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Biomedical subjects

M Cantz

Publications and source records attributed to M Cantz.

At least 37 records · Page 2Linked to original sources
PubMed1996

Methylamine accumulation in cultured cells as a measure of the aqueous storage compartment in the laboratory diagnosis of genetic lysosomal diseases.

Intracellular accumulation of the lysosomotropic compound [14C]methylamine was used to estimate the size of the lysosomal compartment in fibroblasts cultured from patients with a variety of lysosomal storage diseases. In previous work from our laboratory, it was shown that methylamine accumulation was significantly increased in diseases with infantile or juvenile onset and storage of predominantly water-soluble material such as in the mucopolysaccharidoses, mucolipidoses, and oligosaccharidoses. In the present study, methylamine incorporation was abnormally increased in cells from patients with glycogenosis type II and with Niemann-Pick type C disease, whereas it was normal in other sphingolipidoses and in the late-infantile and juvenile forms of neuronal ceroid lipofuscinoses. The methylamine test was also checked regarding its potential use for prenatal diagnostic testing. In model systems with cultured amniotic or chorionic villus cells, lysosomal storage was experimentally induced by the cathepsin inhibitor leupeptin and was readily detected when compared to untreated controls. Cultured amniotic cells from a fetus with mucopolysaccharidosis II were found to incorporate significantly higher amounts of [14C]methylamine than the normal controls. The results indicate that the methylamine accumulation method is an additional tool in the diagnosis and prenatal diagnosis of lysosomal diseases with abnormal storage of water-soluble material.

Amnion↗
PubMed1996

Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells.

Gangliosides of the plasma membrane are important modulators of cellular functions. Previous work from our laboratory had suggested that a plasma membrane sialidase was involved in growth control and differentiation in cultured human neuroblastoma cells (SK-N-MC), but its substrates had remained obscure. We now performed sialidase specificity studies in subcellular fractions and found ganglioside GM3 desialylating activity in presence of Triton X-100 to be associated with the plasma membrane, but absent in lysosomes. This Triton-activated plasma membrane enzyme desialylated also gangliosides GD1a, GD1b, and GT1b, thereby forming GM1; cleavage of GM1 and GM2, however, was not observed. Sialidase activity towards the glycoprotein fetuin with modified C-7 sialic acids and towards 4-methylumbelliferyl neuraminate was solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in gangliosides desialylation of living cells was examined by following the fate of [3H]galactose-labelled individual gangliosides in pulse-chase experiments in absence and presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. When the plasma membrane sialidase was inhibited, radioactivity of all gangliosides chased at the same rate. In the absence of inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degraded at a considerably faster rate in confluent cultures, whereas the GM1-pool seemed to be filled by the desialylation of higher gangliosides. The results thus suggest that the plasma membrane sialidase causes selective ganglioside desialylation, and that such surface glycolipid modification triggers growth control and differentiation in human neuroblastoma cells.

Carbohydrate Sequence↗
PubMed1994

Role of plasma membrane ganglioside sialidase of human neuroblastoma cells in growth control and differentiation.

In cultured human neuroblastoma cells (SK-N-MC), a plasma membrane-bound besides a lysosomal ganglioside GM3 sialidase was detected. Both activities can be distinguished by the specific activation with detergents, as well as differential inhibition by Cu++. Plasma membrane and lysosomal sialidase specific activities showed strikingly different behaviour during the growth phase of neuroblastoma cells. Thus, the plasma membrane sialidase increased about 15-fold and mirrored cell growth, it differed from the kinetics of ornithine decarboxylase, an early marker of cell proliferation. The lysosomal sialidase, on the other hand, exhibited constant specific activities during growth of the cells, as did lysosomal and plasma membrane marker enzymes. When the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was included in the culture medium, a profound change in proliferation kinetics was observed, indicating a release from density-dependent control of cell division. Additionally, the inhibitor abolished the increase of the biochemical differentiation marker acetylcholinesterase. The results suggest an important role of the ganglioside sialidase of the plasma membrane in the processes of proliferation control and differentiation in this neuronal cell system.

Acetylcholinesterase↗
PubMed1994

[14C]Methylamine accumulation in cultured human skin fibroblasts--a biochemical test for lysosomal storage and lysosomal diseases.

Incorporation of the lysosomotropic amine [14C]methylamine by fibroblasts cultured from patients with lysosomal storage diseases and from controls was used to estimate the size of the lysosomal compartment. All cell lines from patients with infantile and juvenile forms of mucopolysaccharidoses, mucolipidoses and oligosacharidoses showed markedly increased radioactivity compared with the normal range of controls. In cells from patients with sphingolipidoses and adult forms of storage diseases, however, methylamine accumulation was not significantly increased. Experimentally induced lysosomal storage by enzyme inhibitors (leupeptin, suramin) also caused increased methylamine accumulation. When the lysosomal pH was determined with fluorescein isothiocyanate-dextran, it was in the range of normal controls (pH 4.7-5.0) in patients cells. Thus, [14C]methylamine accumulation should depend on the volume rather than differences in acidity of the lysosomal compartment and be a measure of its eventual pathological enlargement. We conclude that the determination of [14C]methylamine accumulation in fibroblasts provides a valuable tool in the screening for a variety of lysosomal storage disorders.

Cells, Cultured↗
PubMed1994

Further evidence that human lysosomal sialidase is not derived from prosaposin. Prosaposin biosynthesis and ganglioside sialidase studies in prosaposin- and sialidase-deficient fibroblast lines.

Human lysosomal sialidase has been considered by Potier et al. (Potier M., Lamontagne S., Michaud L., Tranchemontagne J. (1990) Biochem Biophys Res Commun 173, 449-456) to be a processing product of prosaposin, the common precursor of the saposin proteins A, B, C, and D that function as activators in the lysosomal degradation of sphingolipids. We tested this hypothesis on cultured fibroblasts of patients with prosaposin deficiency, a neurolipidosis caused by a complete lack of synthesis of the prosaposin protein, by determining their lysosomal and, for comparison, their plasma membrane sialidase activities. Using both the natural substrate ganglioside GM3 and the synthetic compound 4-methylumbelliferyl neuraminate, we found the lysosomal sialidase activity in the prosaposin-deficient cells to be in the normal control range; normal values were also found for the plasma membrane sialidase. In fibroblasts from patients with a genetic deficiency of the lysosomal sialidase (sialidosis), on the other hand, biosynthesis and processing of prosaposin were unimpaired. Our findings therefore show no precursor/product relationship between prosaposin and the lysosomal or the plasma membrane sialidase.

Cell Line↗
PubMed1993

Protein catabolism in fibroblasts cultured from patients with mucolipidosis II and other lysosomal disorders.

Protein catabolism in fibroblasts cultured from the skin of normal individuals and of patients with mucolipidosis II (I-cell disease) and several other lysosomal storage diseases was examined by metabolic labelling with [3H]leucine and following the fate of radioactive proteins in pulse-chase experiments. In mucolipidosis II cells, overall protein degradative rates were found to be distinctly lower than in normal control cells. To distinguish lysosomal from non-lysosomal degradation, labelling experiments were carried out in the presence and absence of 10 mM NH4Cl, an inhibitor of lysosomal function. It was found that mucolipidosis II fibroblasts exhibited a markedly reduced rate of lysosomal protein degradation, whereas the rate of nonlysosomal degradation appeared normal. Serum and amino acid starvation led to a marked increase in lysosomal protein degradation in normal cells, but had only a minimal effect on that in mucolipidosis II fibroblasts. The specific activities of cathepsins B, H and L were profoundly diminished in all mucolipidosis II cell lines tested. Lysosomal protein degradation in a mucolipidosis III cell line was impaired to a similar degree as in mucolipidosis II cells, whereas it was decreased to a lesser extent in fibroblasts from patients with mucopolysaccharidoses I and VI, galactosialidosis and GM1-gangliosidosis. We conclude that fibroblasts from patients with mucolipidosis II and III have a severely compromised capacity for endogenous lysosomal protein degradation that appears to result from multiple cathepsin deficiency. This lysosomal defect is likely to have pathophysiological consequences.

Cathepsin B↗
PubMed1991

Lysosomal and plasma membrane ganglioside GM3 sialidases of cultured human fibroblasts. Differentiation by detergents and inhibitors.

Cultured human fibroblasts contain two sialidases that degrade gangliosides such as GM3: a lysosomal activity that appears identical with the activity towards water-soluble substrates and that is deficient in the genetic lysosomal disorder sialidosis, and another enzyme that seems localized on the external surface of the plasma membrane. In this report we show that both enzymes can be differentiated in the presence of each other by choice of the detergent used for activation, and also by the inhibitory action of some polyanionic compounds such as sulphated glycosaminoglycans. The lysosomal ganglioside GM3 sialidase is greatly stimulated by sodium glycodeoxycholate and, to lesser degrees, by sodium glycocholate and sodium cholate. The ganglioside GM3 sialidase of the plasma membrane is not measurably active under the conditions of the lysosomal enzyme but is specifically activated by the non-ionic detergent Triton X-100. The glycodeoxycholate-stimulated, but not the Triton-activated, ganglioside GM3 sialidase activity was profoundly diminished in cell lines from patients with the lysosomal disorders sialidosis and galactosialidosis; however, both activities were normal in fibroblasts from patients with mucolipidosis IV, previously thought to be a ganglioside sialidase deficiency disorder. Both the lysosomal and the plasma membrane ganglioside GM3 sialidases were inhibited by sialic acids, suramin, dextran sulphate and sulphated glycosaminoglycans. Among the latter, heparin and heparan sulphate showed a much higher inhibitory potency towards the plasma membrane ganglioside GM3 sialidase than towards the lysosomal onw.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Metabolism, Inborn Errors↗
PubMed1990

Disorders of glycoprotein degradation.

The intracellular degradation of glycoproteins occurs predominantly in the lysosomes through the concerted action of proteases and glycosidases. Genetic defects in any of the enzymes cleaving the oligosaccharide side chains lead to specific diseases because of an excessive lysosomal accumulation of partially degraded material, mostly oligosaccharides. This paper presents an overview of the biochemistry and the clinical spectrum of this group of diseases including sialidosis, galactosialidosis, alpha- and beta-mannosidosis, fucosidosis, aspartylglucosaminuria, and alpha-N-acetylgalactosaminidase deficiency (Schindler disease). In addition, the sialic acid storage disorder (Salla disease) which is caused by a defect in the lysosomal transport of this acidic monosaccharide is included because of functional and clinical correlations.

Carbohydrate Conformation↗
PubMed1989

Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and and some properties.

Sensitive assays for the determination of the ganglioside sialidase activity of fibroblast homogenates were established using ganglioside GM3, 3H-labelled in the sphingosine moiety, as a substrate. Ganglioside GM3 sialidase activity was greatly stimulated by the presence of the non-ionic detergent Triton X-100 and was further enhanced by salts such as NaCl; the optimal pH was 4.5. The subcellular localization of this activity was determined by fractionation using free-flow electrophoresis and found to be exclusively associated with the marker for the plasma membrane, but not with that for lysosomes. This Triton-stimulated ganglioside sialidase activity was selectively inhibited by preincubating intact cells in the presence of millimolar concentrations of Cu2+, suggesting that the activity resides on the external surface of the plasma membrane. In normal fibroblasts homogenates, ganglioside GM3 sialidase was also greatly stimulated by sodium cholate. In contrast to the Triton X-100-activated reaction, however, it was not diminished by prior incubation of intact cells in the presence of Cu2+. Only after cell lysis was Cu2+ inhibitory. the cholate-stimulated ganglioside sialidase activity thus paralleled the behaviour of the lysosomal 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4-MU-NeuAc) sialidase. In fibroblasts from sialidosis patients, the cholate-stimulated ganglioside GM3 sialidase activity, but not that of the Triton-activated enzyme, was profoundly diminished. In fibroblasts from patients with mucolipidosis IV (ML IV), both the Triton X-100- and the cholate-stimulated ganglioside GM3 sialidase activities were in the range of normal controls. The Triton-activated enzyme was associated with the plasma membrane in the same manner as in normal cells. Our findings suggest that, in human fibroblasts, there exist two sialidases that degrade ganglioside GM3: one on the external surface of the plasma membrane, and another that is localized in lysosomes and seems identical with the activity that acts on sialyloligosaccharides and 4-MU-NeuAc. As neither activity was found to be deficient in ML IV fibroblasts, our results argue against the hypothesis of a primary involvement of a ganglioside GM3 sialidase in the pathogenesis of ML IV.

Cell Membrane↗
PubMed1988

Defective lysosomal release of glycoprotein-derived sialic acid in fibroblasts from patients with sialic acid storage disease.

Fibroblasts from patients with sialic acid storage disease (SASD), sialidosis, mucolipidosis II, and from normal controls, were incubated in the presence of the glycoprotein fetuin that was tritium-labelled in its sialic acid residues by the periodate/[3H]borohydride reduction method, and the fate of the intracellular radioactive sialic acid (C7-sialic acid) followed in pulse-chase experiments. The model glycoprotein was readily endocytosed and degraded, more than 90% of the radioactivity being trichloroacetic acid (TCA)-soluble after 4 days of incubation. In all of the patients' fibroblasts, there was an increased accumulation of TCA-soluble radioactivity and, upon chase, a much lower rate of elimination than in normal controls. Gel chromatography of the material from the chase experiment showed that, in normal cells, most of the radioactivity at zero time behaved as free C7-sialic acid. This, as well as material of larger size (sialyloligosaccharides), was very much diminished by 48 h. In cells from two patients with SASD, there were large peaks both in the sialic acid and oligosaccharide positions; whereas the oligosaccharides were somewhat decreased by the end of the chase period, the sialic acid was essentially unchanged. In sialidosis fibroblasts, the radioactive material consisted of oligosaccharides, but very little C7-sialic acid; the elimination of the oligosaccharides was retarded. In normal cells, about 80% of the radioactivity released into the medium after 48 h chase behaved as free C7-sialic acid upon gel chromatography and t.l.c. Subcellular fractionation in Percoll gradients showed that the radioactive C7-sialic acid remaining in normal cells after 48 h of chase was mainly localized in the cytosol. In SASD cells, on the other hand, it was associated with lysosomal fractions which, unexpectedly, exhibited an abnormally low density. Our findings demonstrate that SASD fibroblasts degrade the sialoglycoprotein but, unlike normal cells, accumulate the liberated C7-sialic acid along with sialyloligosaccharides in their lysosomes. The results therefore support the concept of a defective transport system for sialic acid in the lysosomal membrane of patients with SASD.

Biological Transport↗
PubMed1987

Lysosomal sialidase deficiency: increased ganglioside content in autopsy tissues of a sialidosis patient.

Organs obtained at autopsy from a patient with sialidosis were analyzed for 'bound' sialic acid and their ganglioside and neutral glycolipid patterns determined. The water-soluble bound sialic acid was increased between 10- and 17-fold in visceral organs, but only about 2-fold in the brain, when compared to normal controls. Lipid-bound sialic acid was increased up to 8-fold in visceral organs due to elevated amounts of gangliosides GM3, GD3 and probably GM4 and LM1, whereas the brain showed no deviation from controls. An alteration of the neutral glycolipid pattern was also observed. The results indicate an impaired catabolism of gangliosides in sialidosis in addition to that of sialyloligosaccharides and sialoglycoproteins.

Adult↗
PubMed1986

Normomorphic sialidosis in two female adults with severe neurologic disease and without sialyl oligosacchariduria.

Two female patients of German origin, aged 38 and 21 years, with myoclonus epilepsy and cerebellar ataxia, but without dysmorphic signs and dementia, were found to excrete normal amounts of sialyl oligosaccharides in their urine. The younger patient showed cherry red spots in her ocular fundi. The older patient had a brother with an autopsy-proven neuronal storage disease compatible with sialidosis, and in her rectal biopsy lamellar inclusion bodies were detected. Enzyme assays in cultured fibroblasts of both patients revealed a profound but incomplete deficiency of oligosaccharide sialidase activity and normal beta-galactosidase activity. Adult sialidosis was diagnosed in both patients. In their fibroblasts, moderate elevations of bound sialic acid could also be measured. The small residual sialidase activity, which in the older patient had a normal KM value, is considered responsible for the late onset and slow clinical course of the disease. It is concluded that in adult sialidosis the extraneural storage process can be difficult to demonstrate in terms of metabolite accumulation or excretion during the course of intraneuronal storage.

Adult↗
PubMed1985

N-Acetylneuraminic acid storage disease.

Increased amounts of free sialic acid were found in body fluids, leukocytes, cultured fibroblasts, and liver tissue of a four-year-old boy with mental retardation, ataxia, and clinical and radiologic findings of a mild mucopolysaccharidosis. A diagnosis of Salla disease was made though in contrast to earlier reports, recurrent upper respiratory infections and hepatosplenomegaly were present already in infancy, and skeletal abnormalities of dysostosis multiplex were found in early childhood. Free sialic acid in the urine was identified as N-acetylneuraminic acid by 1H-NMR spectroscopy. Sialidase activities were normal. Increased amounts of bound sialic acid were found in liver and cultured fibroblasts and were attributed to an intracellular inhibition of sialyloligosaccharide-degrading neuraminidase by excessive amounts of free neuraminic acid. The molecular basis of N-acetylneuraminic acid storage disease is unknown but may be related to a defective transport mechanism preventing neuraminic acid from leaving the lysosomal compartment.

Cells, Cultured↗
PubMed1985

Familial lysosomal storage disease with generalized vacuolization and sialic aciduria. Sporadic Salla disease.

Two eight- and sixteen-year-old children with severe progressive neurologic disease revealed an ultrastructural finding of lysosomal vacuolization in mesenchymal or parenchymal cells of different organ biopsies (skin, muscle, nerve and liver), which may be very suggestive of mucolipidosis. However, in our patients biochemical tests available for these diseases yielded negative results, except for increased excretion of free sialic acid in urine and sialic acid storage in cultured fibroblasts. The clinical picture and the ultrastructural and biochemical findings were compatible with Salla disease, a rare lysosomal storage disease originally observed in Finland.

Adolescent↗
PubMed1984

Specificity studies on the oligosaccharide neuraminidase of human fibroblasts.

Competition and thermal inactivation experiments with different potential natural substrates indicated that in homogenates of human fibroblasts one single enzyme is acting on both (alpha 2-3) and (alpha 2-6) sialosyl linkages of oligosaccharides and glycoproteins, but not of the ganglioside GM3. N-Acetylneuraminic and 2-deoxy-2,3-dehydro-N-acetylneuraminic acids are competitive inhibitors, whereas chondroitin 4-sulphate and the drug Suramin are potent inhibitors of undefined type.

Chondroitin Sulfates↗
PubMed1984

Pattern of collagen synthesis and chemotactic response of fibroblasts derived from mucopolysaccharidosis patients.

For comparative studies on the migratory potential we screened fibroblast strains derived from mucopolysaccharidosis (MPS) patients regarding their differential response to chemotactic stimuli and analysed their production of extracellular matrix components. Indirect immunofluorescence staining of MPS-fibroblasts showed the same distribution of type I and type III collagen and of fibronectin as in controls. Biochemical quantification of type I and type III collagen demonstrated an unaltered ratio of these collagen types, although the total amount of newly synthesized collagens was slightly reduced in fibroblasts from MPS patients. Whereas the synthesis of major extracellular matrix components was close to normal, the response of the MPS cells to chemotactic stimuli was greatly affected. Chemotactic migration was improved when fibroblasts were pretreated with medium conditioned by normal fibroblasts, although they never reached normal levels.

Cells, Cultured↗