Search PubMed⌕ Search

Biomedical subjects

K Sandhoff

Publications and source records attributed to K Sandhoff.

At least 73 records · Page 4Linked to original sources
PubMed1990

The complete amino-acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein.

The complete amino-acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein have been established by Edman degradation. The GM2 activator is composed of 162 amino acids, the first two serine residues being present in only 20% of the material. A single carbohydrate chain is N-glycosidically linked to Asn32. Three hydrophobic alpha-helices may contribute to its lipid-binding site. Three amino acids differ from those found by cDNA sequencing which may be due to a polymorphism. The cerebroside sulfate activator consists of 80 amino acids and carries one N-linked carbohydrate chain at Asn21. The C-terminal valine residue is lacking in about 80% of the material. In spite their similar functions, both activator proteins show no sequence or structural similarities.

Amino Acid Sequence↗
PubMed1990

Modulation of sphingolipid biosynthesis in primary cultured neurons by long chain bases.

Sphingolipid biosynthesis was studied in cultured murine cerebellar cells in the absence and presence of exogenous sphingosine homologues with different alkyl chain lengths (12, 18, and 24 carbon atoms). Labeling of cells with [14C]serine for 24 h indicated that endogenous sphingosine biosynthesis with incorporation of radiolabeled serine was inhibited by these long chain bases (0.5-50 microM) in a concentration-dependent manner; the inhibition was fully reversible after removal of the long chain bases from the culture medium. Metabolic labeling of neurons with [14C]galactose provided strong evidence that the cells were able to use the exogenous sphingosine homologues, irrespective of their alkyl chain length, as substrates for the biosynthesis of glycosphingolipids. When the biosynthetically inert sphingoid, azidosphingosine (5-50 microM), was fed to the cells, de novo sphingosine and glycosphingolipid biosynthesis were both strongly inhibited.

Animals↗
PubMed1990

Evidence for the presence of water within the hydrophobic core of membranes.

The photoreactive ganglioside derivative N-diazirinyl-lyso-GM1 was incorporated into liposomes and calf brain microsomes. After photoactivation at 350 nm it was found to dimerize with phospholipids such as phosphatidylcholine and phosphatidylserine and with cholesterol. The predominant covalent reaction product, however, was the alcohol, resulting from the reaction with water. It amounted to about 45% of the covalent reaction products in calf brain microsomes and to about 58% in pure phosphatidylcholine liposomes. Based on the temperature dependence of the photoreaction of N-diazirinyl-lyso-GM1 in liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphoryl-choline or 1,2-distearoyl-sn-glycero- 3-phosphorylcholine and on affinity labeling experiments with cholera toxin we propose that the predominant reaction of N-diazirinyl-lyso-GM1 with water is due to the presence of water within the hydrophobic core of artificial and biological membranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

The determination of phytosphingosine-containing globotriaosylceramide from human kidney in the presence of lactosylceramide.

Globotriaosylceramide, the natural substrate of alpha-galactosidase A (the enzyme deficient in Fabry's disease) was prepared from human kidney by repeated medium pressure chromatography on Lichroprep Si 60 (E. Merck) before and after peracetylation. The apparently homogeneous preparation migrating as a single band on HPTLC was analysed by fast atom bombardment mass spectrometry and 1H-NMR at 500 MHz. It was found that in this fraction two major molecular species were comigrating: Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide with nervonic and lignoceric acid linked to phytosphingosine and Gal beta 1-4Glc beta 1-1 ceramide with palmitic acid linked to sphingosine.

Antigens, CD↗
PubMed1990

pH-dependent changes of ganglioside biosynthesis in neuronal cell culture.

Ganglioside biosynthesis was studied in primary cultured murine cerebellar cells after labeling with [14C]galactose. A shift in biosynthesis from "a"-series to "b"-series gangliosides was observed after lowering the pH of the culture medium from 7.4 to 6.2; this effect was fully reversible on changing back to pH 7.4. The observed regulatory effect of pH is in accordance with a recent model of ganglioside biosynthesis. Sialyltransferase II (ST II), the first enzyme for biosynthesis of "b"-series gangliosides, is more active at pH 6.2 than Gal-NAc-transferase, the first enzyme for synthesis of "a"-series gangliosides, which is more active than sialyltransferase II at pH 7.4.

Animals↗
PubMed1990

GM2-gangliosidosis B1 variant: analysis of beta-hexosaminidase alpha gene abnormalities in seven patients.

A single nucleotide transition within exon 5 of the beta-hexosaminidase alpha chain gene was identified in a Puerto Rican patient with GM2-gangliosidosis B1 variant as the mutation responsible for the unusual enzymological characteristics of this variant (G533----A; Arg178----His) (the DN-allele). A total of seven patients with enzymological characteristics of B1 variant have since been studied. They were Puerto Rican (DN), Italian, French, Spanish, two patients of mixed ethnic origin (English/Italian/Hungarian and English/French/Azores), and a Czechoslovakian. In confirmation of our earlier finding based on screening with allele-specific probes, all patients except the one from Czechoslovakia carried the same DN-allele. A new point mutation found in this patient changed the same codon affected in the DN-allele (C532----T; Arg178----Cys). An asymptomatic Japanese individual included as a control also carried one allele with the DN-mutation. Site-directed mutagenesis and expression studies in COS I cells demonstrated that either of the two point mutations abolishes the catalytic activity of the alpha subunit. The Spanish patient was homozygous for the DN-allele, but others were all compound heterozygotes. The Puerto Rican patient was a compound heterozygote with the DN-mutation in one allele and with the four-base insertion in exon 11, one of the two mutations found in the classical Ashkenazi Jewish Tay-Sachs disease, in the other allele. Abnormalities of the other allele were not identified in all other compound heterozygous patients. In these patients, the level of mRNA derived from the other allele was variable, ranging from being undetectable to being much lower than normal. This series of studies uncovered a new B1 variant mutation, confirmed our preliminary finding that the DN-allele has a surprisingly wide geographic and ethnic distribution, and pointed out the highly complex nature of the molecular genetics of this rare disorder. They also support our working hypothesis that mutations responsible for the unique enzymological characteristics of the B1 variant should be located in or near exon 5 of the gene and that this region of the enzyme protein is critical for its catalytic function.

Alleles↗
PubMed1990

Uncoupling of ganglioside biosynthesis by Brefeldin A.

We have studied the effect of Brefeldin A (BFA), an antiviral antibiotic, on glycosphingolipid metabolism in primary cultured cerebellar cells. Cells were labeled metabolically with [14C]galactose, or pulse-labeled with precursors of glycosphingolipid biosynthesis; i.e., [14]serine, [3H]palmitic acid or [3H]sphingosine. In all cases BFA (1 microgram/ml) strongly inhibited (75-95%) ganglioside biosynthesis beyond the stage of GM3 and GD3, that is the formation of GM1, GD1a, GT1b and GQ1b. Simultaneously an accumulation of GlcCer, LacCer, GM3 and GD3 was observed (up to 2000%). These effects could be reversed fully by removal of the BFA from the culture medium. These results indicate that the LacCer-, GM3- and GD3-synthases of murine cerebellar cells are localized together on the proximal site of the Golgi apparatus, probably in the cis-Golgi compartment. It is probable that sphingomyelin synthase and some of the other glycosyltransferases involved in ganglioside biosynthesis are localized in distinct compartments beyond the cis Golgi.

Animals↗
PubMed1989

Identity of GD1C, GT1a and GQ1b synthase in Golgi vesicles from rat liver.

Competition experiments using GM1b, GD1a and GT1b as substrates, and as mutual inhibitors for ganglioside sialyltransferase activity in preparations of Golgi vesicles derived from rat liver, suggested that sialyl transfer to these three respective compounds, leading to gangliosides GD1C, GT1a and GQ1b, respectively, is catalyzed by one enzyme. These results are incorporated into a model for ganglioside biosynthesis and its regulation.

Animals↗
PubMed1989

Isolation of a cDNA encoding the human GM2 activator protein.

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.

Amino Acid Sequence↗
PubMed1989

Identity of GA1, GM1a and GD1b synthase in Golgi vesicles from rat liver.

Synthesis of ganglioside GD1b from ganglioside GD2 was demonstrated using Golgi membranes isolated from rat liver. Competition experiments using gangliosides GA2, GM2 and GD2 as substrates, and as mutual inhibitors for ganglioside galactosyltransferase activity in preparations of Golgi vesicles derived from rat liver, suggested that galactosyl transfer to these three compounds, leading to gangliosides GA1, GM1a and GD1b respectively, is catalyzed by one enzyme. These results strengthen the hypothesis that the main site for the regulation of ganglioside biosynthesis occurs within the reaction sequence LacCer----GA3----GD3----GT3.

Animals↗
PubMed1989

Isolation of cDNA clones encoding human acid sphingomyelinase: occurrence of alternatively processed transcripts.

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine and 12 tryptic peptides were microsequenced (128 residues). Based on regions of minimal codon redundancy, four oligonucleotide mixtures were synthesized and oligonucleotide mixture 1 (20mer; 256 mix) was used to screen 3 X 10(6) independent recombinants from a human fibroblast cDNA library. Putative positive clones (92) were purified and analyzed by Southern hybridization with oligonucleotide mixtures 2-4. These studies revealed two groups of clones; group 1 (80 clones; inserts ranging from approximately 1.2 to 1.6 kb) hybridized with oligonucleotides mixtures 1-4, while group II (12 clones; inserts ranging from approximately 1.2 to 1.4 kb) hybridized with oligonucleotide mixtures 1-3. Several group II clones had larger inserts than those in group I, but did not hybridize with oligonucleotide mixture 4. Screening of a human placental cDNA library with a 450 bp group I fragment, also resulted in the isolation of group I and II clones. Representative clones from group I (pASM-1) and group II (pASM-2) were sequenced. pASM-1 contained a 1879 bp insert which was colinear with 96 microsequenced amino acids, while the pASM-2 1382 bp insert was colinear with 78 microsequenced residues. Notably, pASM-2 did not have an internal 172 bp sequence encoding 57 amino acid residues, but had instead an in-frame 40 bp sequence encoding 13 amino acids which was not present in pASM-1. These findings demonstrate the presence of two distinct acid sphingomyelinase transcripts in human fibroblasts and placenta and suggest the occurrence of alternative processing of the mRNA encoding this lysosomal hydrolase.

Amino Acid Sequence↗
PubMed1989

Structure of full-length cDNA coding for sulfatide activator, a Co-beta-glucosidase and two other homologous proteins: two alternate forms of the sulfatide activator.

Full-length cDNA clones have been isolated for an mRNA which codes for four different but homologous proteins--a sulfatide activator protein, a co-beta-clucosidase, and two other proteins of similar structures. The primary structure as deduced from the nucleotide sequence is highly homologous to the precursor of the rat Sertoli cell sulfated glycoprotein 1. The full-length clone was 2,734-bp long, starting from 8 bases above the initiator ATG and terminating with a poly A tail. The nucleotide sequence confirmed an earlier prediction based on the amino acid sequence that a previously published sequence contained errors. On the other hand, the amino acid sequence now closely agrees with the recent revised sequence published by the same group except for several amino acids near the N-terminus. Two alternate forms of the sulfatide activator were detected, differing from each other by the presence or absence of 3-amino acid insertion.

Base Sequence↗
PubMed1989

Modulation of ganglioside biosynthesis in primary cultured neurons.

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.

Animals↗
PubMed1988

Ganglioside-protein interactions: spin-label electron spin resonance studies with (Na+,K+)-ATPase membranes.

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from Squalus acanthias have been studied using spin-labeled derivatives of the mono- and disialogangliosides GM1, GM2, GM3, and GD1b, in conjunction with electron spin resonance (ESR) spectroscopy. Ganglioside-protein interactions are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component that corresponds closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled gangliosides whose chain motion is significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A small selectively for the motionally restricted component associated with the protein is found in the order GD1b greater than GM1 approximately equal to GM2 approximately equal to GM3. Comparison with previous results from spin-labeled phospholipids in the same system [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] shows that the spin-labeled monosialogangliosides GM1, GM2, and GM3 display little selectivity in the lipid-protein interaction relative to spin-labeled phosphatidylcholine. The spectral characteristics of both the fluid and motionally restricted spin-labeled components differ very significantly, however, between the gangliosides and the phospholipids. The outer hyperfine splitting of the motionally restricted component is smaller for the gangliosides than for the phospholipids, indicating a smaller degree of motional restriction on interaction of the ganglioside lipid chains with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Probable metachromatic leukodystrophy/pseudodeficiency compound heterozygote at the arylsulfatase A locus with neurological and psychiatric symptomatology.

Metachromatic leukodystrophy (MLD) is an autosomal recessive progressive demyelination disorder caused by the deficiency of arylsulfatase A (ASA). However, there exist individuals with low ASA activity without clinical symptoms. This state is described as ASA pseudodeficiency (PD). A number of patients with low ASA activity and various neuropsychiatric symptoms have been observed. It is controversial to what extent low ASA activity predisposes for neurological and/or psychiatric symptomatology. Therefore, persons with low ASA activity who were collected from a large-scale screening among neuropsychiatric patients and healthy controls are presently being extensively evaluated using biochemical, genetic, and clinical methods. Here we present a female patient, who had been first hospitalized with the diagnosis encephalomyelitis disseminata. Her ASA activity determined in fibroblast extracts is intermediate between adult MLD and PD. Sulfatide degradation in cultured fibroblasts is diminished. The subunit pattern obtained after SDS-polyacrylamide gel electrophoresis and immunoblotting was determined in the index patient and 2 sibs. It is compatible with a compound genotype ASA-/ASAp in the index case. It appears probable that in this patient low ASA activity leads to the accumulation of sulfatide and either causes the appearance of neuropsychiatric symptoms or at least contributes to the demyelination process.

Adult↗
PubMed1988

[Sphingolipid storage diseases of the central nervous system: bases of biochemical and clinical heterogeneity].

Lysosomal storage disorders are progredient and often fatal diseases most of which result from a pronounced enzyme deficiency. In the case of sphingolipidoses, usually enzymes of sphingolipid catabolism are missing, or only a few percent of normal activity are detectable. For many sphingolipidoses, damage of the central nervous system is characteristic, but neurological and other symptoms can vary greatly, especially in adult variants. This variability is mainly caused by different allelic mutations of the structural genes, resulting in different levels of residual enzyme activity.

Central Nervous System Diseases↗
PubMed1988

Axonal transport of intraocularly injected [3H-Sph]-GD1a in the chicken optic system and the fate of the exogenous ganglioside distributed by blood.

Twelve-day-old chicks (White Leghorn) received an injection of 481 kBq (8.1 nmol) of [3H-Sph]-GD1a, which was labeled in its sphingoid, into the right eye. Structures of the injected and the non-injected (control) optic system (retinae, optic nerves, chiasm, optic lobes), the cerebrum, blood liver, kidney, and fly-muscle were analyzed 1, 4, 8 and 14 days later, with respect to total non-volatile radioactivity and to that bound to lower-phase lipids and gangliosides. It was demonstrated that exogenous [3H-Sph]-GD1a was taken up by the retina and mainly catabolized. 3H-label, reincorporated into the lower-phase lipids and gangliosides as well as authentic exogenous [3H-Sph]-GD1a were transported rapidly anterogradely in the entire optic system. [3H-Sph]-GD1a, distributed via the blood stream, was taken up by liver, kidney and muscle and was metabolized faster in these organs than in the retina. The cerebrum and the brain structures of the control optic system incorporated 3H-radioactivity to a much lower extent than the non-neural organs.

Animals↗