David F. Smith

LAMP-1 in cho cells is a primary carrier of poly-N-acetyllactosamine chains and is bound preferentially by a mammalian S-type lectin

Recent studies indicate that some mammalian S-type lectins bind preferentially to oligosaccharides containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. We report here our investigation on the distribution of these sequences in glycoproteins in Chinese hamster ovary (CHO) cells and the interaction of glycoproteins containing PL chains with an immobilized S-type lectin (L14) from calf heart tissue. Our results demonstrate that PL chains are carried by a few high molecular weight glycoproteins which are bound by tomato-lectin Sepharose and one of these was precipitated by antibody to LAMP-1 (a lysosomal-associated membrane glycoprotein). More importantly, these high molecular weight glycoproteins, including LAMP-1, were bound with high affinity by L14. These results indicate that mammalian S-type lectins are highly specific in their interactions with glycoproteins and that LAMPs carry important recognition sequences for these lectins.

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing α-linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Galβ1-4[Fucα1-3]GlcNAcβ1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAcβ1-4[Fucα1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Galβ1-3[Fucα1-4]GlcNAc-R. Although 2′-fucosyllactose Fucα1-2Galβ1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fucα1-2Galβ1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Galβ1-3GlcNAcβ1-3Galβ1-3Glc; LNnT, Galβ1-4GlcNAcβ1-3Galβ1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5′-diphosphate-β-L-fucose

Expression of Human H-type α1,2-Fucosyltransferase Encoding for Blood Group H(O) Antigen in Chinese Hamster Ovary Cells EVIDENCE FOR PREFERENTIAL FUCOSYLATION AND TRUNCATION OF POLYLACTOSAMINE SEQUENCES

The human H(O) blood group is specified by the structure Fucα1-2Galβ1-R, but the factors regulating expression of this determinant on cell surface glycoconjugates are not well understood. To learn more about the regulation of H blood group expression, cDNA encoding the human H-type GDPFuc:β-D-galactoside α1,2-fucosyltransferase (α1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(α1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(α1,2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(α1,2)FT cells bound to the immobilized H blood group-specific lectin Ulex europaeus agglutinin-I (UEA-I), whereas none from parental CHO cells bound to the lectin. The glycopeptides from CHO(α1,2)FT cells binding to UEA-I contained polylactosamine [3Galβ1-4GlcNAcβ1-]n with the terminal sequence Fucα1-2Galβ1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(α1,2)FT cells caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in diantennary complex-type N-glycans. The O-glycans and glycolipids were not fucosylated by the H-type α1,2FT. Two major high molecular weight glycoproteins, one of which was shown to be the lysosome-associated membrane glycoprotein LAMP-1, preferentially contained the H-type structure and were bound by immobilized UEA-I. These results demonstrate that in CHO cells the expressed H-type α1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans containing polylactosamine and dramatically alters their length and sialylation.

A solid-phase assay for β-1,4-galactosyltransferase activity in human serum using recombinant aequorin

We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.

A microplate assay for analysis of solution-phase glycosyltransferase reactions: Determination of kinetic constants

We have developed a sensitive and simple method for assaying glycosyltransferase activities. This method makes use of solution-phase transferase reactions followed by capture to a microplate well coated with a substrate-specific monoclonal antibody. Sugar incorporation is quantitated by binding a saccharide-specific lectin and using bioluminescent aequorin for a reporter molecule. We demonstrate this method using the glycoprotein hormone-specific GalNAc-transferase and its acceptor substrate, agalacto-hCG. As little as 20 ng of agalacto-hCG with 32 nU of GalNAc-transferase gives a detectable signal with less than 10% of the acceptor sites substituted. In addition to this high sensitivity, by doing the transferase reactions in solution, we can assay up to 10 micrograms of agalacto-hCG. We show that this allows the determination of Km and Vmax kinetic constants that compare well to those obtained with radiolabeled nucleotide sugars.

A solid-phase assay for the activity of CMPNeuAc:Gal β1-4GlcNAc-R α-2,6-sialyltransferase

Abstract A solid-phase assay for the activity of CMPNeuAc:Gal β1-4GlcNAc- R α-2,6-sialyltransferase (2,6ST) has been developed. In the assay an acceptor glycoprotein is immobilized onto microtiter plate wells. The two glycoprotein acceptors used were asialofetuin (ASF), which contains oligosaccharides terminating in the sequence Gal β1-4GlcNAc- R , and a neoglycoprotein of bovine serum albumin containing covalently attached Gal β1-4GlcNAc- R units. Samples containing the donor CMPNeuAc and the 2,6ST were incubated with the immobilized acceptor to generate the product NeuAc α2-6Gal β1-4GlcNAc- R . The product was detected by a biotin-streptavidin system using the biotinylated plant lectin Sambucus nigra agglutinin (SNA), which binds to sialic acid in α-2,6, but not in α-2,3, linkage. The biotinylated SNA bound to the product was then detected with streptavidin and biotinylated forms of either alkaline phosphatase or the recombinant bioluminescent protein aequorin. The assay was optimized with respect to the commercially available 2,6ST and shown to be dependent on the concentration of acceptor and CMPNeuAc and proportional to the 2,6ST activity in the range of 20 to 400 μU in a 1-h assay. The solid-phase assay also allows for the selective detection of 2,6ST activity in human and fetal bovine serum, where the activity was proportional in the range of 0.1 to 2 μl of serum.

Special Considerations for Glycolipids and Their Purification

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.

Decreased biosynthesis of Forssman glycolipid after retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells. Lectin-affinity chromatography of the glycolipid-derived oligosaccharide

Glycolipids synthesized by the mouse teratocarcinoma F9 cells and F9 cells (RA/F9 cells) induced to differentiate by a 3-day treatment with 0.1 microM all-trans-retinoic acid were analyzed. Both F9 cells and RA/F9 cells were incubated in media containing either D-[6-3H]galactose or D-[6-3H]glucosamine; the metabolically-radiolabeled glycolipids were isolated and the oligosaccharides were released from the glycolipids by ozonolysis and alkali fragmentation. From both cells, a single major pentasaccharide was isolated from the mixture of neutral [3H]oligosaccharides by affinity chromatography on a column of immobilized Helix pomatia agglutinin. The structure of this oligosaccharide was analyzed by methylation analysis and specific exoglycosidase treatments and identified as the Forssman pentasaccharide alpha-D-GalpNAc-(1----3)-beta-D-GalpNAc-(1----4)-alpha-D-Galp-(1----4)-b eta-D- Galp-(1----4)-D-Glc. There was a 3-4-fold decreased amount of the Forssman pentasaccharide from RA/F9 cells relative to F9 cells. In contrast, there were no major differences between these cells in the levels of globoside, the precursor to Forssman glycolipid. To investigate the basis for the decline in Forssman glycolipid synthesis upon differentiation, the activity of UDP-D-Gal-NAc:GbOse4Cer alpha-(1----3)-N-acetyl-D-galactosaminyltransferase (Forssman synthase) was determined in extracts of both the F9 and RA/F9 cells. The specific activity of Forssman synthase was approximately 70% lower in differentiated relative to the nondifferentiated cells. These data demonstrated that F9 cells synthesize authentic Forssman glycolipid, and that its expression and the activity of Forssman synthase were decreased following induced cellular differentiation.