Publications

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.

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.

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.

We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with [6- 3H]galactose or [2- 3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 microM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of [3H]mannose- and [3H]galactose-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid-treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine-linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.

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.

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.

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.

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.