The
monoclonal antibody FDC-6 defines a structure specific to oncofetal
fibronectins (onf-FN) isolated from fetal and malignant cells and tissues. The absence of this structure is characteristic of normal
fibronectin (nor-FN) isolated from plasma and adult normal tissue (Matsuura, H., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6517-6521). The minimum structure required for FDC-6 reactivity was determined to be Val-Thr-
His-Pro-
Gly-Tyr (VTHPGY) with alpha-N-
acetylgalactosamine (alpha-GalNAc) at Thr, although alpha-GalNAc per se is not involved in the FDC-6
epitope (Matsuura, H., Takio, K., Titani, K., Greene, T., Levery, S. B., Salyan, M. E. K., and Hakomori, S. (1988) J. Biol. Chem. 263, 3314-3322). Thus, a single glycosylation on the normally occurring
peptide of FN may induce conformational changes in the
peptide to form the specific oncofetal
epitope recognized by FDC-6 antibody. The FDC-6-nonreactive synthetic
peptide containing the VTHPGY sequence was converted into FDC-6-reactive form on incubation with alpha-N-acetylgalactosaminyltransferase and
UDP-[3H]GalNAc in the homogenate of
hepatoma cell HUH-7, human fetal fibroblast cell line WI-38, or human
epidermoid carcinoma cell line A431. Such a conversion did not take place when the same
enzyme fraction of normal adult tissue was incubated with the VTHPGY
peptide under the same conditions. Thus, the occurrence of alpha-
GalNAc transferase recognizing the VTHPGY
peptide sequence (
UDP-GalNAc:VTHPGY alpha-GalNAc transferase) is specific for fetal and
cancer tissues, and absent in normal adult tissues. However, a similar alpha-
GalNAc transferase activity capable of transferring the GalNAc residue to other Ser or Thr
hydroxyl groups of nor-FN, and presumably located at the type III connecting segment region, was detectable in homogenate of various normal tissues. Such
enzyme activity was determined with the use of enzymatically de-O-glycosylated nor-FN. Thus, the enzymatic basis of FDC-6
epitope formation is a subtle change in the substrate specificity of alpha-
GalNAc transferase. The normal
enzyme is incapable of transferring alpha-GalNAc from
UDP-GalNAc to the Thr residue of the VTHPGY sequence, but is capable of transferring alpha-GalNAc to other Ser or Thr residues of FN. In contrast, alpha-
GalNAc transferase of fetal and
cancer tissues may have broader specificity and the capability to transfer GalNAc to Thr or Ser residues, including those of the VTHPGY sequence.