Biomedical subjects

K Sandhoff

Publications and source records attributed to K Sandhoff.

At least 19 recordsLinked to original sources

PubMed1999

GM2 gangliosidosis AB variant: clinical and biochemical studies of a Japanese patient.

OBJECTIVE: To determine the clinical features and biochemical basis of the first Japanese patient with the GM2 gangliosidosis AB variant. METHODS: The clinical manifestations and laboratory findings in the patient were investigated. Cultured fibroblasts from the patient were analyzed by means of immunofluorescence staining with an anti-GM2 ganglioside monoclonal antibody and thin-layer chromatography and immunostaining. GM1 ganglioside catabolism in cultured cells was analyzed by pulse labeling, and the amount of GM2 activator in cells was determined by Western blot analysis. Gene analysis was performed according to standard protocols. RESULTS: The patient showed progressive neurologic manifestations of quite early onset. Muscular weakness and hypotonia became evident by 1 month of age, and the patient then developed a startle reaction, severe psychomotor retardation, and myoclonic seizures. Immunocytochemical analysis clearly revealed the accumulation of GM2 ganglioside in cultured fibroblasts from the patient, and thin-layer chromatography confirmed it. Western blot and metabolic studies showed a complete deficiency of GM2 activator. Gene analysis did not reveal any mutations in the protein coding region of the GM2 activator gene. CONCLUSION: The clinical features and biochemical basis of this Japanese patient with GM2 gangliosidosis AB variant were determined. Immunocytochemical analysis using cultured fibroblasts as samples is available for the diagnosis of this disease.

Anticonvulsants↗

PubMed1999

Recombinant GM2-activator protein stimulates in vivo degradation of GA2 in GM2 gangliosidosis AB variant fibroblasts but exhibits no detectable binding of GA2 in an in vitro assay.

The interaction between glycosphingolipids and recombinant human GM2-activator was studied in a microwell binding assay. A-series gangliosides like GM3, GM2 and GM1 were strongly bound by the recombinant human GM2 activator. A weak binding was observed to GD1b and sulfatide, while neutral glycolipids were not bound. Optimal binding occurred at pH 4.2 and was inhibited by increasing concentrations of citrate buffer and NaCl. In contrast with these in vitro results the recombinant human GM2-activator is able to restore the degradation of GA2 in fibroblasts from patients with the AB variant of GM2 gangliosidosis in vivo.

Biotin↗

PubMed1999

A genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder.

Inherited defects in the degradation of glycosphingolipids (GSLs) cause a group of severe diseases known as GSL storage disorders. There are currently no effective treatments for the majority of these disorders. We have explored a new treatment paradigm, substrate deprivation therapy, by constructing a genetic model in mice. Sandhoff's disease mice, which abnormally accumulate GSLs, were bred with mice that were blocked in their synthesis of GSLs. The mice with simultaneous defects in GSL synthesis and degradation no longer accumulated GSLs, had improved neurologic function, and had a much longer life span. However, these mice eventually developed a late-onset neurologic disease because of accumulation of another class of substrate, oligosaccharides. The results support the validity of the substrate deprivation therapy and also highlight some limitations.

Animals↗

PubMed1998

Lysosomal degradation on vesicular membrane surfaces. Enhanced glucosylceramide degradation by lysosomal anionic lipids and activators.

According to a recent hypothesis (Sandhoff, K., and Kolter, T. (1996) Trends Cell Biol. 6, 98-103), glycolipids, which originate from the plasma membrane, are exposed to lysosomal degradation on the surface of intralysosomal vesicles. Taking the interaction of membrane-bound lipid substrates and lysosomal hydrolases as an experimental model, we studied the degradation of glucosylceramides with different acyl chain lengths by purified glucocerebrosidase in a detergent-free liposomal assay system. Our investigation focused on the stimulating effect induced by lysosomal components such as sphingolipid activator protein C (SAP-C or saposin C), anionic lysosomal lipids, bis(monoacylglycero)phosphate, and dolichol phosphate, as well as degradation products of lysosomal lipids, e.g. dolichols and free fatty acids. The size of the substrate-containing liposomal vesicles was varied in the study. Enzymatic hydrolysis of glucosylceramide carried by liposomes made of phosphatidylcholine and cholesterol was rather slow and only weakly accelerated by the addition of SAP-C. However, the incorporation of anionic lipids such as bis(monoacylglycero)phosphate, dolichol phosphate, and phosphatidylinositol into the substrate carrying liposomes stimulated glucosylceramide hydrolysis up to 30-fold. Dolichol was less effective. SAP-C activated glucosylceramide hydrolysis under a variety of experimental conditions and was especially effective for the increase of enzyme activity when anionic lipids were inserted into the liposomes. Glucosylceramides with short acyl chains were found to be degraded much faster than the natural substrates. Dilution experiments indicated that the added enzyme molecules associate at least partially with the membranes and act there. Surface plasmon resonance experiments demonstrated binding of SAP-C at concentrations up to 1 microM to liposomes. At higher concentrations (2.5 microM SAP-C), liposomal lipids were released from the liposome coated chip. A model for lysosomal glucosylceramide hydrolysis is discussed.

Caproates↗

PubMed1998

Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).

Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may participate in sphingolipid transport and signaling events. Re-uptake of the secreted precursor is mediated by high-affinity cell surface receptors that are apparently distinct from the M-6-P receptor. We found that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor that is expressed on most cells, can mediate cellular uptake and lysosomal delivery of SAP precursor. Additional in vivo experiments in mice revealed that the mannose receptor system on macrophages also participates in precursor internalization. We conclude that SAP precursor gains entry into cells by at least three independent receptor mechanisms including the M-6-P receptor, the mannose receptor and LRP.

Animals↗

PubMed1998

Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization.

Biochemical and structural studies on human acid sphingomyelinase (haSMase) depend on the access to homogeneous biologically active enzyme. Due to the low abundance of native haSMase (n-haSMase) in human tissue, conventional purification strategies are not suitable for the isolation of preparative amounts of the enzyme. We describe a novel approach to the functional expression and purification of haSMase employing the baculovirus expression vector system. Infection of Spodoptera frugiperda 21 cells with recombinant baculovirus encoding haSMase leads to the expression of a glycosylated 75 kDa precursor protein, which is subsequently processed to an enzymatically active secreted 72 kDa haSMase. Variations in N-glycosylation and proteolytic maturation account for the difference in molecular mass between mature recombinant (72 kDa) and human placental haSMase (75 kDa). N-terminal amino acid sequencing of recombinant haSMase (r-haSMase) reveals a 23-residue N-terminal extension compared to the placental enzyme. The apparent K(m) and Vmax values for sphingomyelin degradation by r-haSMase in a micellar assay system are 32 microM and 0.56 mmol h-1 mg-1, respectively. In conclusion, the established baculovirus expression vector system provides an efficient tool for the expression and functional characterization of haSMase.

Animals↗

PubMed1998

Sphingolipid metabolism. Sphingoid analogs, sphingolipid activator proteins, and the pathology of the cell.

Sphingolipid metabolism and function was investigated using sphingoid analogs, cells from human sphingolipidoses patients, and knockout animals. Treatment of primary cultured murine cerebellar cells with the structurally modified sphingosine base cis-4 methylsphingosine resulted in decreased sphingolipid biosynthesis accompanied by significant morphological changes. Plasma-membrane-derived glycosphingolipids (GSLs) destined for digestion are internalized through the endocytic pathway and delivered to lysosomes. There, GSLs are degraded by the action of exohydrolases, which are supported, in the case of GSLs with short oligosaccharide chains, by sphingolipid activator proteins (SAPs or saposins). The inherited deficiency of activators give rise to sphingolipid storage diseases. The analysis of cultured fibroblasts from corresponding patients suggests a new model for the topology of endocytosis and lysosomal digestion. Mice with disrupted genes for activator proteins and for GM2 degrading hexosaminidases turned out to be useful models for human diseases.

Animals↗

PubMed1998

Cloning and characterization of the full-length cDNA and genomic sequences encoding murine acid ceramidase.

The full-length cDNA and genomic sequences encoding murine acid ceramidase (AC; E.C. 3.5.1.23) have been isolated and characterized. The 2176-bp cDNA was approximately 80% identical to the human cDNA (Koch et al., 1996) and predicted a 394-amino-acid polypeptide that was approximately 90% identical to the human protein. A fluorescence-based assay system was developed to determine AC enzymatic activity, and transfection of COS-1 cells with the full-length mouse cDNA led to increased AC activity, demonstrating its functionality. The murine AC gene, which spanned approximately 38 kb, consisted of 14 exons separated by 13 introns. The exons ranged in size from 46 to 1038 bp and were flanked by exon/intron junctions that adhered closely to known donor and acceptor splice site consensus sequences. Exon 1 encoded the putative translation start site and the signal peptide region, while exon 14 encoded the carboxy end of the AC polypeptide and all of the 3' untranslated region. Sequence analysis of a 497-bp region upstream from the first in-frame ATG revealed several features of a housekeeping promoter, as well as several tissue-specific and/or hormone-inducible regulatory sites. Insertion of this sequence into a chloramphenicol acyltransferase (CAT) expression vector led an approximately fivefold increase in CAT activity after transfection into NIH3T3 cells. Northern blot analysis and enzymatic assays also were carried out on various murine tissues to examine AC expression. Of the tissues studied, the highest AC activity and mRNA levels were found in the kidney, followed by the brain; almost no AC activity or mRNA was found in the testis or skeletal muscle. These latter studies provided clear evidence that despite the housekeeping function of AC, its expression was tissue-specific.

Acid Ceramidase↗

PubMed1998

Bone marrow transplantation prolongs life span and ameliorates neurologic manifestations in Sandhoff disease mice.

The GM2 gangliosidoses are a group of severe, neurodegenerative conditions that include Tay-Sachs disease, Sandhoff disease, and the GM2 activator deficiency. Bone marrow transplantation (BMT) was examined as a potential treatment for these disorders using a Sandhoff disease mouse model. BMT extended the life span of these mice from approximately 4.5 mo to up to 8 mo and slowed their neurologic deterioration. BMT also corrected biochemical deficiencies in somatic tissues as indicated by decreased excretion of urinary oligosaccharides, and lower glycolipid storage and increased levels of beta-hexosaminidase activity in visceral organs. Even with neurologic improvement, neither clear reduction of brain glycolipid storage nor improvement in neuronal pathology could be detected, suggesting a complex pathogenic mechanism. Histological analysis revealed beta-hexosaminidase-positive cells in the central nervous system and visceral organs with a concomitant reduction of colloidal iron-positive macrophages. These results may be important for the design of treatment approaches for the GM2 gangliosidoses.

Animals↗

PubMed1998

Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure.

Gaucher disease is caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC). Three clinical types of Gaucher disease have been defined according to the presence (type 2 and 3) or absence (type 1) of central nervous system disease and severity of clinical manifestations. The clinical course of the disease correlates with the mutation carried by the GC gene. To produce mice with point mutations that correspond to the clinical types of Gaucher disease, we have devised a highly efficient one-step mutagenesis method-the single insertion mutagenesis procedure (SIMP)-to introduce human disease mutations into the mouse GC gene. By using SIMP, mice were generated carrying either the very severe RecNciI mutation that can cause type 2 disease or the less severe L444P mutation associated with type 3 disease. Mice homozygous for the RecNciI mutation had little GC enzyme activity and accumulated glucosylceramide in brain and liver. In contrast, the mice homozygous for the L444P mutation had higher levels of GC activity and no detectable accumulation of glucosylceramide in brain and liver. Surprisingly, both point mutation mice died within 48 hr of birth, apparently of a compromised epidermal permeability barrier caused by defective glucosylceramide metabolism in the epidermis.

Amino Acid Sequence↗

PubMed1998

1-Methylthiodihydroceramide, a novel analog of dihydroceramide, stimulates sphinganine degradation resulting in decreased de novo sphingolipid biosynthesis.

1-Methylthiodihydroceramide (10 microM) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time- and concentration-dependent manner by up to 80%. Complex sphingolipid synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine biosynthesis, as well as its N-acylation to form dihydroceramide, revealed that they were also not affected. In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase. Reduction of de novo sphingolipid biosynthesis by 1-methylthiodihydroceramide is therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 microM) antagonized the inhibition of growth. Interestingly, the D-erythro and the L-threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.

Animals↗

PubMed1998

Complete localization of disulfide bonds in GM2 activator protein.

Lysosomal degradation of ganglioside GM2 by hexosaminidase A requires the presence of a small, non-enzymatic cofactor, the GM2-activator protein (GM2AP). Lack of functional protein leads to the AB variant of GM2-gangliosidosis, a fatal lysosomal storage disease. Although its possible mode of action and functional domains have been discussed frequently in the past, no structural information about GM2AP is available so far. Here, we determine the complete disulfide bond pattern of the protein. Two of the four disulfide bonds present in the protein were open to classical determination by enzymatic cleavage and mass spectrometry. The direct localization of the remaining two bonds was impeded by the close vicinity of cysteines 136 and 138. We determined the arrangement of these disulfide bonds by MALDI-PSD analysis of disulfide linked peptides and by partial reduction, cyanylation and fragmentation in basic solution, as described recently (Wu F, Watson JT, 1997, Protein Sci 6:391-398).

Amino Acid Sequence↗

PubMed1998

Bone marrow transplantation in metachromatic leukodystrophy caused by saposin-B deficiency: a case report with a 3-year follow-up period.

A 2-year-old child had a metachromatic leukodystrophy-variant phenotype mainly involving the peripheral nervous system (PNS) that was caused by saposin-B deficiency. Bone marrow transplantation resulted in transient deterioration then continuous improvement of PNS functions. These findings were supported by nerve conduction velocity measurements, but the symptoms ultimately worsened. Magnetic resonance imaging showed persistent white matter lesions and progressive pontocerebellar atrophy.

Bone Marrow Transplantation↗

PubMed1998

Glycosphingolipid degradation and animal models of GM2-gangliosidoses.

Glycosphingolipids form cell type-specific patterns on the surface of eukaryotic cells. Degradation of glycosphingolipids requires endocytic membrane flow of plasma membrane-derived glycosphingolipids into the lysosomes as the digesting organelles. The inherited deficiencies of lysosomal hydrolases and of sphingolipid activator proteins both give rise to sphingolipid storage diseases. Recent research has focused on the mechanisms leading to selective membrane degradation in the lysosomes and on the mechanism and physiological function of sphingolipid activator proteins. The GM2-degrading system is a paradigm for activator protein-dependent lysosomal degradation. Three polypeptide chains contribute to the in vivo degradation of ganglioside GM2: the alpha- and beta-chains of the beta-hexosaminidases and the GM2 activator. Mouse models of Tay-Sachs disease (alpha-chain deficiency), Sandhoff disease (beta-chain deficiency) and GM2 activator deficiency have been described. While the phenotypes of these variants of GM2-gangliosidoses are only slightly different in humans, the animal models show drastic differences in severity and course of the diseases. The reason for this is the specificity of sialidase, which is different between mouse and human. A double-knockout mouse lacking beta-hexosaminidases A, B and S shows a phenotype of mucopolysaccharidosis and gangliosidosis. A substrate deprivation approach to therapy is discussed with respect to animal models of the GM2-gangliosidoses.

Animals↗

PubMed1998

The generation and characterization of a rat neural cell line overexpressing the alpha2,6(N) sialyltransferase.

In order to examine the effects of altered protein sialylation on neural cell function, B104 rat neuroblastoma cells were stably transfected with the cDNA coding for alpha2,6(N) sialyltransferase (ST(6)N). Lectin blot analysis of the clones demonstrated an increase in staining of the Sambucus nigra lectin, which detects alpha2,6 linked sialic acid, in parallel with enzyme activity. There was a concomitant decrease in staining by the Maackia amurensis lectin which labels alpha2,3-linked sialic acid, indicating that the individual sialyltransferase enzymes may compete for penultimate galactose acceptor sites. While there was an initial increase in protein-bound sialic acid in parallel with enzyme activity, the sialylation of the cells was demonstrated to be saturable. There was an inverse relationship between cell adhesion to a fibronectin substrate and ST(6)N activity suggesting that the negatively charged sugar acts to modulate cell-substrate interaction. These cells will provide an ideal model system with which to further investigate the effect of altered sialic acid on neural cell function.

Animals↗

PubMed1998

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT.

In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N-thioacetylsphingosine (C2-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N-acetylsphingosine (C2-Cer=O), as well as the new compound C2-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC50) of 20 microg/ml and 10 microg/ml, respectively, whereas FS-5 has been potent with an IC50>40 microg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.

Amides↗

PubMed1998

Recent advances in the biochemistry of sphingolipidoses.

Glycosphingolipids are ubiquitous membrane components of eukaryotic cells. They participate in various cell recognition events and can regulate enzymes and receptors within the plasma membrane. Sphingolipidoses are due to an impaired lysosomal digestion of these substances. Glycosphingolipids are degraded by the action of exohydrolases, which are supported, in the case of glycosphingolipids with short oligosaccharide chains, by sphingolipid activator proteins. Five sphingolipid activator proteins are known so far, the GM2-activator and the SAPs, SAP-A to D (also called saposins). Degradation of glycosphingolipids requires endocytic membrane flow of plasma membrane derived glycosphingolipids into the lysosomes. Recent research focused on the topology of this process and on the mechanism and physiological function of sphingolipid activator proteins. Limited knowledge is available about enzymology and topology of glycosphingolipid biosynthesis. Recently, intermediates of this metabolic pathway have been identified as novel signalling molecules. Inhibition of glycosphingolipid biosynthesis has been shown to be beneficial in the animal model of Tay-Sachs disease. Mice with disrupted genes for lysosomal hydrolases and activator proteins are useful models for known human diseases and are valuable tools for the study of glycosphingolipid metabolism, the pathogenesis of sphingolipidoses and novel therapeutic approaches.

Animals↗

PubMed1998

Processing of sphingolipid activator proteins and the topology of lysosomal digestion.

Plasma membrane derived glycosphingolipids (GSLs) destined for digestion are internalized through the endocytic pathway and delivered to the lysosomes. There, GSLs are degraded by the action of exohydrolases, which are supported, in the case of GSLs with short oligosaccharide chains, by sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins) are synthesized from a single precursor protein (pSAP). Intracellular routing of pSAP and of the GM2 activator protein is only in part dependent on mannose-6-phosphate residues. Their endocytosis occurs in a carbohydrate-independent manner. The inherited deficiencies of individual activators, the GM2 activator, SAP-B, and SAP-C, as well as the deficiency of the precursor pSAP give rise to different, neuronal, white matter or visceral sphingolipid storage diseases. The analysis of cultured fibroblasts from corresponding patients suggests a new model for the topology of endocytosis and lysosomal digestion. It supports the hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures, that are then digested within the lysosomes. In combined activator protein deficient cells nondegradable GSLs on the surface of intralysosomal vesicles protect them against lysosomal digestion. Mice with disrupted genes for activator proteins (SAP precursor -/-, GM2A -/-) as well as disrupted genes for ganglioside GM2 degrading hexosaminidases (HEXA -/-, HEXB -/-) turned out to be useful models for known human diseases whereas double knock out mice (HEXA -/- and HEXB -/-) show a new phenotype of both mucopolysaccharidosis and gangliosidosis.

Animals↗