The effects of delipidation on the major antigenic determinant of purified human intestinal mucin

Abstract

To investigate whether the antigenicity of purified human intestinal mucin was dependent on the presence of associated lipid, native mucin (purified by equilibrium density gradient centrifugation in CsCl (twice) and gel filtration on Sepharose 2B) was extracted five times with organic solvents to remove any noncovalently bound lipid and, subsequently, treated with hydroxylamine to release any covalently bound fatty acids. The first organic extract contained cholesterol, phosphatidyl-ethanolamine, and phosphatidylserine, with lesser amounts of phosphatidylcholine, triglycerides, fatty acids, sphingomyelin, and glycolipids. In total, this noncovalently bound lipid amounted to < 5% by weight of the native mucin preparation. Further organic extracts were free of lipid. Removal of noncovalently bound lipid had essentially no effect on mucin antigenicity, as assessed by radioimmunoassay. Treatment of the delipidated mucin with hydroxylamine caused no detectable changes in mucin antigenicity or composition and the release of covalently (ester) bound fatty acids could not be demonstrated. We therefore conclude that although purified human intestinal mucin contains small amounts of noncovalently bound lipid this lipid is not involved in mucin antigenicity.