N‐Linked sugar chains of normal mammary gland, mammary carcinomas (primary lesion), and axillary lymph node metastases of mammary carcinomas were released from their membrane preparations by hydrazinolysis and their structures were analyzed. Fractionation using a Datura stramonium agglutinin (DSA)‐Sepharose column revealed that the metastasized carcinomas contain more than twice as much DSA‐binding oligosaccharides as the normal gland, and the primary carcinomas contain an intermediate amount. These oligosaccharides were elucidated to have tri‐ and tetraantennary structures containing the GlcNAcβ1 → 6(GlcNAcβ1 → 2) Man group with and without N‐acetyllactosamine repeating units. Lectin blot analysis of membrane glycoproteins and histochemical staining of tissues using biotinylated DSA indicated that these glycosylation changes predominantly occur in a limited number of glycoproteins with apparent molecular weights of 90,160, and 210 kilodaltons, and mammary carcinomas are distinguishable from normal gland by their intense intracytoplasmic staining.

Our previous studies showed that expression of the GalNAc␤134GlcNAc group on N-linked oligosaccharides is associated with functional differentiation of the bovine mammary gland. In the present study, the occurrence of the GalNAc␤134GlcNAc group was established in human milk proteins and membrane glycoproteins from a human breast cancer cell line, MRK-nu-1, by structural analysis of oligosaccharides released by hydrazinolysis. Whether the expression level of the disaccharide group is affected upon malignant transformation was examined in human breast cancer specimens using Wistaria floribunda agglutinin ( Accumulating information on the structures of N-linked oligosaccharides has revealed that the GalNAc␤134GlcNAc structure is expressed on the outer chain moieties of N-linked oligosaccharides of glycoproteins from a variety of animal species and organs (reviewed in refs. 1-3). The expression of this disaccharide group was initially found on mammalian pituitary glycoprotein hormones 4 and was considered to be unique to them since ␤-1,4-Nacetylgalactosaminyltransferase, which transfers N-acetylgalactosamine from UDP-GalNAc to N-acetylglucosamine-terminated oligosaccharides in the pituitary, showed specificities not only to the acceptor oligosaccharide but also to Pro-Xaa-Arg/Lys motifs, which are strictly preserved in mammalian pituitary glycoprotein hormones. [5][6][7] Our previous studies, however, showed that the occurrence of ␤-N-acetylgalactosaminylated N-linked oligosaccharides is more universal, as most glycoproteins in the bovine mammary gland contain the GalNAc␤134GlcNAc structure in their outer chain moieties, and several glycoproteins in bovine liver, kidney, spleen and thymus appear to contain such oligosaccharides. 8 -12 In fact, ␤-N-acetylgalactosaminyltransferase in the mammary gland shows no peptide specificity, 3,13 which is also the case in some other tissues. 14 Interestingly, the expression of the GalNAc␤134GlcNAc structure on N-linked oligosaccharides in the mammary gland appears to be under the control of lactogenic hormones since its expression level increases during lactation. 9 Furthermore, the functional differentiation of primary cultured epithelial cells established from bovine mammary gland by induction with lactogenic hormones stimulates expression of the GalNAc␤134GlcNAc structure on N-linked oligosaccharides. 15 These results suggest that the GalNAc␤134GlcNAc group is a new differentiation-associated carbohydrate antigen in mammary gland. Therefore, it is expected that its expression will be suppressed by malignant transformation of cells. In the present study, the prevalence of the GalNAc␤134GlcNAc structure on N-linked oligosaccharides in human breast cancer was investigated by biochemical and histochemical studies using Wistaria floribunda agglutinin (WFA), and the clinical significance of WFA staining of tissue specimens was evaluated.

Expression of beta 1-6 branched oligosaccharides in human breast cancer cells was investigated in vivo and in vitro. Lectin histochemical and lectin blotting analyses of surgically resected specimens were performed using L-PHA (phaseolus vulgaris leukoagglutinin) lectin, which binds to beta 1-6 oligosaccharides. The glycoproteins bearing beta 1-6 oligosaccharides of breast cancer tissues were found to be 170 kD and 120 kD in molecular weight, and the former appeared to be an epitope of carcinoembryonic antigen (CEA). The beta 1-6 oligosaccharides were expressed in both cancer cell lines at the outer layer of the colonies when cultured in type I collagen, but not in agarose gel. No correlation was observed between beta 1-6 expression and cell cycle. The beta 1-6 oligosaccharides did not coincide with breast cancer-associated antigens, such as CEA, MUC1, and cathepsin D. The beta 1-6 oligosaccharides of these cell lines were markedly inhibited when swainsonine, a mannosidase II inhibitor, was added to the culture medium. The 120 kD molecule, which was obtained from MCF-7 cells cultured in type I collagen gel, was consistent with that of breast cancer tissues and was similar to lysosome-associated membrane glycoproteins (LAMPs). The results suggest that the glycoproteins bearing beta 1-6 branched oligosaccharides in human breast cancer incorporate an epitope of CEA and human LAMPs and that the expression of LAMPs may depend on their surrounding matrices and may play an important role in cancer invasion or metastasis.

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