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S Kornfeld

Publications and source records attributed to S Kornfeld.

At least 109 records · Page 6Linked to original sources
PubMed1984

Identification of methylphosphomannosyl residues as components of the high mannose oligosaccharides of Dictyostelium discoideum glycoproteins.

Lysosomal enzymes isolated from the slime mold Dictyostelium discoideum bind to the mannose 6-phosphate receptor which is present in many mammalian cells. While binding to the receptor suggests that the slime mold enzymes possess the same mannose 6-phosphate recognition marker as their mammalian counterparts, initial structural studies of the phosphorylated oligosaccharides have indicated that the phosphate is attached to high mannose-type units via an unusual phosphodiester linkage (Freeze, H.H., Yeh, R., Miller, A.L., and Kornfeld, S. (1983) J. Biol. Chem. 258, 14874-14879). To identify the components of the phosphodiester group we have isolated the phosphorylated high-mannose oligosaccharides from D. discoideum AX3 cells labeled with [2-3H]mannose or [6-3H]glucosamine and from the differentiation medium of unlabeled cells. The major phosphorylated species contain one or two phosphodiester groups and an average of 6 or 7 mannose residues. The phosphodiesters are relatively stable to both acid and base hydrolysis, but upon strong acid hydrolysis (conditions that completely hydrolyze the oligosaccharide) mannose 6-phosphate residues are liberated. Through a combination of techniques, including fast atom bombardment and direct chemical ionization mass spectrometry, it is shown that the mannose 6-phosphate residues of the intact oligosaccharide are diesterified to methyl groups. This indicates that slime mold possesses a different biosynthetic pathway for the formation of phosphorylated high mannose-type oligosaccharides than is utilized by higher organisms.

Chromatography, High Pressure Liquid↗
PubMed1984

Beta-linked N-acetylgalactosamine residues present at the nonreducing termini of O-linked oligosaccharides of a cloned murine cytotoxic T lymphocyte line are absent in a Vicia villosa lectin-resistant mutant cell line.

The O-linked oligosaccharides of the cloned, murine cytotoxic T cell line B6.1.SF.1 were compared with the corresponding oligosaccharides from a Vicia villosa lectin-resistant mutant of B6.1.SF.1 called VV6 (Conzelmann, A., Pink, R., Acuto, O., Mach, J.-P., Dolivo, S., and Nabholz, M. (1980) Eur. J. Immunol. 10, 860-868). The VV6 mutant cells are deficient in binding sites for this GalNAc-specific lectin. Cells were grown in the presence of [3H]glucosamine and [3H] galactose to label the glycoproteins, and the desialyzed, alkaline borohydride-released oligosaccharides were isolated and characterized. The VV6 cells contained a series of O-linked oligosaccharides ranging in size from a disaccharide to a pentasaccharide. These were composed of galactose, N-acetylglucosamine, and N-acetylhexosaminitol, the latter sugar being derived from the reducing terminus. The predominant oligosaccharide had the partial structure Gal beta GlcNAc beta-(Gal beta)N-acetylhexosaminitol. In contrast, the analogous oligosaccharides of the parental cells contained additional beta-linked GalNAc residues located at nonreducing termini. The smallest of these had the structure GalNAc beta 1,4Gal beta-N-acetylhexosaminitol. Neither cell line contained significant amounts of terminal GalNAc linked to Ser/Thr which is the main binding site for the V. villosa B4 lectin on Tn erythrocytes (Tollefsen, S. R., and Kornfeld, R. (1983) J. Biol. Chem. 258, 5172-5176). These findings suggest that the major binding sites for the V. villosa lectin on the parental cytotoxic T cell line consist of structures containing beta 1,4-linked GalNAc residues at the nonreducing ends of conventional O-linked structures. The VV6 cells lack these beta-linked GalNAc residues, and this may account for their deficiency of V. villosa lectin-binding sites. In the following paper (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12536-12542), we demonstrate that the VV6 cells are missing the N-acetylgalactosaminyltransferase that is responsible for the synthesis of these unusual oligosaccharides.

Acetylgalactosamine↗
PubMed1984

A murine cytotoxic T lymphocyte cell line resistant to Vicia villosa lectin is deficient in UDP-GalNAc:beta-galactose beta 1,4-N-acetylgalactosaminyltransferase.

We have reported the presence of N-acetylgalactosamine linked beta 1,4 to galactose on O-linked oligosaccharides of a cloned murine cytotoxic T cell line and the absence of these residues from the O-linked structures of a Vicia villosa lectin-resistant mutant line, VV6, derived from parental B6.1.SF.1 cells (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12528-12535). This study shows that B6.1.SF.1 cells contain an enzyme which transfers N-acetylgalactosamine from UDP-GalNAc onto the O-linked tetrasaccharides of human glycophorin A, giving rise to pentasaccharides which contain beta-glycosidically linked N-acetylgalactosamine. Desialylated glycophorin was inactive as an acceptor. The enzyme also transfers N-acetylgalactosamine to the N-linked oligosaccharides of the Tamm-Horsfall glycoprotein. This glycoprotein is known to contain N-linked oligosaccharides with beta-linked N-acetylgalactosamine residues which constitute the Sda blood group determinant. This N-acetylgalactosaminyltransferase could not be detected in VV6 cells which can account for the lack of beta-linked N-acetylgalactosamine residues on its O-linked oligosaccharides. The two cell lines have comparable levels of UDP-GalNAc:apomucin N-acetylgalactosaminyltransferase, demonstrating that the enzyme deficiency in VV6 cells is selective. Both cell lines have a similar glycolipid content, with the major component being asialo-GM1. Since this glycolipid contains N-acetylgalactosamine linked beta 1,4 to galactose, it would appear that the N-acetylgalactosyltransferase involved in the biosynthesis of glycolipids is different from the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase. An independently derived murine CTL line also contains the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase, suggesting that the expression of this enzyme is a common characteristic of this type of cell line.

Animals↗
PubMed1984

The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1.

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.

Acetylglucosamine↗
PubMed1984

Cell surface antigens on avian erythroid cells. Their expression at various stages of cell differentiation.

Chicken erythroblasts transformed by a temperature-sensitive erythroblastosis virus (tsAEV) can terminally differentiate at the non-permissive temperature (42 degrees C). Morphological changes, as well as production of hemoglobin were shown to occur during maturation. The above system, as well as fractions of normal bone marrow cells were used to study the expression of cell surface antigens on erythroid cells. Four distinct reactivities were found with monoclonal antibodies (MAbs) and polyvalent sera: The MAb 4.5A5 was found to react with erythroblasts only. The MAb 4-M-12-26 reacted with tsAEV erythroblasts shifted to the non-permissive temperature (42 degrees C). The MAb 4.6C1 reacted weakly with erythroblasts and strongly with reticulocytes. The anti-Ery sera reacted with reticulocytes and erythrocytes. In addition the reactivity of MAb 4-M-12-26 was found only on virus-producing erythroblasts, though this MAb was found to react with myeloid cells which produce a helper virus of the same class.

Alpharetrovirus↗
PubMed1984

Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells.

The murine plasma cell line MOPC 315 efficiently targets newly synthesized acid hydrolases to lysosomes in spite of a marked deficiency in the level of the mannose 6-phosphate receptor (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). To better understand the routing of lysosomal enzymes in this cell line, pulse-chase experiments were performed with [2-3H]mannose and [35S]methionine followed by immunoprecipitation of beta-glucuronidase and IgA. By 3 h of chase, essentially all of the newly synthesized beta-glucuronidase had undergone proteolytic processing, suggesting that the molecules had reached lysosomes. At this time 30% of the pulse-labeled IgA was still intracellular. The oligosaccharides on the intracellular IgA were of the high mannose-type, while the secreted IgA contained processed, complex-type oligosaccharides. This indicates that the intracellular IgA was still in the endoplasmic reticulum or an early region of the Golgi complex when all of the beta-glucuronidase had reached lysosomes. Therefore, beta-glucuronidase and IgA must exit from the endoplasmic reticulum or the early Golgi complex at different rates, a finding that is inconsistent with bulk phase movement of these proteins from the endoplasmic reticulum to the trans Golgi complex. The addition of the ionophore monensin greatly slows the rate of IgA secretion from MOPC 315 cells and the molecules secreted have incompletely processed oligosaccharides. In contrast, monensin only slightly delays the transport of newly synthesized beta-glucuronidase to lysosomes and causes no significant alteration in the extent of oligosaccharide phosphorylation, a process that appears to occur in the early (cis) Golgi complex. However, the labeled beta-glucuronidase was deficient in sialylated, phosphorylated hybrid oligosaccharides whose biosynthesis requires the action of late stage oligosaccharide processing enzymes assumed to be localized in the trans Golgi complex.

Animals↗
PubMed1984

Two enzymes involved in the synthesis of O-linked oligosaccharides are localized on membranes of different densities in mouse lymphoma BW5147 cells.

Microsomal membranes from mouse lymphoma BW5147 cells were fractionated on a continuous sucrose gradient and assayed for two enzymes involved in the synthesis of O-linked oligosaccharides. Both enzymes were recovered in membranes that were less dense than the membranes containing the endoplasmic reticulum marker enzymes, glucosidase I and II. UDP-Gal:N-acetylgalactosamine-beta 1, 3-galactosyltransferase had a distribution that coincided with that of the galactosyltransferase that acts on asparagine-linked oligosaccharides. This latter enzyme has been immunolocalized to the trans Golgi elements. The UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase was recovered in a membrane fraction of intermediate density, between the endoplasmic reticulum and trans Golgi markers. These findings are consistent with the assembly of O-linked oligosaccharides occurring in at least two different Golgi compartments.

Animals↗
PubMed1983

Structural analysis of the asparagine-linked oligosaccharides from three lysosomal enzymes of Dictyostelium discoideum. Evidence for an unusual acid-stable phosphodiester.

Lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-phosphate and bind with high affinity to the phosphomannosyl receptor of human fibroblasts. In this study, we have partially characterized the Asn-linked oligosaccharide units present on these enzymes. [3H]Mannose-labeled alpha-D-mannosidase, beta-D-glucosidase, and beta-D-N-acetylglucosaminidase were purified from the spent growth medium of strain AX3 and glycopeptides were prepared by pronase digestion. Approximately 75% of the glycopeptides contained sulfate residues. These could be removed by solvolysis without degrading the underlying oligosaccharide. Following solvolysis (but not before), the oligosaccharides could be released by endo-beta-N-acetylglucosaminidase H, indicating the presence of high mannose-type units. Greater than 85% of the oligosaccharides contained one or two mannose 6-phosphate residues in the form of an unusual acid-stable phosphodiester. About 3% of the oligosaccharides contained phosphomonoesters and only 6% were neutral species. The major neutral oligosaccharide eluted in the position of Man9GlcNAc when analyzed by high performance liquid chromatography whereas the minor species appeared to be 1-2 residues larger. Acetolysis of the major phosphorylated fractions revealed that molecules with a single mannose 6-phosphate contained the phosphomannosyl residue on the branch linked alpha 1,6 to the beta-linked mannose whereas molecules with two phosphomannosyl residues had the residues on this branch as well as the branch linked alpha 1,3 to the beta-linked mannose. The mechanism of mannose phosphorylation in the slime mold must differ from that of mammalian cells since the phosphomannosyl residues are present as acid-resistant phosphodiesters rather than acid-labile phosphodiesters.

Asparagine↗
PubMed1983

Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor.

In human fibroblasts, the receptor for low density lipoprotein (LDL) is synthesized as a precursor of apparent Mr = 120,000 which is converted to a mature form of apparent Mr = 160,000, as determined by migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels (Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724). The current paper describes the relationship of N- and O-glycosylation to this post-translational modification. Oligosaccharides were analyzed from precursor and mature forms of LDL receptors that had been immunoprecipitated from cells grown in media containing radioactive sugars. In human epidermoid carcinoma A-431 cells, the receptor precursor appears to contain one N-linked high mannose oligosaccharide and approximately 6-9 N-acetylgalactosamine residues linked O-glycosidically to Ser/Thr residues. In the mature receptor, the O-linked oligosaccharides are mono- and disialylated species having the core structure of galactose leads to N-acetylgalactosamine leads to Ser/Thr. The single N-linked oligosaccharide of the mature receptor can either be a tri- or tetraantennary complex-type species. Similar results were obtained with normal human fibroblast receptor except that the O-linked oligosaccharides on the precursor are neutral disaccharides, of which one component is GalNAc and the N-linked complex type unit on the mature receptor is less branched. Since the addition of GalNAc residues to Ser/Thr residues precedes the conversion of N-linked high mannose-type oligosaccharides to complex-type structures, the transfer of N-acetylgalactosamine must occur prior to the entry of glycoproteins into the region of the Golgi containing the processing enzyme alpha-mannosidase I. We also studied the receptor from tunicamycin-treated cells and after treatment with neuraminidase. In addition, we analyzed the receptor synthesized by a lectin-resistant clone of Chinese hamster ovary cells that is deficient in adding galactose residues to both N- and O-linked oligosaccharides. These studies suggest that the apparent differences in molecular weight between the precursor and mature forms of the LDL receptor are largely, if not entirely, due to the addition of sialic acid and galactose residues to the O-linked GalNAc residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex↗
PubMed1983

The mod A mutant of Dictyostelium discoideum is missing the alpha 1,3-glucosidase involved in asparagine-linked oligosaccharide processing.

The recessive mutation, mod A, in the Dictyostelium discoideum strain M31 results in an alteration in the post-translational modification of lysosomal enzymes. We now report studies which indicate that mod A is deficient in glucosidase II, an enzyme which is involved in the processing of asparagine-linked oligosaccharides. [2-3H]Mannose-labeled glycopeptides were prepared from three purified mod A lysosomal enzymes and compared to the equivalent glycopeptides from parental enzymes. The mod A glycopeptides were deficient in high mannose oligosaccharides containing two phosphomannosyl residues and accumulated oligosaccharides with one phosphomannosyl residue. The phosphate was present in the form of an acid-stable phosphodiester in both instances. There was also an increase in the amount of nonphosphorylated high mannose oligosaccharides mod A and these were larger than the corresponding material from the parental enzymes. In addition, the nonphosphorylated oligosaccharides were only partially degraded by alpha-mannosidase, indicating the presence of a blocking moiety. In vitro enzyme assays demonstrated that the mod A cells cannot remove the inner 1 leads to 3-linked glucose from a glucosylated high mannose oligosaccharide. The cells are also deficient in membrane-bound neutral p-nitrophenyl-alpha-D-glucosidase activity. This activity has been attributed to glucosidase II in other systems. Removal of the outer 1 leads to 2-linked glucose from Glc3Man9Glc-NAc2 is normal, demonstrating the presence of glucosidase I activity. We conclude from these data that M31 cells are deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the glucosylated oligosaccharides of newly glycosylated proteins. This defect can explain the mod A phenotype and is proposed to be the primary genetic defect in these cells.

Asparagine↗
PubMed1983

Demonstration of the enzymatic mechanisms of alpha-N-acetyl-D-glucosamine-1-phosphodiester N-acetylglucosaminidase (formerly called alpha-N-acetylglucosaminylphosphodiesterase) and lysosomal alpha-N-acetylglucosaminidase.

An enzyme that is capable of removing the outer N-acetylglucosamine residues from phosphodiesters present on the high-mannose-type oligosaccharides of newly synthesized lysosomal enzymes has been described. This enzyme has been called an alpha-N-acetylglucosaminylphosphodiesterase, based upon its substrate specificity and on inhibitor studies. In this study it is demonstrated by the 18O enrichment method that the enzyme cleaves the C-O bond rather than the O-P bond, and therefore acts by a glycosidase type of mechanism. In addition, the enzyme has no significant activity toward alpha-N-acetylglucosamine 1-phosphate, and therefore requires an underlying phosphodiester for activity. In accordance with the IUB recommendations for enzyme nomenclature, it is therefore suggested that the enzyme be renamed alpha-N-acetyl-D-glucosamine-1-phosphodiester N-acetylglucosaminidase (systematic name, 2-acet-amido-2-deoxy-alpha-D-glucose 1-phosphodiester acetamidodeoxyglucohydrolase). For convenience, the trivial name phosphodiester glycosidase is proposed. Lysosomal alpha-N-acetylglucosaminidase also has a glycosidase type of mechanism but it is active toward alpha-N-acetylglucosamine 1-phosphate as well as phosphodiesters with outer N-acetylglucosamine residues.

Acetylglucosaminidase↗
PubMed1983

The spectrum of anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from glycoproteins. Structural studies and interactions with the phosphomannosyl receptor.

We have performed a detailed analysis of all the anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from the whole cell glycoproteins of P388D1 mouse macrophage-like cells labeled for 14 h with [2-3H]mannose. The major anionic species consisted of phosphorylated high mannose-type oligosaccharides containing one or two phosphomonoesters or phosphodiesters in several different positions. In addition we identified hybrid-type molecules containing one, two, or three sialic acid residues. A subset of the latter molecules also contained phosphodiesters or phosphomonoesters on another branch of the same oligosaccharide. Unlike previously reported hybrid-type molecules, these do not have a "bisecting" N-acetylglucosamine residue on the beta-linked mannose. Some of these oligosaccharides contained an unidentified acid-labile group on the core N-acetylglucosamine or the beta-linked mannose. The glycoproteins secreted by these cells were greatly enriched in hybrid oligosaccharides containing one sialic acid and one phosphomonoester. The interaction of the isolated oligosaccharides with bovine liver phosphomannosyl receptor immobilized on Affigel was analyzed. Oligosaccharides with phosphomonoesters were the only species that interacted with high affinity with the receptor, and molecules with two phosphomonoesters showed the best binding. The location of the phosphomonoester on the oligosaccharide influenced the degree of interaction with the receptor. Removal of accessible nonphosphorylated mannose residues improved the binding in some cases. These findings indicate that the generation of the physiological phosphomannosyl ligand on lysosomal enzymes involves removal of the blocking N-acetylglucosamine residues, trimming of certain mannose residues, and correct positioning of the phosphate esters.

Acetylglucosaminidase↗
PubMed1983

Detection of avian hematopoietic cell surface antigens with monoclonal antibodies to myeloid cells. Their distribution on normal and leukemic cells of various lineages.

The isolation and characterization of monoclonal antibodies reacting with cell surface antigenic determinants of normal and leukemic avian hematopoietic cells is described. The antibodies were produced by immunizing mice with normal macrophages, as well as with myeloid cells transformed with the avian acute leukemia viruses MC29, AMV and E26. Eleven antibodies were characterized for their reactivity with a variety of normal and leukemic cells of the myeloid, B- and T-lymphoid and of the erythroid cell lineage. Using several methods, they could be subdivided into five distinct types: I. Four antibodies were specific for the myeloid lineage, predominantly reacting with immature myeloid cells. II. One antibody reacted with mature and immature myeloid cells as well as with T-lymphoid cells. III. Four antibodies reacted with myeloid, erythroid and T-lymphoid cells. IV. One antibody reacted with myeloid as well as with T- and B-lymphoid cells. V. One antibody reacted with all kinds of chicken hematopoietic cells except erythrocytes. The first type of antibodies detected glycoproteins with MWs of 170 and 130 kD. The pattern of antigens precipitated varied with the different monoclonal antibodies of this group. The antibody of the fourth type precipitated a 30 kD polypeptide from extracts of myeloid and lymphoid cells. None of the other antibodies precipitated any detectable proteins.

Animals↗
PubMed1983

Identification and characterization of cells deficient in the mannose 6-phosphate receptor: evidence for an alternate pathway for lysosomal enzyme targeting.

Newly synthesized lysosomal enzymes acquire phosphomannosyl units, which allow binding of the enzymes to the mannose 6-phosphate receptor and subsequent translocation to lysosomes. In some cell types, this sequence of events is necessary for the delivery of these enzymes to lysosomes. Using a slime mold lysosomal hydrolase as a probe, we have identified three murine cell lines that lack the receptor and one line that contains very low (3%) receptor activity. Each of these lines synthesizes the mannose 6-phosphate recognition marker on its lysosomal enzymes, but, unlike cell lines with high levels of receptor, the cells accumulate oligosaccharides containing phosphomonoesters. The receptor-deficient lines possess high levels of intracellular acid hydrolase activity, which is contained in dense granules characteristic of lysosomes. The data suggest that intracellular mechanisms independent of the mannose 6-phosphate receptor must exist in some cells for the delivery of acid hydrolases to lysosomal organelles.

Animals↗