The
breast cancer-associated
epitope (mammary serum
antigen or MSA) defined by
monoclonal antibody (Mab) 3E1.2 is a
neuraminidase-sensitive
carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by
protease treatment of the
mucin, which suggests that 3E1.2 binds to multimers of the sialylated
carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic
acid (5-acetylneuraminic acid) is not involved in the
epitope, since
lectins specific for 5-acetylneuraminic
acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic
acid (Neu5Gc), forms a major part of the
epitope since both free Neu5Gc and porcine stomach
mucin (greater than 90% neuraminic
acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary
mucins,
fetuin, bovine
gangliosides, and other
carbohydrates do not. Indeed, the presence of Neu5Gc on human
tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked
carbohydrate, since the expression of MSA by MCF-7
breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide, but not by the N-glycosylation inhibitor
tunicamycin, and the
epitope is removed by treatment with
O-glycanase but not
N-glycanase F,
endoglycosidase F, or
endoglycosidase H, which are specific for N-linked
glycans. This is likely to be a core
glycan since 3E1.2 reacts
after treatment of the
mucin with
trifluoromethanesulfonic acid, which removes most backbone and peripheral
carbohydrates. Treatment with
galactosidase or N-acetyl
glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to
galactose or N-acetyl
galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter
urea-faciens
neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific
lectins after
periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from
periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to
peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core
glycan, which represents a unique
tumor-associated
epitope not previously identified on human
mucins.