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E F Neufeld

Publications and source records attributed to E F Neufeld.

At least 55 records · Page 3Linked to original sources
PubMed1985

Human beta-hexosaminidase alpha chain: coding sequence and homology with the beta chain.

We have isolated a cDNA clone, p beta H alpha-5, from an adult human liver library that contains the entire coding sequence of the alpha chain of beta-hexosaminidase. The cDNA insert of p beta H alpha-5 is 1944 base pairs long and contains a 168-base-pair 5' untranslated region, a 186-base-pair 3' untranslated region, and an open reading frame of 1587 base pairs corresponding to 529 amino acids (Mr, 60,697). The first 17-22 amino acids satisfy the requirements of a signal sequence. A striking sequence homology with a published partial amino acid sequence for the beta chain [O'Dowd, B. F., Quan, F., Willard, H. F., Lamhonwah, A. M., Korneluk, R. G., Lowden, J. A., Gravel, R. A. & Mahuran, D. J. (1985) Proc. Natl. Acad. Sci. USA 82, 1184-1188] suggests that both chains may have evolved from a common ancestor. A shorter alpha-chain cDNA was found to hybridize to the long arm of chromosome 15, the known location for the alpha-chain gene. In addition, we isolated another alpha-chain cDNA clone, p beta H alpha-4, from a simian virus 40-transformed human fibroblast library that contained an extra 453-base-pair piece at its 3' end. A probe consisting of this additional sequence hybridized exclusively to a single mRNA species (2.6 kilobases) in mRNA preparations from cultured human fibroblasts. In contrast, p beta H alpha-5 hybridized to both a 2.1-kilobase major and a 2.6-kilobase minor mRNA species in these same mRNA preparations, indicating the presence of two distinct alpha-chain mRNA species differing at the 3' end. Fibroblasts from an Ashkenazi Jewish patient with classic Tay-Sachs disease were deficient in both species of mRNA, confirming their genetic relationship.

Amino Acid Sequence↗
PubMed1985

Neurochemical characterization of canine alpha-L-iduronidase deficiency disease (model of human mucopolysaccharidosis I).

This report presents the neurochemical findings on the first dog to die with deficiency of alpha-L-iduronidase (mucopolysaccharide alpha-L-iduronohydrolase; EC 3.2.1.76). The principal findings were (a) markedly increased glycosaminoglycan content in all neural tissues examined (from threefold in sciatic nerve to 15-fold in brainstem), (b) a modest increase in levels of gangliosides GM2, GM3, and GD3, particularly in gray matter, (c) excessive accumulation of glycosaminoglycans in the CSF, (d) the increased glycosaminoglycans were dermatan sulfate and heparan sulfate, and (e) the molecular weights of the liver glycosaminoglycans were shifted toward smaller sizes, indicating partial degradation. The canine disorder thus resembles mucopolysaccharidosis I in all aspects.

Animals↗
PubMed1984

Faulty association of alpha- and beta-subunits in some forms of beta-hexosaminidase A deficiency.

We have previously described the kinetics of association of the alpha- and beta-subunits of beta-hexosaminidase A in intact cultured human fibroblasts, using biosynthetic labeling and immunoprecipitation with antisera that distinguish between monomeric and associated alpha-chains (Proia, R. L., d'Azzo, A., and Neufeld, E. F. (1984) J. Biol. Chem. 259, 3350-3354). We now show lack of alpha-beta association in fibroblasts of several individuals deficient in beta-hexosaminidase A (5 patients with nonclassic forms of Tay-Sachs disease and 2 asymptomatic siblings). Defective association was accompanied by markedly reduced (less than one-tenth of normal) conversion of the alpha-chain precursor of Mr = 67,000 to the mature lysosomal form of Mr = 54,000. Analysis by hybridization with fibroblasts lacking the alpha- or beta-chain showed that the association defect resided in the alpha-chain. Most of the cell strains studied also had decreased synthesis of the alpha-chain, suggesting compound heterozygosity with the Ashkenazi Tay-Sachs (no synthesis) allele. An unusual feature of the association defect is the variability in the resulting clinical manifestations, even within families, implying that other factors determine the adequacy of the residual associated beta-hexosaminidase A in vivo.

Adolescent↗
PubMed1984

Association of alpha- and beta-subunits during the biosynthesis of beta-hexosaminidase in cultured human fibroblasts.

Subunit association of beta-hexosaminidase was studied in intact fibroblasts using antisera that discriminate between free and associated alpha-chains. These were anti-beta-hexosaminidase A (anti-alpha beta), which precipitated all alpha-chains, free or associated; anti-beta-hexosaminidase B (anti-beta beta), which precipitated those alpha-chains that were associated with beta; and anti-alpha-chains, which recognized only monomeric alpha-chains. After biosynthetic labeling, beta-hexosaminidase or its free alpha-subunit were immuno-precipitated from extracts of cells and medium with the aid of protein A-bearing Staphylococcus aureus, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Pulse-chase labeling showed that the alpha-chains existed predominantly in the monomeric precursor form during the first 5 h, and then began to accumulate in the mature (lysosomal) associated alpha beta form. Precursor alpha beta complexes were secreted, along with some precursor alpha monomers; the latter were catalytically inert. Both alpha- and beta-chains were phosphorylated (a Golgi modification) prior to association. Thus alpha-beta association probably occurred in the Golgi area before transfer to lysosomes and before secretion. Cycloheximide inhibited the association and subsequent maturation of preformed alpha-chains, perhaps by causing a depletion of a pool of beta-chain precursor upstream from the site of subunit association. In fibroblasts from a patient with Sandhoff disease, that produced no beta-chains, the alpha-chains self-associated but their maturation was markedly decreased. We suggest that association with beta-chains is necessary not only for acquisition of catalytic activity but also for transport of alpha-chains to lysosomes.

Cell Line↗
PubMed1984

Morphologic and biochemical studies of canine mucopolysaccharidosis I.

This report presents the necropsy and biochemical findings on the first dog to die with alpha-L-iduronidase deficiency (mucopolysaccharidosis I, MPS I). Gross pathologic features, light- and electron-microscopic findings, and tissue enzyme, glycosaminoglycan (GAG), and sphingolipid levels are compared with the human disease counterpart and the previously described feline model. Results lend further support for the similarities of the canine disease and human MPS I.

Animals↗
PubMed1983

Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells.

The natural history of the mannose 6-phosphate receptor was examined by radiolabeling cells in monolayers or in suspension; the receptor was isolated by immuno- or affinity precipitation followed by polyacrylamide gel electrophoresis. The receptor was found to contain asparagine-linked oligosaccharide chains and phosphorylated serine residues. Newly made receptor was sensitive to endo-beta-N-acetylglucosaminidase H (endo-H) and was slowly converted to a mature endo-H resistant form; phosphate was found on the mature receptor only. The receptor had an apparent molecular weight of 215,000 at all times, as determined under reducing and denaturing conditions; unreduced receptor had a greater electrophoretic mobility, suggesting the presence of intrachain disulfide linkages. The synthesis of immunoreactive receptor occurred with a lag of 50 min and of functional receptor with a lag of 70 min, indicating a requirement for some post-translational event(s) for acquisition of immunoreactivity and binding activity. Maturation of asparagine-linked oligosaccharides was not the requisite modification, since endo-H sensitive or deglycosylated receptor bound to both antibody and to insoluble phosphomannan; however, much less immunoreactive and functional receptor was detected in the presence of tunicamycin. Immunoprecipitable [3H]leucine-labeled receptor was degraded with a t1/2 of 16 h and 6 h for cells in monolayers and suspension, respectively, whereas 32P was lost with a corresponding t1/2 of 2.3 and 4 h. A pool of cell surface mannose 6-phosphate receptor was identified by separation on Percoll gradients as well as by iodination of cells with 125I; receptor in this pool was resistant to endo-H and had a t1/2 similar to that of the total [3H]leucine-labeled receptor, even in the presence of a saturating concentration of ligand. During endocytosis, ligand (beta-galactosidase) and 125I-receptor separated, the ligand accumulating within lysosomes. These results are consistent with current concepts of recycling of the mannose 6-phosphate receptor.

Animals↗
PubMed1983

Synthesis and maturation of cross-reactive glycoprotein in fibroblasts deficient in arylsulfatase A activity.

The biosynthesis of arylsulfatase A was studied in cultured fibroblasts by pulse-chase labeling with [2-3H]mannose; the enzyme was isolated by immunoprecipitation and denaturing polyacrylamide gel electrophoresis. In normal fibroblasts, and in fibroblasts from a patient with multiple sulfatase deficiency, the enzyme was synthesized as a glycoprotein of apparent molecular weight of 59,000; half of it was processed over a period of 4 days to Mr = 57,000. The precursor chain of Mr = 59,000 was secreted in the presence of 10 mM NH4Cl. An immunoprecipitable glycoprotein of normal size was synthesized by fibroblasts from two unrelated patients with metachromatic leukodystrophy, but this material disappeared within twenty hours. In fibroblasts from an individual with pseudo-deficiency of arylsulfatase A, the immunoprecipitable precursor glycoprotein was smaller (Mr = 56,000). The synthesis of cross-reactive proteins with altered properties supports the concept of allelic mutations as the genetic basis of metachromatic leukodystrophy and of arylsulfatase A pseudo-deficiency.

Alleles↗
PubMed1983

A canine model of human alpha-L-iduronidase deficiency.

A disease discovered in three Plott Hound littermates was found to be associated with a profound and specific deficiency of alpha-L-iduronidase (mucopolysaccharide alpha-L-iduronohydrolase; EC 3.2.1.76) in fibroblasts and leukocytes. The pedigree was consistent with autosomal recessive inheritance. A markedly increased amount of dermatan sulfate and heparan sulfate was excreted in urine. Fibroblasts cultured from the skin of the affected dogs accumulated excessive 35S-labeled mucopolysaccharide; this accumulation could be decreased to a normal level by exogenous human high-uptake alpha-L-iduronidase (Hurler corrective factor) as well as by secretions of normal human or canine fibroblasts. The correction was inhibited by mannose 6-phosphate. Maturation of alpha-L-iduronidase in normal canine fibroblasts followed the pathway previously observed in human fibroblasts; no cross-reactive material was observed in the cells or in secretions from the fibroblasts of the affected dogs. The canine disorder thus resembles mucopolysaccharidosis I in all biochemical parameters tested; the clinical appearance of the animals is closest to Hurler-Scheie syndrome, a form of alpha-L-iduronidase deficiency of intermediate severity. The animal model should prove valuable for therapeutic experiments.

Animals↗
PubMed1982

Administration of iduronate sulfatase by plasma exchange to patients with the Hunter syndrome: a clinical study.

The Hunter syndrome (MPS II) is the only mucopolysaccharidosis in which there is appreciable activity of the deficient enzyme in normal plasma. We performed enzyme-replacement treatment by plasma exchange in five Hunter syndrome children. Carefully monitoring the cardiovascular status, we administered monthly single plasma volume exchanges for a 3 to 8 mo period. The results indicate a substantial gain of enzyme activity, persisting with a t50% = 19 +/- 5 hr. The maximal level and persistence of increased enzyme activity did not change after repeated exchanges, suggesting that immune responses were not elicited. Despite this, no demonstrable clinical benefit was apparent when the study group was compared with an age-matched control group of Hunter syndrome patients.

Adolescent↗
PubMed1982

Synthesis of beta-hexosaminidase in cell-free translation and in intact fibroblasts: an insoluble precursor alpha chain in a rare form of Tay-Sachs disease.

RNA was isolated from human term placenta or cultured fibroblasts and translated in a rabbit reticulocyte system in the presence of [35S]methionine; the translation products were immunoprecipitated with antisera made against beta-hexosaminidase or its isolated alpha and beta chains and analyzed by polyacrylamide gel electrophoresis. The largest translated alpha and beta chain polypeptides had Mrs of 65,000 and 59,000, respectively. These are approximately equal to 2,000 greater than the Mrs of precursor chains synthesized by intact fibroblasts and deglycosylated with endo-beta-N-acetylglucosaminidase H suggesting the presence of a signal sequence. RNA of fibroblast cultures from two patients with Sandhoff disease did not direct the translation of immunoprecipitable beta chain; RNA of fibroblast cultures from four patients with Tay-Sachs disease (three of Ashkenazi Jewish descent and one of non-Jewish descent) did not direct the translation of immunoprecipitable alpha chain. In contrast, a normal amount of alpha chain was made in the presence of RNA from the fibroblast culture of another non-Jewish Tay-Sachs patient (GM 1110). Intact fibroblasts from this patient also synthesized the alpha chain as shown by labeling with [3H]leucine; however, strong detergent was required for extraction. The alpha chain could be labeled with [3H]mannose but not with [32P]phosphate; it was neither secreted nor accumulated in the proteolytically processed form, and it disappeared within a day of synthesis. A plausible though not unique explanation is that the insoluble alpha chain is not transported from the endoplasmic reticulum (the site of glycosylation) to the Golgi apparatus (the site of phosphorylation) nor to further points of destination--lysosomes and the exterior of the cell.

Cells, Cultured↗
PubMed1982

Nonuniform deficiency of hexosaminidase A in tissues and fluids of two unrelated individuals.

Serum samples from two unrelated, clinically normal individuals lacked detectable hexosaminidase A by heat inactivation and electrophoretic analysis. In contrast, 15 and 17% of the hexosaminidase in their leukocytes and 23 and 26% of the hexosaminidase of their cultured fibroblasts had the heat stability and electrophoretic properties of the A form of this enzyme. An in vitro measurement of fibroblasts GM2 ganglioside-beta-galactosaminidase was in the range expected for Tay-Sachs disease (TSD) heterozygotes (2.5 and 3.1 versus a normal mean of 3.7). In contrast, fibroblasts from a patient with TSD, analyzed in an identical fashion, contained no detectable activity. Ten days after addition of labeled GM2 ganglioside to the medium of the cultured fibroblasts, 43 and 59% of the radioactivity taken up by the cells of these patients remained as unhydrolyzed ganglioside as compared with 94% in TSD fibroblasts and 42% in control cells. An analysis of sphingolipid composition by high performance liquid chromatography although the endogenous level of GM2 was elevated in TSD fibroblasts (0.39 nmoles/mg protein) there was no increase in the cells of these patients (0 and 0.12 versus control of 0.17 nmoles/mg protein). Finally, the synthesis of hexosaminidase was examined by an electrophoretic analysis of immunoprecipitates of the enzyme precursors that had been radiolabeled by culturing fibroblasts in medium containing [3H]-leucine. These studies revealed a normal pattern of biosynthesis, processing and secretion of the alpha and beta chains. The ratio of the alpha chain to the beta chain, however, was in the range expected for TSD heterozygotes.

Adult↗
PubMed1982

Pleiotropic mutations of lysosomal function in human patients and in Chinese hamster ovary cells.

The biosynthesis of lysosomal enzymes entails a complex series of events. The nascent proteins enter the endoplasmic reticulum for glycosylation. Phosphorylation of the enzymes subsequently occurs in the Golgi area. Equipped with a phosphomannose recognition marker, the enzymes are bound to specific receptors and translocated to lysosomes. Mutations are known in the following steps: early glycosylation (CHO cells), phosphorylation (patients with I-cell disease and pseudo-Hurler polydystrophy), and receptor binding (CHO cells).

Animals↗