A Series of Disialogangliosides with Binary 2-3 Sialosyllactosamine Structure, Defined by Monoclonal Antibody NUHB, Are Oncodevelopmentally Regulated Antigens*

A mouse IgM monoclonal antibody, NUH2, was raised after immunization of mice with the disialogan- glioside fractiod of human colonic adenocarcinoma. This antibody reacts specifically with disialogangliosides having the Structure 1 shown below. not with structures the sialic acid at either the 8143 or 8146 side chain (Table I, structure GS), nor with a binary structure having unequal chain lengths (Structure 2 below), nor with a binary type 2 chain structure having a trimannosyl core as found in the side chain of N-linked complex type oligosaccharides (Structure 3

The structural concept of oncodevelopmentally regulated antigens has been well established since the MoAb' approach * This work was supported by National Institutes of Health Outstanding Investigator Grant CA42505 and funds from The Biomembrane Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Gal@l4GlcNAcfl1~3Gal@l4Glcfll+lCer N e u A c a 2 4 G a l f l l 4 G l c N A c~l \ NeuAca2-r3Gal@l4GlcNAc@l 3 / 6 G a l~l 4 G l c N A c @ l~3 G a l @ l~G l c @ l -r~C e r +and CC2 (5), and ACFH18 (6). Accumulation of these glycolipids in human cancer, particularly adenocarcinoma, is due to a predominance in synthesis of /31+3GlcNAc transferase over that of /31+6GlcNAc transferase, thus leading to an unbranched core structure (7). In contrast to the major trends of aberrant glycosylation as described above, we have found an additional change in type 2 chain glycolipids of human colonic adenocarcinoma, defined by the new MoAb NUH2. This antibody was originally raised after immunization of mice with the disialoganglioside fraction of human colonic adenocarcinoma. NUHZ stained glycolipid fractions of various colonic cancers and was directed to an antigen characterized as having two NeuAca2+3Gal/314GlcNAc side chains /3l+ 3 and /314 linked together to the terminal Gal of polylactosamine. A series of glycolipids reacting with NUHZ was also isolated from human placenta. The structures of these highly complex sialyl type 2 chain glycolipids, and their reactivity with NUH2, have defined the specificity of this new MoAb.

MATERIALS AND METHODS' RESULTS
Establishment of MoAb NUH2 after Immunization of Mice with Disialoganglioside Fraction of Human Colonic Aderwcar-Portions of this paper (including "Materials and Methods," part of "Results," Figs. 6-9, and Table 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
On the other hand, NUHZ did react with a major ganglioside component of human colonic cancer and a series of gangliosides present in human placenta. These gangliosides were structurally identified as G10 (DSI) or GI2 (fractions 90-102) ( Table I). NUH2 did not react with gangliosides G8 or G11 (Table I). Characterization of these structures is described in subsequent sections and in part in the Miniprint.
Further characterization of disialoganglioside A by FAB-I I MS of permethylated derivatives and 'H NMR spectroscopy is described in the Miniprint. All available data indicate that the structure is V13NeuAcI~NeuAccr2+3Gal~l+4GlcNAc-nLcs (Table I,  purified from fractions 72-77 of HPLC step I1 (see Miniprint). Two other higher gangliosides, G12 (trisialoganglioside) and G13 (tetrasialoganglioside), were also isolated to homogeneity (lanes 5 and 6, respectively, in Fig. 1, A-C). Structures of G12 and G13, both of which were strongly reactive with NUH2, will be described elsewhere (42) (Fig. 1, A and C, lanes 1   and 3 ) , but gave a doublet in CMW 505519 (0.05% CaC12) (Fig. lB, lanes 1 and 3 ) . The reactivity of this ganglioside with MoAb NUH2 was highly sensitive to treatment with Clostridium perfringens sialidase (Sigma). The neutral glycolipid obtained after sialidase treatment had the same TLC motility as lactoisooctaosylceramide (iso-nLce; IV6Ga1@1+ 4GlcNAc@nLc6) (Fig. 2 A , lane 2 ) . The component after sialidase treatment was strongly positive with 1B2 antibody (Fig.  2 0 , lane 2 ) , indicating the presence of a GalS14GlcNAc residue at the terminus. The compound was also reactive with MoAb C6, which is known to react only with branched I GI2 and G13 are, respectively, tri-and tetrasialogangliosides having three and four NeuAca2~3Gal~14GlcNAc side chains linked together.
These results, together with data from methylation analysis  Table I for structure; isolation and characterization will be described el~ewhere)~; 0, sialidase-treated ganglioside A; A, sialidase-treated ganglioside c; W, G8 ganglioside (14) (Table I) (e.g. transferrin, a-1 acid glycoprotein, fetuin), or glycoproteins with highly sialylated polylactosaminoglycan (e.g. from placenta), were prepared.'50 pl of each glycoprotein solution was serially diluted, placed in 96-well plastic plate (Costar Vinyl 2595), incubated at 4 "C for 18 h, washed with PBS, and incubated with MoAb NUHZ culture supernatant (29 pg/ml) for 18 h. Each well was then washed again with PBS, incubated with rabbit anti-mouse IgM (diluted 1:1000), incubated for 2 h, washed with PBS, supplemented with 12sII-labeled protein A (50,000 cpm/ well), incubated for 2 h, washed three times with PBS, excised, and counted. Abscissa, serial dilution of glycoprotein solutions incubated in each well; well 1 contained 100 pglml, well 2 contained 50 pg/ml, etc. Ordinate, count of antibody binding. V, transferrin; A, a-1 acid glycoprotein; 0, fetuin; 0, placenta glycoprotein. and 'FAB-MS of G11 ganglioside (see Miniprint) clearly confirm its structure as shown in Table I. Quantitative Reactivity of NUH2 Antibody with Various Gangliosides and Glycoproteins-In addition to G10 and G11 gangliosides (fractions 72-77), G8 ganglioside previously iso-lated and characterized from human erythrocytes (14) ( Table   I ) , higher trisialoganglioside G12 (fractions 90-102; see Table  I): and various lacto-series and ganglio-series gangliosides were compared by solid-phase antibody binding assay. The results (Fig. 3) clearly indicate that NUHZ bound only to those gangliosides having binary NeuAca2~3Galfil-+ 4GlcNAc with the same chain length and linked fi1+6/fi1+ 3 to the Gal residue of type 2 chain; Le. it reacted only with G10 and G12, but not with G8 or G11 gangliosides. Other gangliosides so far tested were all nonreactive.
Fetuin, transferrin, and a-1 acid glycoprotein (orosomucoid), which has the binary NeuAca2+3Galfi1-*4GIcNAc structure but is linked to the trimannose core structure of the complex-type N-linked carbohydrate chain of glycoprotein, were all nonreactive (Fig. 4).

Glycolipid Profiles of Human Cancer, Placenta, and Normal
Tissue Reacting with MoAb NUH2-TLC immunostaining of ganglioside fractions prepared from various human cancer cases showed the presence of a common doublet, which was ~" ,~r~" l " -l l r l~s .~~Y " . V~I " . . . " " I V . identified as disialosyl I (GI0 ganglioside) (Table I). 56 cases of various types of cancer showed the presence of this ganglioside and other higher gangliosides through positive staining with NUH2 (Fig, 5). Likewise, human placenta showed a series of bands with extremely strong reactivity. In contrast, ganglioside fractions from normal kidney, brain, and large intestine showed no reactivity, while normal liver gave a weak doublet band (Fig. 5). DISCUSSION Human gastrointestinal and bronchopulmonary tumors have been characterized by a preponderance of unbranched type 2 chain (i type) having extensive al+3 fucosylation at the penultimate as well as internal GlcNAc. The fucosylation is coupled with terminal a2+3 sialylation or a1+2 fucosylation, resulting in a variety of tumor-associated epitopes defined by MoAbs showing preferential reactivity with tumors over corresponding normal tissues (1). A branched type 2 chain based on a /31-&GlcNAc linkage has been shown to be a relatively minor component in various tumors, and the branching process is regarded as developmentally regulated (15,16). However, this assumption is based on lack of branched type 2 chain ABH and I antigens in gastrointestinal tumors and fetal tissues. The results of the present study indicate that the majority of terminal Gal residues of essentially all branched type 2 chain structures in tumors could be a 2 4 3 sialylated, thus resulting in accumulation of NUH2positive antigens in a large variety of human cancers. Therefore, branched type 2 chain appears to be a relatively minor component in human tumors, yet its fully sialylated derivative may be a tumor-associated antigen. This situation could result from greatly enhanced a 2 4 3 sialyltransferase activity. Fucosylation, a prerequisite to formation of branched ABH determinant, could be competitively inhibited.
Distribution of the NUH2-defined antigen in normal tissue is limited. It is virtually absent in various parenchymatous organs (brain, kidney, lung, liver, or spleen) as well as in normal gastrointestinal tissues. In contrast, high quantities of the antigen were detected in human placenta (as shown in this paper), human sperm: myelocytic leukemia cells, megakaryocytes,b and trophoblast? NUHZ reacts strongly with ejaculated sperm and induced immobilization of sperm in the presence of complement, as we described (17). In colonic tissues, it is completely absent in normal mucosa regardless of location (distal or proximal regions). A strong focal expres-* T. Kaizu, Y. Tsuji, and s. Hakomori, unpublished data. ' H. Nojiri, and S. Hakomori, unpublished data. Y. Tsuji, and S. Hakomori, unpublished data. sion is seen in tissues from many cases of colonic cancer?
Thus, it appears that the disialosyl I structures defined by NUH2 constitute an important new group of oncodevelopmental markers, although their functional role is unknown.