Summary Most secretory and membrane-bound proteins produced by mammalian cells contain covalently linked sugar chains. Alterations of the sugar chain structures of glycoproteins have been found to occur in various tumours. Because the sugar chains of glycoproteins are essential for the maintenance of the ordered social behaviour of differentiated cells in multicellular organisms, alterations to the sugar chains are the molecular basis of abnormal social behaviours in tumour cells, such as invasion into the surrounding tissues and metastasis. In this review, the structure and enzymatic basis of typical alterations of the N -linked sugar chains, which are found in various tumours, are introduced. These data are useful for devising diagnostic methods and immunotherapies for the clinical treatment of tumours. Three β -N -acetylglucosaminyltransferases, GnT-III, -IV and -V, play roles in the structural alteration of the complex-type sugar chains in various tumours. In addition, transcriptional changes in various glycosyltransferases, together with the transporters of sugar nucleotides and sulfate, which are responsible for the formation of the outer chain moieties of complex-type sugar chains, are the keys to inducing the alterations.

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG’ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.

We made a comparative study of the structures of the oligosaccharides on the glycoproteins from Caco-2 human colonic adenocarcinoma cells, before and after differentiation. Enterocytic differentiated Caco-2 cells highly express H type 1 blood group antigen on the cell surface as well as activities of brush border membrane hydrolases, such as dipeptidyl peptidase IV and alkaline phosphatase. A strong correlation was observed between the amounts of H type 1 blood group antigen and the degrees of differentiation. Structural analysis with use of lectin affinity high performance liquid chromatography revealed that typical mucin-type sugar chains of the glycoproteins from undifferentiated cells have H type 2 group, linear polylactosamines, and core 1 structure. On the other hand, differentiated cells newly contain H type 1 and Le b groups and core 2 structure. Mucins with H type 1 group make contact with brush border membrane enzymes on differentiated cells. Furthermore benzyl 2-acetamide-2-deoxy-␣-D-galactopyranoside inhibited both expression of H type 1 group on the cell surface and enhancement of brush border membrane enzyme activities even in the presence of a differentiating inducer. These results suggest that the mucintype sugar chains with H type 1 group have important functions regarding differentiation of Caco-2 cells.Caco-2 cells derived from a human colonic adenocarcinoma differentiate into enterocytes-like cells spontaneously (1) or by induction with sodium butyrate (2). During enterocytic differentiation, in addition to morphological change with acquisition of a brush border, various biological changes have been noted, for example, expression of brush border-associated enzymes (1), mucin synthesis (3), and glycosylation. However, little is known of detailed oligosaccharide structures before and after differentiation of Caco-2 cells. Decrease in polylactosaminoglycans of lysosomal membrane glycoprotein h-Lamp-1 was observed in spontaneously differentiated Caco-2 cells (4), but, unexpectedly, the glycosyltransferases directly involved in polylactosaminoglycan biosynthesis remain essentially unchanged (5). Findings that increased activities of branching enzymes and decreased activity of mucin-type sugar chain core 1 enzyme were also obtained (5). Although these results suggest a change in glycosylation with differentiation of Caco-2 cells, no information on outer chains that contain blood group antigens and interact with other cells has been available. Only an increase in the ␣2,6-sialylation after differentiation has been reported (6). Differentiated Caco-2 cells are used for a model of adherence of bacteria to the intestinal epithelium (7,8). This protein-carbohydrate interaction is critical for bacterial infection and involves microbial lectin-like adhesins and specific oligosaccharides present on the intestinal epithelium. To elucidate oligosaccharide structures on the surface glycoproteins of Caco-2 cells is essential for a better understanding of the mechanism of microbial infections. For this purpose ...

We prepared neutral oligosaccharide fraction from milk of a woman (blood type A, Le(b+)) by anion-exchange column chromatography after the removal of lipids and proteins. Further fractionation was performed by means of Aleuria aurantia lectin-Sepharose column chromatography and reverse-phase HPLC after labeling with a pyrene derivative. This pyrene labeling allowed identification by negative-MALDI-TOFMS(n) analysis of 22 oligosaccharides with decaose cores, among which 21 had novel structures. Negative ions could not be produced from neutral oligosaccharides without labeling on MALDI. Mono-, di-, tri-, and tetrafucosylated decaose fractions contained three, nine, six, and four isomers, respectively. Our method enables easy determination of fucosylated structures on the N-acetyllactosamine branches of these isomers. On negative-MS(n) the fragment ions included several A and D ions, from which fucosylation on the branches could be elucidated. Other characteristic ions were also detected. Y-type cleavage at the reducing side of -3GlcNAc indicated the occurrence of type 1 chain. Specific fragment ions were produced from H, Le(a), and Le(x) antigens. Linkage-specific exoglycosidase digestion confirmed the structures. The results indicate that the diversity of the oligosaccharides is due to combinations of type 1 H, Le(a), Le(x), and Le(b)/Le(y) on branched decaose cores. In typical oligosaccharides, 6-branches always consist of type 2 chain, while 3-branches, such as beta and gamma chains, are fucosylated type 1 chains. From the viewpoint of biosynthesis, the presence of fucosylation and type 1 chain may halt elongation of the N-acetyllactosamine and promote formation of branched structures.

Oligosaccharides have many isomers and MALDI-QIT-TOFMS(n) analysis is effective for determining their structures. However, it is difficult to elucidate in detail the structures of fucosylated and/or sialylated oligosaccharides that are known to be disease markers because fucose and sialic acid residues are easily released. We have introduced a technique of labeling oligosaccharides with a pyrene derivative prior to negative-ion MALDI-QIT-TOFMS(n), and we have established a reliable method using this technique for the analysis of neutral oligosaccharides, such as fucosylated oligosaccharides containing blood group antigens H, Le(a), and Le(x). Intense and stable ionization in both positive and negative modes was achieved by derivatization with pyrene. As little as 10 fmol of pyrene-labeled oligosaccharides gave sufficient signals for analysis. Specific A-, D- or Y-type ions that depend on the structures of branching antennae could be detected by MS(n) and were useful for rapid and easy structural determination. These specific fragmentations resulting from collision-induced dissociation can be used to elucidate the structures of unknown oligosaccharides even if authentic oligosaccharides are not available as standards. By using this method, we identified and quantitated isomeric oligosaccharides with different fucosyl linkages from their mixtures. Moreover, sialylated oligosaccharide was converted to the corresponding neutral oligosaccharide by amidation, and the negative-ion spectrum was shown to be more informative than that of the original acidic oligosaccharide. Structural determination of both fucosylated and sialylated isomers, such as sialylfucosyllacto-N-hexaose I and monosialyl monofucosyllacto-N-neohexaose, was successful because fragment ions bearing fucose or amidated sialic acid were obtained on negative-MS(n).