Studies on the structure of the organ-specific determinant of human colonic mucin.

We had previously shown that the human colon produces at least two immunochemically distinct mucins, one neutral and the other a sialomucin [Gold et al. J. biol. Chem. 256, 6354-6358 (1981)]. In addition, the sialomucin was shown to contain an immunodeterminant restricted to colonic epithelium and may thus prove useful as a tissue-specific marker. In the current study we have shown that a specific linkage of sialic acid to the oligosaccharide backbone has a major role in the organ-specific immunodeterminant structure. Treatment of intact colonic mucin with sialidase (Cl. perfringens) cleaved 20-80% of the sialic acid as measured colorimetrically. Immunoreactivity was decreased by 0-42% with respect to the untreated material. Saponification (0.1 N KOH, 20 min at room temp) caused an approximate 90% decrease in immunoreactivity for each mucin. Subsequent to saponification, neuraminidase cleaved most of the sialic acid from the mucins. The majority of sialic acid was observed to be O-acetylated, thus making it sialidase-insensitive. Gas chromatography-mass spectrometric analyses of the trimethylsilyl sialic acid derivatives indicated the presence of NeuNAc; NeuNAc, 9-OAc; and NeuNAc, 7,9 diOAc as the major sialyl derivatives. The radioimmunoassay data appeared to indicate that O-acetylated sialic acid was necessary for immunoreactivity. It should be noted that jejunal mucin and bovine submaxillary mucin also contain O-acetylated sialic acid, but did not inhibit in our radioimmunoassay. This may have been due to differences in the O-acetylation pattern or the linkage of sialic acid to the core carbohydrate. Analyses of the partially methylated alditol acetate derivatives by gas chromatography-mass spectrometry of the untreated, as well as the saponified and neuraminidase treated, mucins revealed that sialic acid was attached to the carbohydrate core either to galactose, N-acetylglucosamine, and/or N-acetylgalactosamine. Linear regression analyses comparing immunoreactivity with specific epitope concns, in conjunction with RIA analyses of known structures, suggested that the organ-specific immunodeterminant was (or was dependent upon the presence of) the structure GlcNAc (1,3)[O-acetylated Neu5Ac(2,6)] GalNAc.