Biomedical subjects

Volkmar Gieselmann

Publications and source records attributed to Volkmar Gieselmann.

At least 19 recordsLinked to original sources

PubMed2005

Oligodendrocyte-specific ceramide galactosyltransferase (CGT) expression phenotypically rescues CGT-deficient mice and demonstrates that CGT activity does not limit brain galactosylceramide level.

Galactosylceramide (GalC) is the major sphingolipid of the myelin membrane. Mice lacking GalC due to ceramide galactosyltransferase (CGT) deficiency form unstable and functionally affected myelin and exhibit a progressive demyelination, accompanied by severe motor coordination deficits. In addition to oligodendrocytes, CGT is also expressed in other cells, e.g., neurons and astrocytes. We examined the possibility that lack of CGT in these cells contributes to the phenotype of CGT-deficient mice. Toward this aim, we generated transgenic mice expressing CGT under the control of oligodendrocyte-specific proteolipid protein (PLP) promoter and examined the possibility of a transgenic rescue of CGT-deficient mice. CGT-deficient mice expressing the PLP-CGT transgene did not show any behavioral abnormalities, normal myelin structure, and MBP levels. CGT activity as well as GalC and sulfatide levels of rescued mice were not significantly different from wild-type controls. Thus, transgenic rescue with the PLP-CGT transgene was apparently complete. In contrast to wild-type and rescued mice, PLP-CGT transgenic mice on a wild-type background exhibited significantly elevated CGT activity which directly correlated with an increase in non-hydroxy fatty acid (NFA)-GalC, but not alpha-hydroxy fatty acid (HFA)-GalC. HFA-GalC decreased in adult transgenic mice, indicating that NFA-GalC, but not HFA-GalC levels are limited by CGT activity. As a consequence, the total amount of GalC is unchanged over a rather wide range of CGT expression levels in the mouse brain. Our results indicate that loss of CGT in oligodendrocytes is exclusively responsible for the myelin structural deficits, demyelination, and behavioral abnormalities in CGT-deficient mice.

Animals↗

PubMed2005

A fast and robust quantitative time-lapse assay for cell migration.

We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.

Animals↗

PubMed2005

Collapsin response mediator protein-4 regulates F-actin bundling.

Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.

Actins↗

PubMed2005

Mutations c.459+1G>A and p.P426L in the ARSA gene: prevalence in metachromatic leukodystrophy patients from European countries.

In this multicentre study, we examined the prevalence of two mutations in the arylsulfatase A (ARSA) gene, i.e., c.459+1G>A and p.P426L, in 384 unrelated European patients presenting with different types of metachromatic leukodystrophy (MLD). In total, c.459+1G>A was found 194 times among the 768 investigated ARSA alleles (25%), whereas p.P426L was identified 143 times (18.6%). Thus, these two mutations accounted for 43.8% of investigated MLD alleles. Mutation c.459+1G>A was most frequent in late-infantile MLD patients (40%), while p.P426L was most frequent in adults (42.5%), which is consistent with earlier observations, although p.P426L was also found in a few late-infantile patients (0.9%), and c.459+1G>A was present in some adults (9%). Mutation c.459+1G>A is more frequent in countries situated at the western edges of Europe, i.e., in Great Britain and Portugal, and also in Belgium, Switzerland, and Italy, which is visible as a strand ranging from North to South, and additionally in Czech and Slovak Republics. Mutation p.P426L is most prevalent in countries assembled in a cluster containing the Netherlands, Germany, and Austria. In other Central European countries, the frequency of both c.459+1G>A and p.P426L ranges from 8 to 37.5%. Our study has confirmed that c.459+1G>A and p.P426L are the most frequently found MLD-causing mutations in Europe. The data about their prevalence reflect the population variability in Europe.

Adolescent↗

PubMed2005

A mammalian fatty acid hydroxylase responsible for the formation of alpha-hydroxylated galactosylceramide in myelin.

Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the alpha-C atom of the fatty acid moiety. alpha-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of alpha-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)-FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid alpha-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2-3 weeks of age. This is in agreement with the reported time course of fatty acid alpha-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of alpha-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of alpha-hydroxylation modification in myelin, skin and other organs.

Amidohydrolases↗

PubMed2005

Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy.

A deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy, which is characterized by accumulation of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide). Sphingolipid storage results in progressive demyelination and severe neurologic symptoms. The disease is lethal, and curative therapy is not available. To assess the therapeutic potential of enzyme replacement therapy (ERT), ASA knockout mice were treated by intravenous injection of recombinant human ASA. Plasma levels of ASA declined with a half-time of approximately 40 min, and enzyme was detectable in tissues within minutes after injection. The uptake of injected enzyme was high into liver, moderate into peripheral nervous system (PNS) and kidney and very low into brain. The apparent half-life of endocytosed enzyme was approximately 4 days. A single injection led to a time- and dose-dependent decline of the excess sulfatide in PNS and kidney by up to 70%, but no reduction was seen in brain. Four weekly injections with 20 mg/kg body weight not only reduced storage in peripheral tissues progressively, but also were surprisingly effective in reducing sulfatide storage in brain and spinal cord. The histopathology of kidney and central nervous system was ameliorated. Improved neuromotor coordination capabilities and normalized peripheral compound motor action potential demonstrate the benefits of ERT on the nervous system function. Enzyme replacement may therefore be a promising therapeutic option in this devastating disease.

Animals↗

PubMed2005

Exocytosis of storage material in a lysosomal disorder.

Lysosomal exocytosis is a ubiquitously occurring process, which has a physiological role in repair of wounds of the plasma membrane. Lysosomal storage disorders are a group of more than 40 different diseases, which are characterized by intralysosomal storage of various substances. Metachromatic leukodystrophy is a lysosomal disease caused by the deficiency of arylsulfatase A, which results in the storage of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide) in, e.g., oligodendrocytes and distal tubule kidney cells. Here we show that sulfatide storing cultured primary kidney cells of arylsulfatase A deficient mice can undergo calcium induced lysosomal exocytosis and that this results in the delivery of storage material to the culture medium. In metachromatic leukodystrophy extracellular sulfatide has been found in urine and cerebrospinal fluid. Lysosomal exocytosis may explain the presence of sulfatide in these body fluids.

Animals↗

PubMed2005

Hepatoma-derived growth factor. Significance of amino acid residues 81-100 in cell surface interaction and proliferative activity.

Hepatoma-derived growth factor (HDGF) has proliferative, angiogenic, and neurotrophic activity. It plays a putative role in the development and progression of cancer. When expressed in cells, the mitogenic activity of HDGF depends on its nuclear localization, but it also stimulates proliferation when added to the cell culture medium. A cell surface receptor for HDGF has not been identified so far. We investigated the interaction of various purified recombinant HDGF fusion proteins with the cell surface of NIH 3T3 fibroblasts. We showed that binding of a HDGF-beta-galactosidase fusion protein to the cell surface of NIH 3T3 fibroblasts was saturable, occurred with high affinity (K(D) = 14 nm), and had a proliferative effect. We identified a peptide comprising amino acid residues 81-100 within the amino-terminal part of HDGF that bound to the cell surface of NIH 3T3 cells with saturation and affinity values similar to those of HDGF. When added to primary human fibroblasts, this peptide stimulated proliferation. Substitution of a single amino acid (K96A) within this peptide was sufficient to abolish its binding to the cell surface and its proliferative activity. In contrast, when expressed transiently in NIH 3T3 cells, a HDGF-beta-galactosidase fusion protein in which amino acid residues 81-100 were deleted still had proliferative activity, whereas a fusion protein containing only the 81-100 peptide did not. Our results suggest the existence of a plasma membrane-located HDGF receptor for which signaling depends on amino acid residues 81-100 of HDGF. This region differs from the one that has been recently identified to be essential for mitogenic activity depending on the nuclear localization of HDGF. Thus, HDGF exerts its proliferative activity via two different pathways.

Animals↗

PubMed2005

Adult onset metachromatic leukodystrophy without electroclinical peripheral nervous system involvement: a new mutation in the ARSA gene.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ARSA). Clinically, the disease is heterogeneous with respect to the age of onset, affection of peripheral and central nervous systems, and progression. OBJECTIVES: To analyze mutations in the ARSA gene of a patient with adult-onset MLD with no signs of peripheral polyneuropathy and to emphasize the clinical, neuroradiologic, neuropathologic, and genetic features of the disease. DESIGN: Case study of a patient clinically presenting with rapidly progressive dementia and behavioral abnormalities. We report the findings of clinical evaluation and neurophysiologic and neuropathologic studies of peripheral nerves; we also performed DNA sequence analysis, transfections, metabolic labeling, and immunoprecipitation of mutant ARSA polypeptides. SETTING: Genetic research and clinical unit, university hospital. RESULTS: Genetic analysis revealed homozygosity for a novel mutation in exon 3 of ARSA (F219V). This substitution leads to a misfolded unstable enzyme with a specific activity less than 1% of normal. There were no clinical or neurophysiologic signs of peripheral nervous system dysfunction. Typical neuropathologic signs for MLD were absent from nerve biopsy specimens. CONCLUSIONS: This novel mutation is associated with progressive psychocognitive impairment without clinical or electrophysiologic signs and only minor morphologic signs of peripheral nerve affection. The F219V substitution causes reduction in enzyme activity to an extent unexpected for an adult patient with MLD.

Adult↗

PubMed2005

Delay of myelin formation in arylsulphatase A-deficient mice.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (ASA). This leads to the accumulation of the sphingolipid 3-O-sulphogalactosylceramide (sulphatide) and progressive demyelination in the nervous system of MLD patients. The mechanisms and development of pathology in the disease are still largely unknown. In this study we investigate how the inability to degrade sulphatide affects the formation of myelin in ASA-deficient (ASA-/-) mice. In mice at 2 weeks of age there was a substantial reduction in myelin basic protein (MBP) mRNA and protein. This was confirmed by an immunohistochemical analysis. MBP mRNA and protein, however, reach normal levels at 3 weeks of age. Proteolipid protein (PLP) and MAL mRNA were also reduced in ASA-/- mice at 2 weeks of age; whereas the level of PLP mRNA was normal at 26 weeks of age, MAL mRNA expression remained reduced up to this age. In situ hybridization revealed no significant changes in the number of myelinating oligodendrocytes or oligodendrocyte precursor cells in ASA-/- mice. These results suggest that oligodendrocyte differentiation was normal in ASA-/- mice. No differences were found in the expression of the sulphatide synthesizing enzymes cerebroside sulphotransferase and UDP-galactose : ceramide galactosyltransferase. Our data demonstrate a delay in myelin formation in ASA-/- mice. This raises the possibility that similar alterations in MLD patients may contribute to the pathology of the disease.

Animals↗

PubMed2005

Reversal of non-hydroxy:alpha-hydroxy galactosylceramide ratio and unstable myelin in transgenic mice overexpressing UDP-galactose:ceramide galactosyltransferase.

The sphingolipids galactosylceramide and sulfatide are important for the formation and maintenance of myelin. Transgenic mice overexpressing the galactosylceramide synthesizing enzyme UDP-galactose:ceramide galactosyltransferase in oligodendrocytes display an up to four-fold increase in UDP-galactose:ceramide galactosyltransferase activity, which correlates with an increase in its products monogalactosyl diglyceride and non-hydroxy fatty acid-containing galactosylceramide. Surprisingly, however, we observed a concomitant decrease in alpha-hydroxylated galactosylceramide such that total galactosylceramide in transgenic mice was almost unaltered. These data suggest that UDP-galactose:ceramide galactosyltransferase activity does not limit total galactosylceramide level. Furthermore, the predominance of alpha-hydroxylated galactosylceramide appeared to be determined by the extent to which non-hydroxylated ceramide was galactosylated rather than by the higher affinity of UDP-galactose:ceramide galactosyltransferase for alpha-hydroxy fatty acid ceramide. The protein composition of myelin was unchanged with the exception of significant up-regulation of the myelin and lymphocyte protein. Transgenic mice were able to form myelin, which, however, was apparently unstable and uncompacted. These mice developed a progressive hindlimb paralysis and demyelination in the CNS, demonstrating that tight control of UDP-galactose:ceramide galactosyltransferase expression is essential for myelin maintenance.

Age Factors↗

PubMed2005

Missense mutations as a cause of metachromatic leukodystrophy. Degradation of arylsulfatase A in the endoplasmic reticulum.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Biosynthesis studies of ASA with various structure-sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epitopes form later, between 10 and 25 min. When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes. Eleven of the 12 mutants show partial expression of the early epitopes, but only six of these show, in addition, incomplete expression of late epitopes. In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes. Thus, data from the wild-type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation of the late epitopes. All mutant enzymes in which the amino acid substitution prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER). In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER. Thus, irrespective of the missense mutation, the expression of epitopes forming late in biosynthesis correlates with the ability of the enzyme to leave the ER. The degradation of ER-retained enzymes can be reduced by inhibitors of the proteasome and ER alpha1,2-mannosidase I, indicating that all enzymes are degraded via the proteasome. Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes.

Amino Acid Substitution↗

PubMed2005

Gene therapy of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease that is caused by a deficiency of arylsulfatase A (ASA). The deficiency results in the intralysosomal accumulation of the acidic sphingolipid 3-O-sulfogalactosyl-ceramide (sulfatide). Patients suffer from progressive demyelination and die from multiple neurological deficits. Curative treatment is not available. ASA bears mannose 6-phosphate residues which function as recognition markers in endosome/lysosome-specific targeting pathways. The endocytic targeting route can be exploited to deliver exogenous ASA to the lysosomes of ASA-deficient cells. ASA knockout mice, which develop a disorder related to MLD, have therefore been treated by ex vivo and in vivo gene therapy. Following transplantation of bone marrow cells overexpressing ASA from a retroviral vector, donor-type cells secrete ASA, which is endocytosed by recipient cells. The enzyme transfer results in the metabolic cross-correction of recipient cells and the improvement of biochemical, histological and clinical parameters. For the transfer of the ASA cDNA to non-dividing cells, adenovirus, adeno-associated virus and lentivirus vectors have been constructed. Such vectors might be particularly advantageous for direct ASA gene delivery to the brain, which is the main site of disease in MLD.

Animals↗

PubMed2004

Novel mutations associated with metachromatic leukodystrophy: phenotype and expression studies in nine Czech and Slovak patients.

Metachromatic leukodystrophy (MLD) is an inherited demyelinating disorder caused by the deficiency of arylsulphatase A (ASA). This defect leads to an accumulation of galactosylceramide I(3)-sulphates (sulphatides) in lysosomes of different tissues. We report on mutations found in a group of nine patients from the Czech and Slovak Republics (former Czechoslovakia). Their diagnosis was confirmed by determination of the activity of arylsulphatase A in leukocytes and by abnormal urinary excretion of sulphatides. All alleles of the patients were identified and eight different mutations were found. They include four novel missense mutations in one infantile (D29N), one juvenile (C294Y), and three adult (C156R, G293S) patients. Four mutations were previously described sequence alterations (459 + 1G > A, G309S, I179S, and P426L). Polymorphisms characteristic for the ASA pseudodeficiency allele were not found in the patients. Substitutions of D29N, C294Y, and G293S in arylsulphatase A caused a severe reduction of enzyme activity in transient expression studies. In contrast, the C156R substitution reduces arylsulphatase A only to 50% of wild type ASA activity. Since no other mutations were found in this patient, the contribution of this mutation to the development of disease remains unclear.

Adolescent↗

PubMed2004

Expression patterns and different subcellular localization of the growth factors HDGF (hepatoma-derived growth factor) and HRP-3 (HDGF-related protein-3) suggest functions in addition to their mitogenic activity.

HDGF (hepatoma-derived growth factor) and the HRPs (HDGF-related proteins) comprise a family of six proteins which display high identity in their N-terminus, but differ at the C-terminus. Here we investigate the patterns of expression of HDGF and HRP-3, by generating antisera specifically recognizing each growth factor. Whereas HRP-3 protein is expressed only in brain, HDGF can be found in a broad range of tissues, with highest levels in brain, testis, lung and spleen. The expression of HDGF and HRP-3 was found to be regulated during brain development, with highest levels around birth, followed by a decline until postnatal day 9. Interestingly, expression of HRP-3 increases again in adult brain. In situ hybridization and immunohistochemistry of cerebellar, cerebral and hippocampal brain slices showed that expression of both growth factors is not limited to areas of high proliferative activity. Both mRNAs and proteins are expressed in neuronal as well as glial cells. Immunocytochemistry of cultured neocortical neurons revealed that HDGF and HRP-3 can be found in the nucleus as well as the cytoplasm. HDGF is restricted to the neuronal soma, whereas HRP-3 can also be found in neurites. Thus the expression of HDGF and HRP-3 in differentiated cells, post-mitotic neurons and primary cultures of rat neocortex points to functions in brain that might not be limited to proliferation. In addition, their simultaneous expression in the same cell and their different subcellular localization in cultured neurons suggest different functions of HDGF and HRP-3 within single cells.

Animals↗

PubMed2004

Three novel mutant arylsulfatase A alleles causing metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. This leads to the accumulation of 3-O-sulfogalactosylceramide, which results in severe demyelination. Here we describe a novel non-sense mutation W124ter and two disease-causing missense mutations E382Q and C500F in arylsulfatase A gene. Another so far unknown allele harbors three sequence alterations: two polymorphisms (N350S, R496H) and a missense mutation (R288H). The R288H substitution and the N350S polymorphism have previously been found on one allele together with a polymorphism in a polyadenylation signal characteristic for the arylsulfatase A pseudodeficiency allele. The R496H has been shown to occur on another allele. The presence of the R288H, N350S, and R496H substitution on one allele in the absence of the polyadenylation site polymorphism shows that this allele has probably arisen by recombination between the nucleotides of codon 350 and 496.

Alleles↗

PubMed2004

Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice.

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA -/-) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA -/- mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV-VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA -/- but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA -/- mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA -/- mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.

Animals↗

PubMed2003

Oligomerization capacity of two arylsulfatase A mutants: C300F and P425T.

Arylsulfatase A (ARSA) is a lysosomal enzyme implicated in most cases of metachromatic leukodystrophy (MLD). The quaternary structure of ARSA is pH-dependent: at neutral pH, ARSA is a homodimeric protein; at lysosomal (acidic) pH, ARSA is homo-octameric. This dimer-octamer transition seems to be of major importance for the stability of the enzyme in the lysosomal milieu. Sedimentation analysis was used to study the oligomerization capacity of C300F and P425T-substituted ARSA, two MLD-associated forms of the enzyme displaying reduced lysosomal half-lives. P425T-ARSA displays a modest reduction in its octamerization capacity. In contrast, the C300F mutation strongly interferes with the octamerization process of ARSA but not with its dimerization capacity. Interestingly, a major fraction of dimeric ARSA-C300F is composed of covalently linked ARSA molecules, through a thiol-cleavable bond that probably involves Cys414 residues from each monomer. Our data support the notion that the reduced lysosomal half-life of some mutated forms of ARSA is related to deficient octamerization.

Amino Acid Substitution↗