C, compare lanes 1 and 3 or lanes 5 and 7; Fig. 1D). There was less EGFR and CD9 (~ 35% and 25% decrease, respectively) immunoprecipitated with the anti-CD82 antibody from the lysates prepared from the NB-DGJ-treated cells (Fig. 1C, lane 1 and 3, first panel from the top) in comparison to the controls, untreated cells. Association of CD82 with α3 integrin subunit was also effected (~20% decrease) (Fig. 1C, lane 1 and 3, second panel from the top; Fig. 1D). Notably, the association of CD82 with the tetraspanin CD151 was diminished by ~ 4 fold (Fig. 1C, lane 1 and 3, second panel from the bottom; Fig. 1D). Western blotting with the anti-CD82 mAb indicated that the amounts of immunoprecipitated CD82 were comparable in the controls and NB-DGJ-treated cells (Fig. 1C, lanes 1 and 3, lower panel). Depletion of gangliosides with PPMP also led to decreases in the association of CD82 with its interacting partners (Fig. 1C, compare lanes 5 and 7 or lanes 6 and 8). To investigate how specific was the effect of ganglioside depletion we also analysed interactions involving tetraspanin CD9. In contrast to CD82-containing complexes, there was no obvious effect of ganglioside depletion on the interactions involving CD9 (Fig. 1C, compare lanes 2 and 4 or lanes 6 and 8; Fig. 1D). We concluded from these data that gangliosides are important for the interactions within CD82-enriched microdomains. We examined the effect of the overexpression of the plasma membrane-bound Neu3 sialidase on the stability of protein complexes within tetraspanin microdomains.

Changes in gangliosides concentration

Sialidases are specific hydrolytic enzymes that remove sialic acid residues from gangliosides and sialated proteins [START_REF] Tettamanti | Ganglioside/glycosphingolipid turnover: new concepts[END_REF]. Sialidase Neu3 is a cell surface enzyme that is characterised by its high substrate specificity towards gangliosides [29;30].

Previous studies showed that Neu3 selectively target gangliosides GD1a and GM3 at plasma membrane [START_REF] Papini | The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-tocell interactions[END_REF].

HB2/CD82 cells were transiently transfected with the HA-tagged form of sialidase

Neu3. It has been shown previously that HA-tag did not affect the activity of the enzyme [START_REF] Monti | Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane[END_REF]. Because HB2/CD82 cells do not have detectable GM3, we expected that the expression of Neu3 in these cells would only affect the level of GD1a. To validate the activity of Neu3 sialidase in transiently transfected HB2/CD82 cells we carried out immunofluorescence staining with the anti-GD1a and anti-HA mAbs.

Figure 2 illustrates that the cells transfected with Neu3 have significantly lower levels of GD1a compared to the surrounding non-transfected cells (Fig. 2, compare A and B). We also tested whether GM1 expression was affected by Neu3 sialidase.

Staining with CT indicated that levels of GM1 were comparable in the Neu3-positive and Neu3-negative cells (Fig. 2, compare C and D). Interestingly, we consistently observed that incubation with CT affected distribution of HA-tagged Neu3 sialidase making it more disperse. This may be due to the multivalent nature of CT. We did not examine GM3 levels since we demonstrated earlier that GM3 could not be detected in HB2 cells (Fig. 1A, lane b). These data provided evidence that of the two main A-series gangliosides expressed on the surface of HB2 cells (i.e. GD1a and GM1) expression of Neu3 sialidase specifically affected GD1a levels. Assembly of CD82-enriched microdomains is not affected by GM3 ganglioside.

We showed that GM3 ganglioside could not be detected in significant quantities in HB2 cells (Fig. 1A). However, there is evidence that this ganglioside may be important for the interactions involving some of the tetraspanins (e.g. CD9) [START_REF] Kawakami | Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility[END_REF][START_REF] Ono | GM3 Ganglioside Inhibits CD9-Facilitated Haptotactic Cell Motility: Coexpression of GM3 and CD9 Is Essential in the Downregulation of Tumor Cell Motility and Malignancy[END_REF][START_REF] Ono | Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation[END_REF].

To examine the contribution of GM3 to the stability of TERM we analysed tetraspanin-enriched microdomains in GM95 and GM95-CGlcT.ER cells, two derivatives from MEB-4 mouse melanoma cell line [33;34]. While GM95 cells are glycosphingolipid-deficient, GM95-CGlcT.ER cells are transfected with ceramide glucosyl transferase and predominantly express ganglioside GM3. Both cell lines were stably transfected to express human CD82 and CD151 proteins.

We used GM95/CD82+CD151 and GM95-CGlcT.ER/CD82+CD151 cells in the immunoprecipitation analysis. When we compared CD82-immunoprecipitates prepared from both cell lines there were no differences in the amounts of CD151 found in each of the samples (Fig. 5, lanes 1 and 4, lower panel). Accordingly, the anti-CD151 mAb co-immunoprecipitated similar amounts of CD82 (Fig. 5, lanes 2 and 5, upper panel). Both cell lines had comparable expression levels of transfected tetraspanins as detected by Western blotting (Fig. 5, lanes 7 and 8) and flow cytometry (data not shown). We concluded from these experiments that CD82 interactions are not affected by GM3 ganglioside.

To examine the links between CD82 and gangliosides further we incubated ganglioside-deficient cells GM95/CD82+CD151 with exogenously added GD1a or GM3. In our experiments we used the non-toxic concentration of gangliosides (50µg/ml) that was determined in pilot experiments. Flow cytometry analysis confirmed incorporation of gangliosides into the plasma membrane of the cells (data not shown). Comparable amounts of CD151 and CD82 were co-immunoprecipated by the anti-CD82 (Fig. 6, lanes 1, 4, 7) and anti-CD151 mAbs (Fig. 6, lanes 2, 5, 8).

Similar results were obtained when we used HB2/CD82 cells (data not shown).

These data showed that exogenous administration of gangliosides does not affect tetraspanin-tetraspanin interactions.

Inhibition of ganglioside biosynthesis has specific effect on the EGFR activity in

Inhibition of ganglioside biosynthesis causes redistribution of CD82-associated

proteins. We hypothesized that the effect of ganglioside depletion on the associations involving CD82 may be due to the alterations in membrane distribution of the proteins. Thus, we examined how treatment of HB2/CD82 cells with NB-DGJ effected protein distribution within the membrane by fractionation of cellular lysates in continuous sucrose gradient. For the preparation of lysates for fractionation we used a modified detergent-free protocol that allowed concentration of the majority of tetraspanin-containing complexes to the light membrane fractions of the gradient (Fig. 8A, c panels in each frame). We avoided detergents in these experiments, as there is evidence that some detergents may induce artifactual formation of microdomains in the membranes [START_REF] Heerklotz | Triton promotes domain formation in lipid raft mixtures[END_REF]. The bulk of CD82 was found in fractions 2-4 together with significant amount of EGFR, α3 integrin subunit and CD151 tetraspanin (Fig. 8A, c panels in each frames, lanes 2-4). Depletion of gangliosides had a dramatic effect on the membrane distribution of all tested proteins (Fig. 8A, i panels in each frame). The most striking fact was the difference in the fractional distribution of CD82 and CD82-associated proteins. The bulk of EGFR, α3 integrin subunit and CD151 were shifted to the heavy fractions (7-10) of the gradient (Fig. 8A, i panels in each frame). The distribution of CD82 has also shifted to the right, to heavier fractions, but this shift was not as pronounced as for the other proteins: most Redistribution of CD82 at the cell membrane was further illustrated by the immunofluorescence staining carried out on the cells depleted of gangliosides by incubation with NB-DGJ inhibitor. In non-permeabilised untreated cells tetraspanin CD82 exhibited typical peripheral staining (Fig. 9,A). However, after incubation with NB-DGJ a significant proportion of CD82 re-localised to large dots randomly scattered on the cell surface (Fig. 9,B). These results indicated that the profound effect of gangliosides depletion on the stability of tetraspanin-enriched microdomains is likely to be a consequence of the re-distribution of proteins at the plasma membrane.

Cholesterol has an important role for the assembly and maintenance of various microdomains at the plasma membrane [START_REF] Silvius | Role of cholesterol in lipid raft formation: lessons from lipid model systems[END_REF]. There are controversial data in the literature about the role of cholesterol in the assembly of tetraspanin-enriched microdomains [7;37]. To compare effects of ganglioside and cholesterol depletion on the distribution of CD82-associated proteins we incubated HB2/CD82 cells with 20 mM MβCD for 1 hour. Typically, after this treatment we removed ~ 40% of total cholesterol (as measured following cell lysis). Lysates after MβCD treatment were subjected to the fractionation in sucrose gradient as described above. Cholesterol depletion led to the re-distribution of CD82-associated proteins in sucrose gradient.

The effect was more or less similar to the effect of ganglioside depletion (Fig. 8B, compare c and d panels). Notably, the fractional distribution of CD82 itself was only marginally affected by the cholesterol depletion. Together these data indicate that gangliosides and cholesterol have distinct roles in maintaining the structural integrity of the CD82-enriched microdomains.

Discussion.

Interactions between tetraspanins provide a structural platform for the assembly of TERM. Our results demonstrate for the first time that gangliosides can stabilise a subset of tetraspanin-tetraspanin interactions. This was shown by using both pharmacological and physiological (i.e. ectopic expression of Neu3) approaches.

Notably, we found that only interactions involving CD82 (e.g. CD82-CD151, CD82-EGFR) are sensitive to changes in the ganglioside content, thus pointing to a diversity of the mechanisms that control the assembly and (or) maintenance of TERM. Extreme sensitivity of CD82-based interactions to gangliosides may also imply that there exist different types of TERM, which are specifically enriched with one (or two) tetraspanins (e.g. CD82-enriched TERM, CD9-enriched TERM, etc.).

This idea is not without a precedent: for example, urothelial tetraspanins UPIa and UPIb are specifically abundant in the asymmetric unit membranes (AUM), which do not seem to include any other tetraspanin proteins [START_REF] Sun | Uroplakins as markers of urothelial differentiation[END_REF]. Further analysis will be necessary to verify this hypothesis.

In contrast to the data obtained with the inhibitor (and results with overexpression of Neu3) exogenous administration of purified gangliosides had no effect on the association of tetraspanins with each other. There may be at least two reasons for this "discrepancy". It is well established that biosynthesis of complex gangliosides takes place in Golgi [START_REF] Tettamanti | Ganglioside/glycosphingolipid turnover: new concepts[END_REF]. Furthermore, it is likely that many of the TERM-specific interactions are also initiated in this compartment [8;11]. Thus, there may be a spatial and temporal co-ordination of these two processes in the Golgi complex. Although exogenously added gangliosides can be internalised and subsequently delivered to Golgi [START_REF] Tettamanti | Ganglioside/glycosphingolipid turnover: new concepts[END_REF], they may be excluded from the "TERM assembly line". Alternatively, exogenous gangliosides may be incorporated into microdomains that are distinct from those containing the endogenous molecules. In this regard, it has been recently demonstrated that added GM1 accumulated mainly in non-DRM compartments whereas the endogenous ganglioside was completely resistant to detergent extraction [START_REF] Crespo | Effect of gangliosides on the distribution of a glycosylphosphatidylinositol-anchored protein in plasma membrane from Chinese hamster ovary-K1 cells[END_REF].

Our data indicate that there is a specific link between ganglioside GD1a and tetraspanin CD82. Indeed, specific depletion of this ganglioside with Neu3 sialidase reveals that GD1a has both positive (association of CD82 with CD151) and negative (interaction between CD82 and EGFR) role in maintaining CD82-enriched microdomains. On the other hand, it is unlikely, that GM3, which is known to facilitate associations of CD9 with integrins [17;18], is involved in regulation of CD82-dependent interactions. Firstly, GM3 could not be detected in HB2 cells (perhaps, because it is quickly converted to GM2 and, subsequently, to GM1). Thus, the effect of Neu3 on the stability of CD82-enriched microdomains in HB2 cells is not mediated by this ganglioside. Secondly, our experiments with GM95 cells (Fig. 5) also excluded the involvement of GM3 in the maintenance of CD82-enriched microdomains. The individual contribution of two other main gangliosides of Aseries, GM2 and GM1 is currently less clear. The fact that the association between CD82 and EGFR is potentiated in the GD1a-depleted cells, but weakened in cells with decreased levels of all gangliosides suggest that GM1 and/or GM2 may stabilise some of the interactions within CD82-enriched microdomains. Further experiments will be necessary to address this issue.

How does GD1a affect the interactions of CD82 with other TERM-associated proteins? Hakomori and collaborators proposed a model in which gangliosides directly bind to a carbohydrate moiety in various tetraspanins and facilitate their interactions with other glycosylated proteins in tetraspanin-containing microdomains [START_REF] Hakomori | Glycosphingolipid-dependent cross-talk between glycosynapses interfacing tumor cells with their host cells: essential basis to define tumor malignancy[END_REF]. Indeed, stability of the complexes involving CD82 was dependent on glycosylation of the protein [18;19]. Hence, one could envisage that binding of GD1a (but not GM3) to the large extracellular loop of CD82 (which has three N-linked glycosylation sites) either strengthens (e.g. CD151) or diminishes (e.g. EGFR) its interactions with other glycosylated proteins. Alternatively, it is possible that the effect of GD1a on the interactions involving CD82 is not dependent on glycosylation of the protein. In this regard, we found that NB-DGJ still had a pronounced effect on the associations of glycosylation-deficient mutant of CD82 (our unpublished data).

Finally, instead of (or in addition to) providing a direct physical link between the protein components GD1a may have a more general role in the spatial organisation of the CD82-enriched microdomains. This could be achieved via the GD1a-dependent recruitment of specific lipid or protein components that help stabilising interactions involving CD82.

The ectopic expression of the membrane-bound sialidase Neu3 specifically affects the interactions involving CD82. Thus, our results provide a first example of a physiological regulator of tetraspanin-tetraspanin interactions. Importantly, we found that Neu3 is not directly associated with TERM in transiently transfected epithelial cells (Fig. 3A). Furthermore, recent data show that the enzyme is localised in the Triton X-100-resistant microdomains (i.e. "classical" GEM) where it interacts with caveolin [41;42]. Then how can one explain the effect of Neu3 on the interactions of the protein, which is excluded from the classical lipid rafts? One possibility is that Neu3, which is abundant at the plasma membrane, targets GD1a, and, consequently, the CD82-containing complexes "in trans" (i.e. on the surface of opposing cells) [START_REF] Papini | The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-tocell interactions[END_REF].

Alternatively, Neu3 may become involved during the assembly of the complexes while passing through Golgi en route to the plasma membrane.

We have previously shown that the CD82-dependent attenuation of signalling via EGF receptor is correlated with the increased level of GD1a in the CD82 expressing cells [START_REF] Odintsova | Tetraspanin CD82 regulates compartmentalisation and ligand-induced dimerization of EGFR[END_REF]. Our current data indicate that the effect of CD82 on signalling via EGFR is even more complex than thought previously. Firstly, we established that only in CD82-expressing cells (HB2/CD82) depletion of gangliosides led to the attenuation of EGFR phosphorylation (Fig. 7). On the other hand, inhibition of ganglioside biosynthesis caused re-distribution of the proteins and weakened interactions of CD82 with the receptor (Fig. 8A and Fig. 1C). While the exact mechanisms remain to be established one possibility is that alterations in glycosphingolipid composition of the membranes triggers activation of the protein(s) that regulate dephosphorylation of EGFR (e.g. tyrosine phosphatase(s)). We propose that this(-ese) protein(s) can be recruited to the membrane by CD82. While presence of gangliosides in the CD82enriched microdomains would prevent activation of this pathway, ganglioside depletion and subsequent re-distribution of the proteins lead to its activation.

Accordingly, in CD82-negative cells (HB2/ZEO) this pathway would be inactive either in the presence or absence of gangliosides because lack of CD82 precludes the recruitment of the regulatory proteins to the membrane.

It has been previously documented that CD9 is directly associated with GM3 [START_REF] Kawakami | Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility[END_REF],

and depletion of GM3 in fibroblasts resulted in a minor increase in the association of CD9 with integrins [START_REF] Toledo | Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor[END_REF]. Thus, various gangliosides may have specific (and opposing) functions in stabilization of TERM. Notably, the pronounced effect of GD1a on the stability of CD82-containing complexes was specific: interactions involving CD9 were not regulated by GD1a. In this regard, we have noticed that HB2 cells do not accumulate GM3 (assayed in TLC and flow cytometry experiments), thereby suggesting that this ganglioside is rapidly converted to GM2 (and subsequently to GM1 and GD1a) in Golgi. This may explain why depletion of gangliosides in these cells had no apparent effect on the interaction between CD9 and α3β1 integrin (Fig. 1c).

In summary, we have established that gangliosides are involved in stabilization of a wide range of interactions within CD82-enriched microdomains. Taken together these and some of our previous results [44;45] indicate that multiple interactions within TERM are controlled at various levels and involve distinct mechanisms (e.g. direct protein-protein interactions, palmitoylation of tetraspanins) [44;46]. Our results also identify Neu3 sialidase as a physiological regulator of these interactions. Further examination of the links between tetraspanins and gangliosides may explain the diverse functions associated with tetraspanin microdomains. and control cells were prepared under the detergent-free conditions as described in the "Materials and Methods". Lysates were fractionated by ultracentrifugation in continuous sucrose gradient (5-45%). Fractions were collected from the top of the gradient. Equal volumes of each fraction were resolved in 10 or 12% SDS-PAGE.

Distribution of proteins in the gradient fractions was assessed by Western blotting using specific antibodies: anti-CD82 (mAb TS82b), anti-EGFR (mAb Ab-15), anti-

.

  of ganglioside biosynthesis. For the inhibition of ganglioside biosynthesis cells were incubated with the appropriate concentration of the inhibitors either NB-DGJ or PPMP for 6-8 days. The inhibitor was added to the media upon replating of cells. After the incubation, the cells were used for lipid extraction, immunoprecipitation, flow cytometry analysis, fractionation in sucrose gradient, immunofluorescense staining or for the incubation with EGF. Control cells were grown in parallel without the inhibitor for the water soluble NB-DGJ or in the presence of equivalent amount of ethanol (0.1%) used for the solubilization of PPMP.

( 1

 1 ml of methanol and 2 ml of methanol:PBS, 1:10) Sep-Pak cartridge, which was subsequently washed with chloroform-methanol-water (2:43:55, v/v) and methanolwater (1:1, v/v) prior to elution of gangliosides in methanol. The gangliosidecontaining fraction was vacuum-dried and resuspended in a small volume of chloroform-methanol (2:1, v/v) for subsequent TLC analysis. Thin layer chromatography was performed on HPTLC plates (Merck, Darmstadt, Germany) using a solvent system of chloroform:methanol: 0.22% CaCl 2 (60:40:5). The gangliosides were visualised with resorcinol reagent. The standard mixture of gangliosides was purchased from Matreya Inc. (PA, USA).Immunofluorescence staining. Cells were grown on glass coverslips in complete media for 24-36 hours. When required the cells grown on coverslips were transiently transfected with Neu3 sialidase cDNA. Spread cells were fixed with 2% paraformaldehyde/PBS for 10 minutes. When required the cells were permeabilized with 0.4% saponin/PBS. Staining with primary and fluorochrome-conjugated secondary antibodies was carried out as previously described[START_REF] Berditchevski | Characterization of integrin/tetraspanin adhesion complexes: role of tetraspanins in integrin signaling[END_REF]. Staining with FITC-conjugated cholera toxin B subunit (CT) was carried out on life cells at 4 0 C for 1 hour. Staining was analysed using Nikon Eclipse E600 microscope. Images were acquired using Nikon digital camera DXM1200 and subsequently processed using a Nikon ACT-1 image processing programme. Biochemical Journal Immediate Publication. Published on 24 Jul 2006 as manuscript BJ20060259ResultsInhibition of glycosphingolipid biosynthetic pathway weakens protein-protein interactions within tetraspanin-enriched microdomains.To elucidate the relationship between gangliosides and tetraspanin-enriched microdomains we used two inhibitors of glucosylceramide synthase (a key enzyme in the glycosphingolipid biosynthetic pathway[START_REF] Andersson | Nbutyldeoxygalactonojirimycin: a more selective inhibitor of glycosphingolipid biosynthesis than N-butyldeoxynojirimycin, in vitro and in vivo[END_REF]), N-butyldeoxygalactonojirimycin (NB-DGJ) and D-threo-1-Phenyl-2-hexadecanoylamino-3-morpholino-1-propanol•HCl (PPMP). In the preliminary experiments we established that after 6 days of incubation with 2mM of NB-DGJ the level of gangliosides in HB2/CD82 cells was beyond detection by TLC (Fig.1A, lane d). Notably, TLC analysis also demonstrated the absence of GM3 in HB2 cells (Fig.1A, lane b). Flow cytometry analysis indicated that in NB-DGJtreated cells the surface expression of gangliosides has been reduced to 4% of GD1a and 7% of GM1 (Fig.1B) in comparison to control cells. The cells were incubated for 6-8 days with 2 mM of NB-DGJ in all subsequent experiments. Similarly, we found that using PPMP at 20 µM for 7 days dramatically decreased the concentration of gangliosides. The efficacy of inhibition of ganglioside biosynthesis in each

  on the cell surface caused by Neu3 sialidase expression had an impact on the stability of CD82-containing complexes. Inhibition of ganglioside biosynthesis does not allow evaluation of the contribution of individual gangliosides to the assembly of TERM. To answer the question what ganglioside(s) is(are) important for the organisation of these domains we applied other methods for the manipulation of ganglioside composition in cells.

  Neu3 transfectants and the control cells were examined in the immunoprecipitation experiments with the anti-CD82, anti-CD9 and anti-HA-tag mAbs. Control cells and Neu3 transfectants expressed comparable levels of tetraspanins CD82, CD9 and CD151 (Fig.3A, lanes 9 and 10). Notably, anti-CD82 mAb co-immunoprecipitated significantly less of CD151 from the cells transfected with Neu3 than from the control cells (~ 50% decrease, Fig.3A, second panel from the top, lanes 1 and 5); the amounts of immunoprecipitated CD82 in both samples were comparable (Fig.3A, third panel from the top, lanes 1 and 5). In contrast, we observed that the expression of Neu3 increased the association of CD82 with EGFR (~ 40% increase over the control sample, Fig.3B, lanes 1 and 4). These data provide additional evidence that microdomains containing CD82 are sensitive to the modulation of ganglioside composition at the cell surface. Importantly, interactions of CD9 with other tetraspanins and EGFR were not affected by the expression of Neu3 sialidase (Fig.3A, lanes 3 and 7, Fig3B, lanes 2 and 5). In additional experiments we found that ectopic expression of Neu3 had no effect on the ability of CD82 to bind SNA lectin (Fig.3C). These data indicated that Neu3 is inactive towards sialic acid residues on CD82 and exclude a possibility that the effect of the sialidase on the interactions involving CD82 was due to changes in glycosylation of the protein.To assess the effect of the Neu3 overexpression on tetraspanin-tetraspanin associations in the absence of CD82 we examined the interactions within TERM in HB2 cells, which have negligible amounts of CD82. As expected, transfection of Neu3 sialidase into HB2 cells decreased surface expression of ganglioside GD1a present in these cells (Fig.4A) indicating that the enzyme was active. We used one of the clones transfected with Neu3 and one of the control clones transfected with the vector in further experiments. The anti-CD151 and anti-CD9 mAbs coimmunoprecipitated comparable amounts of CD9, CD151 and α3 integrin subunit from the lysates (Fig.4B, lanes 1 and 6, and 3 and 8). In these experiments we also examined interactions of the tetraspanin CD81 (Fig.4B, lanes 2 and 7). There were no apparent differences in the immunocomplexes co-immunoprecipitated by the anti-CD81 antibody from Neu3-transfected and control cells. Taken together these data led us to the conclusion that in mammary epithelial cells only CD82-based interactions are sensitive to the modulations in ganglioside composition at the membrane. Furthermore, in the absence of CD82, changes in the concentration of ganglioside GD1a had no effect on the tetraspanin-tetraspanin interactions.

CD82-expressing cells.

  Fig.7B, compare lanes 2 and 5, and lanes 3 and 6). On the other hand, depletion of

  Figure 8C, CD82-CD151 and CD82-CD9 complexes can be detected in these

Figure 1 .

 1 Figure 1. Effect of ganglioside depletion on the interactions in tetraspanin-

Figure 2 .

 2 Figure 2. Immunofluorescence staining of HB2/CD82 cells transiently

Figure 3 .

 3 Figure 3. Expression of Neu3 sialidase affects the interactions involving CD82

Figure 4 .

 4 Figure 4. Expression of Neu3 sialidase does not affect interactions of

Figure 5 .

 5 Figure 5. Ganglioside GM3 has no affect on the interactions of CD82.

Figure 6 .

 6 Figure 6. Exogenous administration of gangliosides does not affect stability of

Figure 7 .

 7 Figure 7. Inhibition of ganglioside biosynthesis has specific effect on the EGFR

Figure 8 .

 8 Figure 8. Effect of inhibition of ganglioside biosynthesis on the

Biochemical Journal Immediate Publication. Published on 24 Jul 2006 as manuscript BJ20060259 Copyright 2006 Biochemical Society

	Immunoprecipitation and Western blotting. Proteins were solubilised into the homogeniser (15 strokes) and a sonicator (4x20 sec bursts, 1 min interval, power 10).
	immunoprecipitation The resulting homogenate was mixed with equal volume of 90% sucrose prepared in buffer containing 1% Brij 98/PBS, 2 mM
	phenylmethylsulphonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin for 2 hours 25 mM MES buffer (pH 6.5), and overlaid with 2 volumes of 35% sucrose and
	at 4 0 C. The insoluble material was pelleted at 7000 g for 10 minutes. The cell lysates followed by 1 volume of 5% sucrose (both in MES). Samples were centrifuged at 55
	were then precleared by incubation for 2 hours at 4 0 C with agarose beads conjugated 000 g for 16-18 hours at 4 0 C. 400 µl aliquots were collected from the top of the tube.
	with goat anti-mouse antibodies (mIgG-beads; Sigma). Immune complexes were The pellet has been dissolved in Laemmli buffer supplemented with protease
	collected using appropriate mAbs prebound to mIgG-beads and washed 4 times with inhibitors. Equal amounts of each fraction were mixed with 4X Laemmli loading
	the immunoprecipitation buffer. In some experiments we incubated an aliquot of the buffer and the distribution of proteins was analysed by SDS-PAGE followed by
	pre-cleared lysate with biotinylated Sambucus Negra (Elderberry) Bark Lectin, SNA Western blotting. Protein concentration has been measured by BioRad kit.
	(Vector Laboratories), that was bound to the immobilized Neutravidin (Pierce). The Cholesterol depletion. To remove cholesterol cells were washed three times in PBS
	complexes were eluted from the beads with Laemmli sample buffer. Proteins were and incubated in serum-free medium containing 20 mM methyl-β-cyclodextrin
	resolved in 10-12% SDS-PAGE, transferred to a nitrocellulose membrane and (MβCD) for 1 hour at 37 0 C. Levels of cholesterol in MβCD-treated cells were
	developed with the appropriate Ab. Protein bands were visualised using horseradish determined using Amplex Red Cholesterol Assay Kit (Molecular Probes). After
	peroxidase-conjugated goat anti-mouse (or goat anti-rabbit) antibodies (Daco) and MβCD treatment the cells were used for the fractionation in sucrose gradient as
	chemiluminescence reagent (PerkinElmer). described above.
	Flow cytometry. Cells were detached with EDTA solution, washed in PBS, diluted
	in 1% heat-inactivated BSA/PBS, and incubated with saturating concentrations of
	primary mouse mAbs for 1 hour on ice. After the incubation, the cells were washed
	twice and then labelled with fluorescein isothiocyanate (FITC)-conjugated goat anti-
	mouse immunoglobulin (Sigma). Stained cells were analysed using Coulter Epics
	programme (Becton Dickinson).
	Fractionation in sucrose gradient. Cells were scraped into 2 ml of PBS, centrifuged
	at 1200 rpm for 3 minutes, washed once with PBS. 100 mM sodium carbonate (pH
	11.0) buffer supplemented with 10 µg/ml aprotinin, 10 µg/ml leupeptin and 2 mM
	PMSF, was mixed with the pelleted cells. Lysates were homogenized using Dounce

Acknowledgements. We are grateful to Drs. O.Yoshie, E.Rubinstein, R.Schnaar and H.Conjeaud for providing mAbs and human cDNA. E.O. wishes to thank Dr T.Pettitt for help and advise. T.D.B. thanks the Glycobiology Endowment for support. Work was supported by a grant from Breast Cancer Campaign (to F.B. and E.O.). Biochemical Journal Immediate Publication. Published on 24 Jul 2006 as manuscript BJ20060259 Copyright 2006 Biochemical Society

α3 integrin subunit (polyclonal sera), anti-CD151 (polyclonal sera), anti-CD9 (mAb C9BB). Shown results of one of the two separate experiments. c -control cells; iinhibitor-treated cells. Note, distribution of proteins in the gradients obtained from the control cells in the experiments shown in A and B are slightly different. This may be due to the differences in the culturing conditions of cells prior to lysis.

Cholesterol depletion was carried out under serum-free conditions and, consequently, the control cells were kept under serum-free conditions (panel B). In the experiments depicted in panel A cells were grown in the serum-containing medium. C. Light fractions (2-5) of sucrose gradients obtained from the control, untreated cells and cells depleted of gangliosides were pooled together, and the CD82-containing complexes were immunoprecipitated using mAb γC11.

Immunoprecipitated material was resolved in 12% SDS-PAGE, transferred to a nitrocellulose membrane and probed with the anti-CD82 (TS82b) mAb, anti-CD9 (C9BB) mAb and anti-CD151 polyclonal Ab.