Publications

The factors regulating modifications of terminal beta-Gal residues in lactosaminyl (Gal beta 1-->4GlcNAc beta-->R) units in intact glycoproteins are not well understood. To examine these factors, rat liver alpha 2,3 sialyltransferase (alpha 2,3ST) and alpha 2,6 sialyltransferase (alpha 2,6ST) and the murine alpha 1,3 galactosyltransferase (alpha 1,3GT) were incubated with a variety of well-defined desialylated glycoproteins and with glycoproteins in extracts of the Lec2 mutant CHO cells. Lec2 cells constitutively synthesize nonsialylated glycoproteins with terminal lactosaminyl sequences. The results demonstrate that each enzyme displays preferences for glycoprotein acceptors and in the types of N-glycans recognized. The alpha 2,3ST, in contrast to the alpha 2,6ST and alpha 1,3GT, prefers more branched N-glycans compared to diantennary N-glycans. However, only the alpha 1,3GT is able to efficiently modify polylactosamines (3Gal beta 1-->4GlcNAc beta 1-->)n in N-glycans. Glycopeptides were also prepared by proteolysis of Lec2 glycoproteins and tested as acceptors compared to intact Lec2 glycoproteins. The alpha 2,6ST and alpha 1,3GT utilized intact glycoproteins and glycopeptides with a 2-fold preference for the former over the latter. In contrast, the alpha 2,3ST showed a 20-fold preference for intact glycoproteins over glycopeptides. These results demonstrate that each of these terminal glycosyltransferases differentially recognizes glycans and glycoprotein acceptors, and that the alpha 2,3ST requires peptide features for efficient utilization of branched N-glycan acceptors.

Schistosoma mansoni is a blood fluke that produces glycoconjugates containing the Lewis x antigen (Le(x)) Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->R. However, Le(x) antigen is also normally expressed in many tissues of adult rodents. We now report that mice and hamsters chronically infected with S.mansoni generate high titers of both IgM and IgG antibodies reactive with Le(x) and that no reactivity is present in sera from uninfected animals. Anti-Le(x) antibodies were detected by ELISA using the Le(x)-containing neoglycoprotein lacto-N-fucopentaoseIII-BSA. The IgG in infected animals consists of IgG1, IgG2a, and IgG2b subclasses and binds to Protein A-Sepharose. The sera of infected animals reacts only with Le(x) antigen and has no reactivity toward either Le(a) or sialyl Le(x). The IgM response to Le(x) is detectable at week 2, whereas the IgG response is detectable at weeks 5-6 following infection of mice. The sera of infected mice and hamsters can mediate the complement-dependent cytolysis (CDC) of cells expressing surface Le(x). This cytolytic activity is exclusively effected by the anti-Le(x) antibodies, since their removal from sera by adsorption depletes the sera of CDC activity. Thus, the abundant expression of the Le(x) antigens by the parasite elicits cytolytic antibodies reactive with a host antigen.

The lacdiNAc sequence GalNAc beta 1-->4GlcNAc beta 1-R occurs in the N- and O-glycans of many glycoproteins in vertebrate and invertebrates. We now report that both human 293 cells and Chinese hamster ovary (CHO) cells contain a UDPGalNAc:GlcNAc beta 1,4 N-acetylgalactosaminyltransferase (beta 1,4GalNAcT) that forms the lacdiNAc sequence. The beta 1,4GalNAcT in CHO cells is distinct from beta 1,4 galactosyltransferase in that the latter enzyme, but not the former, binds to a column of immobilized bovine alpha-lactalbumin. To determine whether endogenous glycoproteins in these cells contain lacdiNAc sequences, glycoproteins from 293 cells, CHO and Lec8 CHO cells were desialylated and passed over immobilized Wisteria floribunda agglutinin (WFA), a plant lectin with affinity for terminal GalNAc residues. WFA bound to approximately 120 and approximately 80 kDa glycoproteins in 293 cells and glycans from these glycoproteins contained lacdiNAc sequences. The approximately 120 kDa glycoproteins in 293 cells bound by WFA is a mixture of both the lysosome-associated membrane glycoproteins LAMPs-1 and -2. WFA bound to two glycoproteins of approximately 47 and approximately 78 kDa in Lec8 CHO cells, but these glycoproteins are not LAMPs and they do not contain the lacdiNAc sequence. Instead, they contain multiple GalNAc alpha-Ser/Thr O-glycans that promote binding to WFA. Thus, the beta 1,4GalNAcT in 293 cells displays a limited specificity in its recognition of acceptors, whereas the beta 1,4GalNAcT in CHO cells fails to promote synthesis of the cognate lacdiNAc sequence. The presence of the beta 1,4GalNAcT may not be sufficient for synthesis of lacdiNAc sequences and other factors may contribute to regulate the functionality of the enzyme.

The human H(O) blood group is specified by the structure Fucalpha1-2Galbeta1-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:beta-D-galactoside alpha1, 2-fucosyltransferase (alpha1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(alpha1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(alpha1, 2)FT cells and the distribution of these glycans on glycoproteins were characterized. Seventeen percent of the [3H]Gal-labeled glycopeptides from CHO(alpha1,2)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(alpha1,2)FT cells binding to UEA-I contained polylactosamine [3Galbeta1-4GlcNAcbeta1-]n with the terminal sequence Fucalpha1-2Galbeta1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(alpha1, 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 alpha1, 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 alpha1,2FT 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 defined set of oligosaccharides and glycopeptides containing alpha-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 (Le(x)) Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc beta 1-4[Fuc alpha 1-3]GlcNAc. The lectin did not bind glycans containing either sialylLe(x) or VIM-2 determinants, nor did it bind the isomeric Le(x), Gal beta 1-3[Fuc alpha 1-4]GlcNAc-R. Although 2'-fucosyllactose Fuc alpha 1-2Gal beta 1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc alpha 1-2Gal beta 1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Le(x) antigen and is useful in analyzing specific fucosylation of glycoconjugates.

This study was prompted by the paradoxical observation that a pair of dinitrophenyl-specific murine monoclonal IgG2a Abs had similar monosaccharide content and yet differed in their binding to lectins. The differential lectin-binding properties were lost when the Abs were denatured, suggesting that variations in lectin binding reflected the conformational accessibility of the N-glycans rather than intrinsic differences in the lectin binding capacity of the glycans themselves. This hypothesis was supported by experiments indicating that the degree to which the N-glycans on the Abs were reactive with beta-1,4-galactosyltransferase or susceptible to peptide N-glycosidase F corresponded directly to their relative accessibility to lectins. Moreover, the relative susceptibility to these enzymes and accessibility to lectins was inversely related to the capacity of the Abs to activate the classical pathway, suggesting that the orientation of the more accessible N-glycan might inhibit C1q binding. This hypothesis was supported by evidence that enzymatic cleavage of the more accessible N-glycan resulted in enhanced Clq, C4b, and C3b deposition. Conversely, removal of the less accessible N-glycan expressed by the other Ab inhibited C1q, C4b, and C3b deposition. The respective increase or decrease in C3b deposition on the two deglycosylated Abs was magnified when complement activation was performed in factor B-depleted serum, suggesting that N-glycan conformation primarily affects the classical pathway. Collectively, these data suggest that the orientation of the N-glycan expressed on Igs can profoundly influence interaction with the complement system.

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity. A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation. The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures. Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.

The density, molecular isoform, and posttranslational modifications of CD44 can markedly influence growth and metastatic behavior of tumors. Many CD44 functions, including some involving tumors, have been attributed to its ability to recognize hyaluronan (HA). However, only certain CD44-bearing cells bind soluble or immobilized HA. We now show that CD44 made by wild-type Chinese hamster ovary (CHO-K1) cells and a ligand-binding subclone differ with respect to N-linked glycosylation. While both bear CD44 with highly branched, complex-type glycoforms, CD44 expressed by the wild type was more extensively sialylated. CHO-K1 cells which failed to recognize HA when grown in culture gained this ability when grown as a solid tumor and reverted to a non-HA-binding state when returned to culture. The ability of CHO-K1 cells to recognize HA was also reversibly induced when glucose concentrations in the medium were reduced. Glucose restriction influenced CD44-mediated HA binding by many but not all, of a series of murine tumors. Glucose concentrations and glycosylation inhibitors only partially influenced CD44 receptor function on resting murine B lymphocytes. These observations suggest that glucose levels or other local environmental conditions may markedly influence glycosylation pathways used by some tumor cells, resulting in dramatic alteration of CD44-mediated functions.