Search PubMed⌕ Search

Biomedical subjects

S Kornfeld

Publications and source records attributed to S Kornfeld.

At least 145 records · Page 8Linked to original sources
PubMed1981

Identification of a variant of mucolipidosis III (pseudo-Hurler polydystrophy): a catalytically active N-acetylglucosaminylphosphotransferase that fails to phosphorylate lysosomal enzymes.

Fibroblasts from patients with I-cell disease (mucolipidosis II) or with pseudo Hurler polydystrophy (mucolipidosis III) are markedly deficient in UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. As a consequence, the common phosphomannosyl recognition marker of acid hydrolases is not regenerated, and these enzymes are not targeted to lysosomes. We have developed a sensitive assay for the transferase that uses alpha-methyl mannoside as an acceptor, and this has allowed us to distinguish between fibroblasts from these two types of patients. The enzyme activity is less in the former than in the latter (less than 0.4-2.0 pmol/mg per hr vs 2.9-39.4). This may provide an explanation for the difference in clinical severity between the two syndromes, However, in two siblings with pseudo Hurler polydystrophy (GM 3392), the enzyme activity was normal when assayed by using alpha-methyl mannoside as acceptor whereas it was low when assayed with endogenous glycoprotein acceptors or with human placental beta-hexosaminidase A. The apparent Km values of the mutant enzyme toward alpha-methyl mannoside, high-mannose oligosaccharides, and UDP-GlcNAc were not different from those of the normal enzyme. Mixing experiments demonstrated that the mutant fibroblasts contained endogenous acceptors and were free of inhibitors. We conclude that the N-acetylglucosaminylphosphotransferase in the mutant fibroblasts has normal catalytic activity but is defective in the ability to recognize lysosomal enzymes as specific substrates for phosphorylation. This variant form of pseudo Hurler polydystrophy demonstrates the biological importance of this recognition mechanism in the generation of the phosphomannosyl marker.

Glucosyltransferases↗
PubMed1981

Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5'-diphosphate-N-acetylglucosamine: glycoprotein N-acetylglucosaminylphosphotransferase activity.

Newly synthesized acid hydrolases, destined for transport to lysosomes, acquire a phosphomannosyl targeting signal by the transfer of N-acetylglucosamine 1-phosphate from uridine 5'-diphosphate (UDP)-N-acetylglucosamine to a mannose residue of the acid hydrolase followed by removal of the outer, phosphodiester-linked N-acetylglucosamine to expose 6-phosphomannose. This study demonstrates that fibroblasts from patients with the lysosomal enzyme storage diseases, I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III), are severely deficient in UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminylphosphotransferase, the first enzyme of the sequence. The N-acetylglucosaminylphosphotransferase activity (assayed using endogenous acceptors) in cultures from six normal subjects ranged from 0.67 to 1.46 pmol N-acetylglucosamine-1-phosphate transferred/mg protein per h, whereas five pseudo-Hurler polydystrophy and five I-cell disease cultures transferred less than 0.02 pmol/mg protein per h. The activity in five other pseudo-Hurler cultures ranged from 0.02 to 0.27 pmol transferred/mg protein per h. The activity of alpha-N-acetylglucosaminyl phosphodiesterase, the enzyme responsible for phosphomonoester exposure, is normal or elevated in cultured fibroblasts from both I-cell disease and pseudo-Hurler polydystrophy patients. The deficiency of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminylphosphotransferase explains the biochemical abnormalities previously observed in I-cell disease and pseudo-Hurler polydystrophy.

Cells, Cultured↗
PubMed1980

Structural studies of phosphorylated high mannose-type oligosaccharides.

Phosphomannosyl residues on acid hydrolases serve as recognition markers which target these enzymes to lysosomes. We have found that the oligosaccharide units of newly synthesized beta-glucuronidase contain phosphate residues in diester linkage between mannose and alpha-linked N-acetylglucosamine residues (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633--6639). To obtain larger amounts of these molecules for structural studies, total cellular glycopeptides were isolated from [2-3H]mannose-labeled mouse lymphoma cells, and the phosphorylated oligosaccharides were released by endo-beta-N-acetylglucosaminidase CII and H and isolated by gel filtration and ion exchange chromatography. The fractions were characterized by alpha-mannosidase digestion before and after removal of the phosphate residues and by acetolysis. We also determined whether the phosphate was present as a phosphomonoester or as a diester. The phosphorylated oligosaccharides consisted of a family or related molecules, all of which contained a high mannose-type oligosaccharide core. The major class consisted of isomers containing a single phosphate in diester linkage to 1 of 3 mannose residues of the underlying oligosaccharide. The second class contained isomers with two phosphodiester groups located at five different positions of the oligosaccharide. In both of these classes, the phosphodiester group could be converted to a phosphomonoester by pig liver alpha-N-acetylglucosaminidase, indicating that the cover is alpha-linked N-acetylglucosamine. The third class was similar to the second except that the 2 phosphate residues were present as monoester groups. The last class contained molecules with a single phosphomonoester group. These molecules differed from those with single phosphodiester groups in that the core oligosaccharides were smaller. This is consistent with their being more mature species which have undergone partial processing. These data demonstrate that phosphorylation can occur at 5 separate mannose residues on the high mannose-type oligosaccharides. Individual molecules can have 1, 2, and perhaps even 3 phosphate residues. The majority of the newly synthesized phosphorylated oligosaccharides contain phosphate groups in diester linkage.

Acetylglucosaminidase↗
PubMed1980

Mouse lymphoma cell lines resistant to pea lectin are defective in fucose metabolism.

Two mutants of the BW5147 mouse lymphoma cell line have been selected for their resistance to the toxic effects of pea lectin. These cell lines, termed PLR1.3 and PHAR1.8 PLR7.2, have a decreased number of high affinity pea lectin-binding sites (Trowbridge, I.S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Intact cell labeling experiments using [2-3H]mannose indicated that PLR1.3 cells have a block in the conversion of GDP-[3H]mannose to GDP-[3H]fucose whereas PHAR1.8 PLR7.2 cells appear to be blocked in the transfer of fucose from GDP-[3H]fucose to glycoprotein acceptors. In vitro experiments with extracts of PLR1.3 cells confirmed the failure to convert GDP-mannose to GDP-fucose and indicated that the defect is in GDP-mannose 4,6-dehydratase (EC 4.2.1.47), the first enzyme in the conversion of GDP-mannose to GDP-fucose. The block in the PLR1.3 cells could be bypassed by growing the cells in the presence of fucose, demonstrating that an alternate pathway for the production of GDP-fucose presumably via fucose 1-phosphate is functional in this line. PLR1.3 cells grown in 10 mM fucose showed normal high affinity pea lectin binding. PHRA1.8 PLR7.2 cells synthesize GDP-fucose and have normal or increased levels of GDP-fucose:glycoprotein fucosyltransferase when assayed in vitro. The fucosyltransferases of this clone can utilize its own glycoproteins as fucose acceptors in in vitro assays. These findings indicate that this cell line fails to carry out the fucosyltransferase reaction in vivo despite the fact that it possesses the appropriate nucleotide sugar, glycoprotein acceptors, and fucosyltransferase. The finding of decreased glycoprotein fucose in two independent isolates of pea lectin-resistant cell lines and the restoration of high affinity pea lectin binding to PLR1.3 cells following fucose feeding strongly implicates fucose as a major determinant of pea lectin binding.

Animals↗
PubMed1980

Identification of a rat liver alpha-N-acetylglucosaminyl phosphodiesterase capable of removing "blocking" alpha-N-acetylglucosamine residues from phosphorylated high mannose oligosaccharides of lysosomal enzymes.

We recently reported that the high mannose-type oligosaccharides of the biosynthetic intermediates of beta-glucuronidase contain phosphate groups in diester linkage between mannose residues and outer alpha-linked N-acetylglucosamine residues (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We now describe an alpha-N-acetylglucosaminyl phosphodiesterase from rat liver that is capable of removing the N-acetyl-glucosamine residues, leaving phosphomonoester groups on the high mannose oligosaccharide units. This activity is greatly enriched in smooth membrane preparations. It can be distinguished from a lysosomal alpha-N-acetylglucosaminidase by several criteria, including subcellular localization and differential inhibition by amino sugars. In addition, human fibroblasts with mutations which lead to a deficiency of the lysosomal activity have normal levels of the alpha-N-acetylglucosaminyl phosphodiesterase. This enzyme may be involved in the "unmasking" of the phosphomannosyl recognition marker on newly synthesized acid hydrolases which could then direct the targeting of these enzymes to lysosomes.

Acetylglucosaminidase↗
PubMed1980

An autosomal dominant gene regulates the extent of 9-O-acetylation of murine erythrocyte sialic acids. A probable explanation for the variation in capacity to activate the human alternate complement pathway.

Nydegger et al. (4) have reported that the difference in susceptibility of erythrocytes from different inbred murine strains to lysis by the human alternate complement pathway is determined by an autosomal locus. We have found a good correlation between the degree of O-acetylation of the erythrocyte sialic acid residues and the susceptibility to complement lysis, whereas there was no correlation between total erythrocyte sialic acid content and complement sensitivity. The major O-acetylated species in all the murine strains is 9-O-acetyl-N-acetylneuraminic acid. We propose that the autosomal dominant locus, which determines complement sensitivity, acts by influencing the extent of 9-O-acetylation of the erythrocyte sialic acid residues. By using recombinant inbred strains, we determined that this genetic locus is probably located on chromosome 9. The nature of the gene product remains unknown.

Acetylation↗
PubMed1980

Characterization of the oligosaccharide units of the fourth component of complement (Ss protein) synthesized by murine macrophages.

The glycosylation of murine C4 (Ss protein) synthesized by peritoneal macrophages has been investigated. Both the intracellular precursor, P-C4 (185), and the C4 alpha- and beta-chains that were secreted into medium were found to be glycosylated; however, no carbohydrate units were detected on the gamma-chain. Analyses of the oligosaccharide units showed that P-C4 (185) appears to contain both a "complex" and a "high mannose" carbohydrate group, the alpha-chain a "complex" group and the beta-chain a "high mannose" carbohydrate unit.

Acetylglucosaminidase↗
PubMed1979

Purification and characterization of a rat liver Golgi alpha-mannosidase capable of processing asparagine-linked oligosaccharides.

Studies in intact cells have shown the following processing reaction to occur during Asn-linked oligosaccharide biosynthesis (M, mannose; GlcNAc, N-acetylglucosamine): Formula: (See Text) We have identified a rat liver Golgi enzyme which catalyzes this reaction in vitro. This alpha-mannosidase has been purified 3,000 to 6,000-fold by subcellular fractionation, Triton X-100 solubilization, and ion exchange and hydroxylapatite chromatography. The purified enzyme has a pH optimum between 6.0 and 6.5 and a Km between 17 and 100 microM for a processing intermediate. The enzyme shows specificity for alpha 1,2-linked mannose residues. Structural analysis of the in vitro reaction products reveal that specific intermediates are formed in the conversion of the (Man)9GlcNAc oligosaccharide to the (Man)5GlcNAc oligosaccharide. Heat inactivation studies are consistent with the possibility that one enzyme activity is responsible for this conversion. The alpha 1,2-specific mannosidase described here appears to be distinct from two other rat liver Golgi alpha-mannosidase activities based on differential substrate specificity, inhibitor susceptibility, and detergent extractability.

Animals↗
PubMed1979

Biosynthesis of lipid-linked oligosaccharides. Isolation and structure of a second lipid-linked oligosaccharide in Chinese hamster ovary cells.

Previous work has shown that vesicular stomatitis virus-infected Chinese hamster ovary cells contain a major high molecular weight lipid-linked oligosaccharide which is transferred en bloc to protein during the formation of the asparagine-linked complex-type oligosaccharides of the vesicular stomatitis virus G protein (Tabas, I., Schlesinger, S., and Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722). We now report the characterization of a second, lower molecular weight lipid-linked oligosaccharide. The oligosaccharide portion of this molecule was isolated and its structure was determined by methylation analysis, digestion with exoglycosidases, acetolysis and Smith periodate degradation to be: (formula: see text). Several lines of evidence are presented which indicate that this lipid-linked oligosaccharide is primarily involved in the assembly of the major lipid-linked oligosaccharide rather than in the direct glycosylation of proteins.

Animals↗