The oncofetal structure of human fibronectin defined by monoclonal antibody FDC-6. Unique structural requirement for the antigenic specificity provided by a glycosylhexapeptide

Previously, monoclonal antibody FDC-6 was established, which defines a structure specific for fibronectins isolated from fetal and malignant cells and tissues. The presence of the FDC-6-defined structure at type III connecting segment (III CS) is characteristic of oncofetal fibronectin (onf-FN), and its absence is characteristic of normal fibronectin (nor-FN) (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). Hepatoma fibronectin was sequentially digested by various proteases, followed by subsequent chromatography on an FDC-6 affinity column and reverse-phase columns at each step of digestion. A single strongly active glycosylhexapeptide (glycopeptide 1) and an inactive glycosylpentapeptide (glycopeptide 3) were isolated from glycopeptide A containing 35 amino acid residues. The minimum essential structure required for the FDC-6 activity was found to be a hexapeptide sequence Val-Thr-His-Pro-Gly-Tyr having NeuAc alpha 2----3Gal beta 1----3GalNAc or its core (Gal beta 1----3GalNAc or GalNAc) linked at threonine. Various synthetic peptides including the Val-Thr-His-Pro-Gly-Tyr sequence and a glycopeptide having the Val-Thr-His-Pro-Gly pentapeptide with the same glycosylation at threonine were all inactive. Elimination of sialic acid slightly increased the activity, and subsequent elimination of galactose did not alter the activity; however, removal of the Gal beta 1----3GalNAc residue by endo-alpha-N-acetylgalactosaminidase from desialylated glycopeptide A resulted in total inactivation of the reactivity with FDC-6 antibody. Thus, a single glycosylation at a defined threonine residue of the III CS region may induce conformational changes in the peptide to form the specific oncofetal epitope recognized by FDC-6 antibody. This finding opens the possibility that a number of other oncofetal epitopes consist of a peptide and a common O-linked carbohydrate and that the combination produces a conformation specific to cancer or to a stage of development.