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At least 289 records · Page 16Linked to original sources
PubMed1982

Degradation of keratan sulfate by beta-N-acetylhexosaminidases in GM2-gangliosidosis.

We have prepared a new substrate from a keratan sulfate-derived-oligosaccharide (2-acetamido-2-deoxyglucosyl-(1--3)-[1-3H] Galactitol), which is necessary to measure beta-N-acetylhexosaminidase activity. This substrate was prepared from a cornea keratan sulfate by digestion with endo-beta-galactosidase, followed by isolation of disaccharide on gel filtration chromatography and chemical desulfation. Using this substrate, we found that a striking deficiency of beta-N-acetylhexosaminidase activity was present in the skin fibroblasts of patients with Sandhoff disease but not in Tay-Sachs disease. Both beta-N-acetyl-hexosaminidase A & H contributed to the catabolism of keratan sulfate.

Cells, Cultured↗
PubMed1982

Impaired degradation of chondroitin sulfate in GM2-gangliosidosis.

We have prepared a new radiolabeled substrate, derived from chondroitin 6-sulfate oligosaccharide, for the assaying of chondroitin sulfate degradation by beta-N-acetylgalactosaminidase. Using this substrate, we found a striking deficiency of beta-N-acetylgalactosaminidase activity in the cultured skin fibroblasts of patients with Sandhoff disease and Tay-Sachs disease. DEAE-cellulose chromatography at pH 6.0 revealed that both isoenzymes A and B of beta-N-acetylgalactosaminidases from normal human liver participated in the catabolism of chondroitin 6-sulfate. However, there were major differences in substrate specificity between isoenzyme A and isoenzyme B.

Cells, Cultured↗
PubMed1984

Usefulness of 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyrano sid e for the diagnosis of GM2 gangliosidoses in leukocytes.

4-Methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized and tested as a substrate for the diagnosis of GM2 gangliosidoses using leukocytes. Less than 2% of normal activity was measured in homogenates from patients with typical Tay-Sachs disease and from a patient with a variant form having 37% Hexosaminidase A by heat denaturation using the usual fluorogenic substrate. An adult patient had 8.5% of normal activity. Three patients with Sandhoff disease were found to have values ranging from 17% to 37% of normal. These values overlap the range found for carriers of Tay-Sachs disease, and suggest that the usefulness of this substrate, while excellent for diagnosing B variants of GM2 gangliosidosis, requires further study. Perhaps when used together with 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside excellent discriminations of patients, carriers and controls will be realized.

Acetylglucosaminidase↗
PubMed1979

A new N-acetyl-beta-D-hexosaminidase disease with late onset of progressive neurological symptoms.

Clinical data are presented on a 30-year-old male with normal early development (4-5 years) but subsequent progressive impairment of psychomotor functions. He has marked kyphoscoliosis and talipes calcaneo-valgus. The organs appear normal and the patient can walk unaided and feed himself although he does not recognize his parents. He has normal fundi oculi. Biochemical data show an absence of mucopolysacchariduria and very low but detectable levels of N-acetyl-beta-D-hexosaminidase in serum and leucocytes. The clinical symptoms are much milder than would normally be expected from such a profound enzyme deficiency (Sandhoff disease).

Adult↗
PubMed1983

Characterization of polypeptides serologically and structurally related to hexosaminidase in cultured fibroblasts.

Human fibroblasts synthesize several polypeptides that assort into the various forms of hexosaminidase (hex). We report here the occurrence of three newly identified, hexosaminidase-related polypeptides resolved by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of immunoprecipitates from [35S]methionine-labeled cell extracts. These polypeptides, called band 2 (75,000), band 3 (70,000), and band 4 (63,000), were immunoprecipitated by an antiserum specific to placental hex I2. They are distinct from pre-alpha- (60,000) and pre-beta- (58,000) precursor polypeptides and the alpha- (56,000), beta a- (27,000), and beta b- (27,000) polypeptides of the mature hex A (alpha beta a beta b) and hex B (2[beta a beta b]). When fibroblast extracts were chromatographed on DEAE-Sepharose, bands 2, 3, and 4 were eluted together in fractions before hex A, in a position characteristic of serum and placental hex I2 and serum hex P. This suggests that bands 2, 3, and 4 might represent the polypeptides of a fibroblast hex I. The analysis of partial proteolytic digests of the radioactively labeled polypeptides revealed that bands 2 and 3, pre-beta, and beta a had several peptides in common, suggesting that they are structurally related to each other. However, bands 2, 3, and 4 were present in extracts of Tay-Sachs (pre-alpha and alpha deficiency) and Sandhoff cells (pre-beta, beta a, and beta b deficiency) and appeared later than pre-beta in pulse-chase experiments. These results suggest that bands 2 and 3 occur independently of pre-beta and beta a and are probably specified by different mRNA, whether from the same gene or distinct but homologous genes.

Animals↗
PubMed1983

Concentrations of an activator protein for sphingolipid hydrolysis in liver and brain samples from patients with lysosomal storage diseases.

The hydrolysis of sphingolipids by lysosomal enzymes requires the presence of additional proteins, which have been called activator proteins. The number of activator proteins, their specificity, exact mechanism of action, and response to a storage process all remain to be determined. In this study, antibodies to an activator protein known to bind sphingolipids and activate the enzymatic hydrolysis of GM1 ganglioside and sulfatide were used to estimate the concentration of this activator protein in small samples of liver and brain from patients with lysosomal storage diseases. By using rocket immunoelectrophoresis, the concentration of cross-reacting material (CRM) was determined. Control livers had an average of 0.95 +/- 0.18 (mean +/- 1 SD) microgram CRM/mg protein in the extracts, and control brains had an average of 0.25 +/- 0.14 microgram CRM/mg protein. Extremely high levels of CRM were found in extracts of livers from patients with type 1 GM1 gangliosidosis (15.1 and 16.9), and type A Niemann-Pick disease (10.7). Extracts of brain samples revealed a large amount of CRM in type 1 GM1 gangliosidosis (14.8), Tay-Sachs disease (5.3 and 8.7), and Sandhoff disease (13.5). Significantly elevated CRM was also measured in brain samples from patients with type 2 GM1 gangliosidosis, type A Niemann-Pick disease, metachromatic leukodystrophy, and Krabbe disease. The highest levels are found in those genetic diseases where the lipids stored, primarily or secondarily to the genetic defect, bind to this activator protein. This activator protein may have an important function in regulating intralysosomal lipid catabolism, and changes in its concentration in certain genetic diseases may be the cause of clinical, biochemical, and pathological heterogeneity found in the patients.

Adolescent↗
PubMed2004

Possible role of autoantibodies in the pathophysiology of GM2 gangliosidoses.

Mice containing a disruption of the Hexb gene have provided a useful model system for the study of the human lysosomal storage disorder known as Sandhoff disease (SD). Hexb(-/-) mice rapidly develop a progressive neurologic disease of ganglioside GM2 and GA2 storage. Our study revealed that the disease states in this model are associated with the appearance of antiganglioside autoantibodies. Both elevation of serum antiganglioside autoantibodies and IgG deposition to CNS neurons were found in the advanced stages of the disease in Hexb(-/-) mice; serum transfer from these mice showed IgG binding to neurons. To determine the role of these autoantibodies, the Fc receptor gamma gene (FcR gamma) was additionally disrupted in Hexb(-/-) mice, as it plays a key role in immune complex-mediated autoimmune diseases. Clinical symptoms were improved and life spans were extended in the Hexb(-/-)FcR gamma(-/-) mice; the number of apoptotic cells was also decreased. The level of ganglioside accumulation, however, did not change. IgG deposition was also confirmed in the brain of an autopsied SD patient. Taken together, these findings suggest that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in SD and therefore provides a target for novel therapies.

Animals↗
PubMed1987

Oligosialogangliosides inhibit GM2- and GD3-synthesis in isolated Golgi vesicles from rat liver.

The effect of end-product gangliosides (GD1a, GT1b, GQ1b) on the activities of two key enzymes in ganglioside biosynthesis, namely GM2-synthase and GD3-synthase in rat liver Golgi apparatus, has been investigated in detergent-free as well as in detergent-containing assays. In detergent-free intact Golgi vesicles, phosphatidylglycerol was used as a stimulant. This phospholipid was earlier shown to stimulate the activity of GM2-synthase without disrupting the vesicular intactness; it has, however, no effect on GD3-synthase (Yusuf, H.K.M., Pohlentz, G., Schwarzmann, G. & Sandhoff, K. (1983) Eur. J. Biochem. 134, 47-54). In the presence of this stimulant, all higher gangliosides inhibited the activity of GM2-synthase, the inhibition being more profound with increasing negative charge of the inhibiting gangliosides. These inhibitions are unspecific, but they do not exclude an end-product regulation of ganglioside biosynthesis. In detergent-solubilized Golgi membranes, on the other hand, the inhibition pattern was completely different. Here, ganglioside GD1a was the strongest inhibitor of GM2-synthase, followed by GM1 and GM2, but GT1b also inhibited this enzyme appreciably, in fact more strongly than GM1 or GM2. On the other hand, GQ1b had no effect at all. Conversely, GD3-synthase activity was most strongly inhibited by GQ1b, followed by GT1b, but GD1a also inhibited this enzyme almost as strongly as GT1b. These latter findings indicate that feed-back control of the a- and the b-series pathways of ganglioside biosynthesis is probably not specific, but the pathways appear to be inhibited more preferably by their respective end-products than by any other gangliosides of the same of the other series.

Animals↗
PubMed1997

The beta-hexosaminidase deficiency disorders: development of a clinical paradigm in the mouse.

Tay-Sachs disease and Sandhoff disease are severe neurodegenerative disorders caused by a deficiency of beta-hexosaminidase A and resultant accumulation of its substrate, GM2 ganglioside, in neuronal lysosomes. The three clinical forms of the disorders (infantile, juvenile and adult) are of varying severity and onset, and have been correlated with the amount of residual GM2 ganglioside-degrading activity present in patients' cells. Through targeted disruption of the murine beta-hexosaminidase genes in embryonic stem cells, we have developed a set of mice that vary in their GM2 ganglioside-degrading capacity and exhibit many of the clinical features of the human diseases. These mice are valuable for the study of pathogenic mechanisms and for devising novel therapeutic strategies in these disorders.

Adult↗
PubMedDate not supplied

I. Abnormalities in cells of the testis, efferent ducts, and epididymis in juvenile and adult mice with beta-hexosaminidase A and B deficiency.

Beta-hexosaminidase (Hex) is a lysosomal enzyme that exists as two major isoenzymes: Hex A (subunit structure, alphabeta) and Hex B (betabeta). The presence of Hex in the testis and epididymis suggests important roles for the enzyme and its substrates in male fertility and reproductive functions. Disruption of the Hexb gene encoding the beta-subunit of Hex has led to the generation of a mouse model of human Sandhoff disease that survives to adulthood, enabling us to analyze the effects of Hex A and Hex B deficiency on epithelial cellular morphology of the male reproductive tract. At 1 and 3 months of age, the testes, efferent ducts, and epididymides of Hex-deficient (Hexb -/-) and wild-type (Hexb +/+) mice were perfuse fixed and analyzed by routine light and electron microscopy (LM and EM, respectively) as well as with immunocytochemistry employing antibodies to lysosomal proteins. In the testis, the morphological appearance and topographical arrangement of the cell types of the seminiferous epithelium of Hexb -/- mice were similar to those of wild-type animals at both ages. Both Sertoli and germ cells appeared to be unaffected. However, at both ages, myoid cells and macrophages showed an increased number of lysosomes in their cytoplasm as compared with the number seen in controls. The epithelial cells of the efferent ducts also showed an accumulation of lysosomes that increased with age as compared with controls. Principal cells of the entire epididymis revealed an increase in the size and number of lysosomes at 1 month of age as compared with those of controls, and by 3 months, these lysosomes often filled the supranuclear and basal regions of the cells. Narrow cells of the distal initial segment and intermediate zone, normally slender cells showing several lysosomes, became greatly enlarged and entirely filled with lysosomes in Hexb -/- mice. Clear cells of the caput, corpus, and cauda regions also showed a progressive increase in the size and number of lysosomes with age as compared with controls; the clear cells of the mutant mice were often enlarged and at times bulged into the lumen. Some basal cells of each epididymal region in Hexb -/- mice were similar to controls at 1 and 3 months, showing few lysosomes, while others showed an accumulation of lysosomes. Lysosomes of all affected epithelial cells were of varying sizes, but many large ones were present, apparently resulting from lysosomal fusion. Although pale stained, their identification as lysosomes was confirmed by EM immunocytochemistry with anti-cathepsin D and anti-Hex A antibodies. Predominantly in the proximal initial segment, large, pale cellular aggregates were noted in the LM analysis at the base of the epithelium, which by EM analysis were identified as belonging to two different cell types, narrow cells and halo cells. Taken together, these data reveal an increase in the size and number of lysosomes in all epithelial cell types lining the efferent ducts and entire epididymis as well as in myoid cells and macrophages of the testis. In the light of data showing epididymal defects restricted predominantly to the initial segment in Hexa -/- (Hex A-deficient) mice, our data on the Hexb -/- mice demonstrate a major role for Hex that can be fulfilled by either Hex A or Hex B in the epididymis.

Aging↗
PubMed1992

A novel exon mutation in the human beta-hexosaminidase beta subunit gene affects 3' splice site selection.

The molecular basis of a dramatically decreased steady state level of beta-hexosaminidase beta subunit mRNA in a patient with juvenile Sandhoff disease was investigated. Nucleotide sequence analysis of the HEXB gene coding for the beta subunit revealed two single base substitutions, one in exon 2 (A to G, a known polymorphism) and the other in exon 11 (C to T). Analysis of the beta subunit mRNA species demonstrated activation of a cryptic splice site in exon 11 as well as skipping of the exon. A transfection assay using a chimeric gene containing intron 10 flanked by cDNA sequences carrying the mutation confirmed that the single base substitution located at position 8 of exon 11 inhibits the selection of the normal 3' splice site. The results demonstrate a new type of exon mutation affecting 3' splice site selection.

Adult↗
PubMed1991

The human GM2 activator protein. A substrate specific cofactor of beta-hexosaminidase A.

Ganglioside GD1a-GalNAc was isolated from Tay-Sachs brain, tritium-labeled in its sphingosine moiety, and its enzymic degradation studied in vitro and in cultured fibroblasts. When offered as micelles, GD1a-GalNAc was almost not hydrolyzed by Hex A or Hex B, while after incorporation of the ganglioside into the outer leaflet of liposomes, the terminal GalNAc residue was rapidly split off by Hex a. In striking contrast to ganglioside GM2, the major glycolipid substrate of Hex A, the enzymic hydrolysis of GD1a-GalNAc was not promoted by the GM2 activator protein, although the activator protein did bind GD1a-GalNAc to form a water-soluble complex. Pathobiochemical studies corroborate these results. After incorporation of [3H]GD1a-GalNAc into cultured skin fibroblasts from healthy subjects and from patients with different variants of GM2 gangliosidosis, its degradation was found to be strongly attenuated in mutant cells with Hex A deficiencies such as variant B (Tay-Sachs disease), variant B1 and variant 0 (Sandhoff disease), while in cells with variant AB (GM2 activator deficiency), its catabolism was blocked only at the level of GM2. In line with these metabolic studies, a normal content of GD1a-GalNAc was found in brains of patients who had succumbed to variant AB of GM2 gangliosidosis whereas in brains from variants B, B1, and 0, its concentration was considerably elevated (up to 19-fold). Together with studies on the enzymic degradation of GM2 derivatives with modifications in the ceramide portion, these results indicate that mainly steric hindrance by adjacent lipid molecules impedes the access of Hex A to membrane-bound GM2 (whose degradation therefore depends on solubilization by the GM2 activator) and in addition that the interaction between the GM2. GM2 activator complex and the enzyme must be highly specific.

Brain↗
PubMed1991

Human acid sphingomyelinase. Isolation, nucleotide sequence and expression of the full-length and alternatively spliced cDNAs.

Two types of partial cDNAs encoding human acid sphingomyelinase (EC 3.1.4.12; ASM) were recently isolated from fibroblast and placental cDNA libraries (Quintern, L. E., Schuchman, E.H., Levran, O., Suchi. M., Ferlinz, K., Reinke, H., Sandhoff, K., and Desnick, R. J. (1989) EMBO J. 8, 2469-2473). The cDNA inserts had identical sequences with the exception of an internal region; type 1 cDNAs (representing approximately 90% of the ASM cDNAs isolated) had 172 in-frame base pairs (bp), which were replaced in the type 2 cDNAs by a 40-bp in-frame sequence. Northern hybridization and RNase protection studies indicated that both type 1 and 2 transcripts were approximately 2.5 kilobases; therefore, efforts were directed to isolate full-length type 1 and 2 cDNAs by screening human placental, testis, hepatoma, and retinal cDNA libraries. In addition to type 1 and 2 cDNAs, a new type of ASM cDNA (type 3), which did not contain the type 1- or 2-specific regions, was isolated and sequenced. The full-length type 1 and the reconstructed full-length type 2 and 3 cDNAs were transiently expressed in COS-1 cells. Only the full-length type 1 transcript encoded catalytically active human ASM, demonstrating its functional integrity. The 2347-bp full-length type 1 placental cDNA (pASM-1FL) had an 87-bp 5'-untranslated region, an 1890-bp open reading frame encoding 629 amino acids, and a 370-bp 3'-untranslated sequence. The predicted location of the signal peptide cleavage site was after alanine 46. Two base differences were identified in codons 322 and 506 and shown to be polymorphisms with the common alleles having frequencies of 0.6 and 0.7, respectively. To determine the genomic organization of the type 1, 2, and 3 sequences, a 1665-bp genomic region containing both the unique type 1 (172 bp) and type 2 (40 bp) sequences was amplified by the polymerase chain reaction and sequenced. The 172-bp sequence was exonic, flanked by 5'- and 3'-intronic sequences of 1052 and 229 bp, respectively. The 40-bp type 2 sequence was intronic, occurring at the 5' end of the 1052-bp intron due to the use of a cryptic 5' donor splice site, which deleted the entire 172-bp exon and both flanking intronic sequences. The type 3 cDNA resulted from an alternative splicing event, which excised the 172-bp exon. These studies demonstrate the occurrence of alternatively splicing of the ASM transcript, but the existence of only one functional mRNA.

Amino Acid Sequence↗
PubMed1991

Sulfatide activator protein. Alternative splicing that generates three mRNAs and a newly found mutation responsible for a clinical disease.

The sulfatide activator protein, also known as SAP-1, is derived from a gene that generates an mRNA coding for four homologous proteins. Its physiological function is to stimulate hydrolysis of sulfatide by arylsulfatase A in vivo. A genetic defect in the sulfatide activator results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is itself caused by a genetic defect in arylsulfatase A. In a patient with sulfatide activator deficiency, a nucleotide transversion G722----C (counted from A of the initiation codon ATG) was found in the mRNA of the sulfatide activator precursor, resulting in the substitution of serine for Cys241 in the mature sulfatide activator. The remainder of the coding sequence was completely normal except for a polymorphism C to T in position 1389, which does not change the amino acid sequence. The patient produces at least three different forms of mRNA for the precursor. Two of them include a stretch of an additional 9 and 6 bases, respectively, within the sulfatide activator coding region. In normal individuals this stretch of additional bases has also been observed. This could be explained by the presence of a small 9-base pair exon which can be introduced, or not, by alternative splicing as a stretch of 9 or 6 bases into the mature mRNA. The shortest form of the mRNA yields an active sulfatide activator (Fürst, W., Schubert, J., Machleidt, W., Meier, H. E., and Sandhoff, K. (1990) Eur. J. Biochem. 192, 709-714).

Base Sequence↗
PubMedDate not supplied

Increased density of the thalamus on CT scans in patients with GM2 gangliosidoses.

In 13 patients, the GM2 gangliosidoses, Sandhoff disease and Tay-Sachs disease, were found to be constantly associated with homogeneously and symmetrically increased CT attenuation within the thalami. In the only patient examined with MR imaging, a T2-weighted sequence showed hypointense thalami. It is suggested that this finding is caused by an accumulation of calcium, associated with the intracellular storage of GM2 ganglioside. The finding of dense thalami may be useful as a specific diagnostic criterion for GM2 gangliosidoses. In a few patients with blocks in adjacent steps in the sphingolipid metabolism, this finding was not present.

Female↗
PubMed1990

[Diagnosis of lysosomal storage diseases. Pathomorphologic and biochemical possibilities].

Optical light and electron microscopy were used in studies into two cases of infantile GM2-gangliosidosis. The results are reported in this paper. The correlation has been evident between histological and ultrastructural findings. Reliable delimitation between two different variants of infantile GM2-gangliosidosis was achieved through biochemical investigation of postmortally cultured skin fibroblasts. A classical form with isolated hexosaminidase-A defect (Tay-Sachs disease) was distinguished from a second variant with complete defect of both isoenzymes of hexosaminidase (Sandhoff's disease). Biochemical investigation of postmortally cultured fibroblasts today has become indispensable to enlargement of autopsy findings from other storage diseases, as well.

Brain↗
PubMed1986

[Detection of homozygotes and heterozygote carriers of GM2-gangliosidosis].

11 patients with Tay-Sachs disease (TSD) and 4 patients with Sandhoff disease were identified using the methods of heat inactivation of hexosaminidase at 50 degrees C (3 and 4 hours) and electrofocusing on PAG-plates in the pH range 3.5-9.5. Ion exchange chromatography on DEAE cellulose DE-52 proved to be reliable for identification of heterozygotes in cases when the proband was not available. The incidence of TSD gene was estimated in 2 population samples--from the cities of Gomel and Kostroma. It was about 0.004 in the Gomel sample. No heterozygotes were detected in Kostroma.

Chromatography, Ion Exchange↗
PubMed1979

Preparation of radiolabeled GM2 and GA2 gangliosides.

GM2 and GA2 gangliosides from the brain of a patient who died of Sandhoff's disease were purified by solvent partition, silicic acid and silica gel column chromatography, and silica gel preparative thin-layer chromatography. They were tritiated in the terminal N-acetylgalactosamine residue using galactose oxidase and sodium [3H]borohydride with the inclusion of catalase and peroxidase into the oxidation reaction. The specific activities were 4.62 X 10(8) dpm/mumol of GM2 ganglioside and 5.54 X 40(7) dpm/mumol of GA2 ganglioside. The addition of catalase and peroxidase to the tritiation procedure is recommended.

Brain Chemistry↗