Our previous studies showed that expression of the GalNAcβ1→4GlcNAc group on N-linked oligosaccharides is associated with functional differentiation of the bovine mammary gland. In the present study, the occurrence of the GalNAcβ1→4GlcNAc 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 (WFA) which interacts with oligosaccharides with N-acetylgalactosamine at their termini. Lectin blot analysis of membrane glycoprotein samples from human breast cancer specimens showed that the number of protein bands reacting with WFA, as well as their intensities, are lower in samples from primary carcinoma lesions compared with samples from surrounding normal tissues. No lectin binding was observed when the blots were treated with jack bean β-N-acetylhexosaminidase or N-glycanase, indicating that WFA-reactive oligosaccharides are N-linked. A histochemical study of tissue specimens from 92 patients with breast cancer revealed that the reduced WFA staining levels in primary carcinoma lesions correlate with advancing clinical stages and prognostic status (i.e., 58% of patients in a group showing reduced/negative staining died of disease recurrence, whereas more than 90% of those in the positive staining group survived for 5 years after surgery). These results indicate that reduced expression of β-N-acetylgalactosaminylated N-linked oligosaccharides on primary carcinoma lesions predicts a poor prognosis for patients with breast cancer. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]