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R D Cummings

Publications and source records attributed to R D Cummings.

At least 109 records · Page 6Linked to original sources
PubMed1989

Characterization of an S-type lectin purified from porcine heart.

We have purified a carbohydrate-binding protein from porcine heart by affinity chromatography on asialofetuin-Sepharose and have characterized this protein with respect to its size, amino acid composition, partial amino acid sequence, and carbohydrate-binding specificity. Porcine heart lectin (PHL) has a subunit molecular mass of 14,700 and is immunologically cross-reactive with a polyclonal antibody raised against a lectin isolated from calf heart. The amino acid composition of PHL is similar to that of lectins that have been isolated from calf heart, bovine brain, and rat lung. Moreover, the primary sequences of four tryptic fragments (52 amino acids total) derived from PHL are closely related to sequences previously determined for 10 other vertebrate-derived lectins. The ability of PHL to agglutinate rabbit erythrocytes was inhibited only by oligosaccharides containing terminal beta-galactosyl residues. These data indicate that PHL is a vertebrate "S-type" lectin and provide further evidence that the structures and carbohydrate-binding specificities of these lectins are highly conserved across diverse vertebrate genera.

Amino Acid Sequence↗
PubMed1989

Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine.

Many studies have shown that the human blood fluke Schistosoma mansoni contains glycoproteins whose oligosaccharide side chains are antigenic in infected hosts. We report here that adult male schistosomes synthesize glycoproteins containing complex-type N-linked chains that have structural features not commonly found in mammalian glycoproteins. Adult male worms were incubated in media containing either [3H]mannose, [3H]glucosamine, or [3H]galactose, and the metabolically radiolabeled oligosaccharides on newly synthesized glycoproteins were analyzed. Schistosomes synthesize triantennary- and biantennary-like complex-type asparagine-linked chains that contain mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Interestingly, none of the complex-type chains contain sialic acid, and few of the chains contain galactose. Since N-acetylgalactosamine is not a common constituent of mammalian-derived N-linked chains, we investigated the position and linkage of this residue in the schistosome-derived glycopeptides. Virtually all of the N-acetylgalactosamine was beta-linked and in a terminal position. The unusual features of the S. mansoni glycoprotein oligosaccharides support the possibility that they may be involved in the host immune response to infection.

Acetylgalactosamine↗
PubMed1989

Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer.

We have developed a genetic approach to isolate cloned cDNA sequences that determine expression of cell surface oligosaccharide structures and their cognate glycosyltransferases. A cDNA library was constructed in a mammalian expression vector by using mRNA from a murine cell line known to express a UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase [(alpha 1-3)GT; EC 2.4.1.151]. This library was transfected into COS-1 cells, which lack expression of (alpha 1-3)GT. Transfected cells containing functional (alpha 1-3)GT cDNAs were detected and isolated with a lectin that recognizes the surface-expressed glycoconjugate product of the (alpha 1-3)GT enzyme. One cloned (alpha 1-3)GT cDNA was rescued from lectin-positive transfected cells. This cDNA contains a single long open reading frame that predicts a 394-amino-acid protein. No significant primary structure similarities were identified between this protein and other known sequences. However, the protein predicts a type II transmembrane topology similar to two other mammalian glycosyltransferases. This topology places the large COOH-terminal domain within the Golgi lumen; this domain was shown to be catalytically active when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. Biochemical analysis confirmed that this enzyme catalyzes a transglycosylation reaction between UDP-Gal and Gal(beta 1-4)GlcNAc to form Gal(alpha 1-3)Gal(beta 1-4)GlcNAc. This cloning approach may be generally applicable to the isolation of cDNAs encoding other mammalian glycosyltransferases.

Amino Acid Sequence↗
PubMed1988

Asparagine-linked oligosaccharides containing poly-N-acetyllactosamine chains are preferentially bound by immobilized calf heart agglutinin.

We have investigated the carbohydrate-binding specificity of a mammalian lectin, calf heart agglutinin, by determining the interaction of the immobilized lectin with a variety of complex-type Asn-linked oligosaccharides. Our results demonstrate that calf-heart agglutinin binds with high affinity to oligosaccharides containing the repeating disaccharide (3Gal beta 1-4GlcNAc beta 1)n or poly-N-acetyllactosamine sequence and that the presence of terminal beta-linked galactosyl residues is neither sufficient nor necessary for high affinity interactions.

Animals↗
PubMed1988

The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues.

We recently reported that the purified leukoagglutinin (designated MAL) from the seeds of the leguminous plant Maackia amurensis is a potent leukoagglutinin for the mouse lymphoma cell line BW5147 (Wang, W.-C., and Cummings, R. D. (1987) Anal. Biochem. 161,80). We and others have shown that this lectin is a weak hemagglutinin of human erythrocytes (Kawaguchi, T., Matsumoto, I., and Osawa, T. (1974) J. Biol. Chem. 249, 2786). We now report that leukoagglutination by MAL is inhibited by low concentrations of 2,3-sialyllactose (NeuAc alpha 2,3Gal beta 1,4Glc), but it is not inhibited by either 2,6-sialyllactose (NeuAc alpha 2,6Gal beta-1,4Glc), lactose, or free NeuAc. To further study the carbohydrate-binding specificity of this lectin, we investigated the interactions of immobilized MAL with glycopeptides prepared from the mouse lymphoma cell line BW5147 and from purified glycoproteins. We found that immobilized MAL interacts with high affinity with complex-type tri- and tetraantennary Asn-linked oligosaccharides containing outer sialic acid residues linked alpha 2,3 to penultimate galactose residues. Glycopeptides containing sialic acid linked only alpha 2,6 to penultimate galactose did not interact detectably with the immobilized lectin. Our analyses indicate that the interactions of complex-type Asn-linked chains with the lectin are dependent on sialic acid linkages and are not dependent on either the branching pattern of the mannose residues or the presence of poly-N-acetyllactosamine sequences.

Agglutination↗
PubMed1988

Retinoic acid-induced differentiation of the mouse teratocarcinoma cell line F9 is accompanied by an increase in the activity of UDP-galactose: beta-D-galactosyl-alpha 1,3-galactosyltransferase.

We report here that both the mouse teratocarcinoma F9 cells and F9 cells induced to differentiate by treatment with retinoic acid contain cell surface glycoconjugates with terminal alpha-linked galactose residues, as shown by agglutination of cells with antisera to blood type B, but not to type A. In addition, both cell types contain high numbers of binding sites for Griffonia simplicifolia-I, a lectin which binds to terminal alpha-linked galactose residues, although differentiated F9 cells contain approximately 50% more binding sites/cell for this lectin. We have also confirmed that differentiation is accompanied by a decrease in the expression of the fucose-containing stage-specific embryonic antigen (SSEA)-1, as evidenced by the fact that F9 cells, but not differentiated F9 cells, are agglutinated by monoclonal antibody to this antigen. Since these results indicate that surface glycoconjugates contain terminal alpha-linked galactose residues, we assayed cell extracts for the enzyme UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase. We have found that F9 cell extracts contain this activity, and differentiation results in a significant increase in the specific activity of the enzyme, from approximately 2 nmol/mg h in F9 extracts to 7 nmol/mg h in RA/F9 extracts. It has been suggested that the loss of the SSEA-1 antigen upon differentiation of F9 cells is due to decreased activity of the enzyme GDP-Fuc:beta-D-GlcNAc-alpha 1, 3-fucosyltransferase. We therefore determined the activities of this fucosyltransferase and several other glycosyltransferases, which included UDP-GlcNAc:beta-D-Gal-beta 1,3-N-acetylglucosaminyltransferase, UDP-Gal:beta-D-GlcNAc-beta 1,4-galactosyltransferase, and GDP-Fuc:beta-D-GlcNAc-alpha 1,6-fucosyltransferase. We have found that extracts from both cell types contain these enzyme activities; differentiation, however, does not result in substantial changes in any of these activities.

Agglutination↗
PubMed1988

Separation of oligosaccharides containing terminal alpha-linked galactose residues by affinity chromatography on Griffonia simplicifolia I bound to concanavalin A-sepharose.

The seeds of Griffonia simplicifolia contain a family of five isolectins (GS-I) (L. A. Murphy and I. J. Goldstein (1977) J. Biol. Chem. 252, 4739-4742) that bind with high affinity to glycoconjugates containing terminal nonreducing alpha-linked galactose residues. Here, we report that GS-I itself is bound via its high mannose-type, Asn-linked sugar chains to immobilized concanavalin A (Con A-Sepharose). The GS-I in the GS-I-Con A-Sepharose complex retains its ability to bind glycoconjugates containing terminal alpha-linked galactose residues. This convenient method to immobilize GS-I is rapid and quantitative. We have exploited this affinity system to separate oligosaccharides based on their number of terminal alpha-linked D-galactose residues.

Animals↗
PubMed1988

Characterization of the high mannose asparagine-linked oligosaccharides synthesized by Schistosoma mansoni adult male worms.

This report describes the structures of the high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult male worms. Adult male schistosomes were incubated in vitro in media containing either [2-3H]mannose, [6-3H]glucosamine or [6-3H]galactose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with Pronase and fractionation by chromatography on concanavalin A-Sepharose. Eleven percent of [3H]mannose incorporated into the schistosome glycopeptides was recovered in high mannose-type Asn-linked oligosaccharides which bound to the immobilized lectin. Upon treatment of [3H]mannose-labeled glycopeptide with endo-beta-N-acetylglucosaminidase H, the high mannose-type chains were released and their structures were determined by high performance liquid chromatography, methylation analysis, acetolysis and exoglycosidase digestion. The major species of high mannose-type chains synthesized by S. mansoni adult males have the composition Man7GlcNAc2, Man8GlcNac2 and Man9GlcNA2. Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by mammalian cells.

Animals↗
PubMed1988

Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex.

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.

Cell Compartmentation↗
PubMed1988

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula.

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.

Animals↗
PubMed1987

Schistosoma mansoni synthesizes glycoproteins containing terminal O-linked N-acetylglucosamine residues.

In this report, we describe our studies on the structures of the O-linked oligosaccharides in glycoproteins synthesized by the human blood fluke Schistosoma mansoni. Adult male schistosomes were incubated with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel newly synthesized glycoproteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were labeled by each of the radioactive precursors. Glycopeptides were prepared from radiolabeled glycoproteins by pronase treatment and fractionated on columns of concanavalin A-Sepharose and pea lectin-agarose. The O-linked oligosaccharides were released from glycopeptides by treatment with mild base/borohydride. All O-linked material was found in glycopeptides not bound by either of the immobilized lectins. The structures of the released chains were then analyzed by a variety of techniques. Our results demonstrate that the schistosomes synthesize glycoproteins containing two major types of simple O-linked sugar chains. One type, which represents a minor fraction of the O-linked oligosaccharides, contains N-acetylgalactosamine linked to peptide. These O-linked chains occur as terminal O-linked N-acetylgalactosamine and the O-linked disaccharide, galactose----N-acetylgalactosamine. Sialic acid was not present in either of these O-linked chains or in any other glycopeptides derived from adult male schistosomes. However, the major type of O-linked chain in glycoproteins synthesized by adult schistosomes is an unusual terminal O-linked N-acetylglucosamine linked to peptide. This latter structure represents approximately 10% of the total radioactive N-acetylglucosamine recovered in all glycopeptides. Our results also suggest the possibility that the O-linked oligosaccharides are highly clustered on the glycopeptides.

Acetylglucosamine↗
PubMed1987

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains.

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.

Animals↗
PubMed1987

An assay for leukoagglutinating lectins using suspension cultured mouse lymphoma cells (BW5147) stained with neutral red.

A sensitive and rapid assay for leukoagglutinating lectins has been developed. This assay utilizes neutral red-stained mouse lymphoma cells from the suspension cultured cell line BW5147. The agglutination of the stained cells can be monitored visually in a manner similar to that for conventional assays for erythroagglutinating lectins using erythrocytes. The activity of lekoagglutinating lectins that are not capable of agglutinating erythrocytes can be quantified by this assay. The utility of the assay was demonstrated using leukoagglutinating and erythroagglutinating lectins from the seeds of Phaseolus vulgaris and Maackia amurensis.

Animals↗
PubMed1985

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues.

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.

Carcinoma, Squamous Cell↗
PubMed1985

Structural characterization of murine Ia antigen N-linked oligosaccharides and localization of specific structures to two unique alpha-chain glycosylation sites.

The sequence of N-linked oligosaccharides of differentially glycosylated murine I-Ak alpha-(alpha 2- and alpha 3-) and beta-chains was determined. I-Ak beta-chains predominantly bear a biantennary complex oligosaccharide with a core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. The I-Ak alpha-chain has two N-linked glycosylation sites at Asn-82 and Asn-122. When Lubrol-insoluble alpha 3-chains are examined they are found to bear high-mannose oligosaccharides of either the Man9GlcNAc2 or Man8GlcNAc2 type at both sites. When Lubrol-soluble alpha 2-chains are examined, in about 85% of the molecules the Asn-82 site bears a biantennary complex oligosaccharide with core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. Interestingly, the Asn-122 site bears a variety of structures. In about 50% of the molecules, the structure at Asn-122 is a biantennary complex oligosaccharide without core fucose and with the peripheral sequence SA----Gal----GlcNAc----Man. In addition, it can bear other complex structures which we did not define further. The apparently restricted addition of fucose to the oligosaccharide at the alpha-Asn-82 site, even when both alpha-sites bear biantennary complex structures with the same peripheral sequence, is a feature unique to this system. The unusual variety of structures present at the alpha-Asn-122 site may indicate differential processing in different cell types.

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↗