Human colonic sulfomucin identified by a specific monoclonal antibody.

Published

Journal Article

Since the 1960s, the loss of sulfomucin from colonic epithelium has been considered to be an indicator of an early stage of carcinogenesis; yet, the biochemical basis for this phenomenon has never been elucidated. We recently prepared a monoclonal antibody (mAb) 91.9H that immunoprecipitates the normal colonic mucins metabolically incorporating [35S]-sulfate. This mouse IgG1 antibody did not cross-react with colon carcinoma mucins that lack sulfate groups. Using normal colonic epithelia unlabeled or radiolabeled with [35S]sulfate and [3H]glucosamine, we purified a high molecular weight glycoprotein that reacts with mAb 91.9H. This was achieved by a combination of DEAE-cellulose anion-exchange chromatography, consecutive treatments with chondroitinase ABC plus heparitinase and with sodium dodecyl sulfate plus 2-mercaptoethanol, and gel filtration on Sepharose CL-2B in the presence of 8 M urea. Antibody reactivity was found in acidic but not neutral high molecular weight glycoproteins. After Sepharose CL-2B fractionation, the mAb 91.9H-reactive fractions consisted of a component with an approximate molecular weight of 500,000-900,000. A purified sulfomucin contained protein, neutral sugar, amino sugar, sialic acid, and sulfate in an approximate ratio of 2.5:1.0:1.1:0.4:0.5. The polypeptide portion was rich in hydrophilic amino acids, particularly threonine. Binding of mAb 91.9H in solid-phase assays was inhibited to 50% by purified normal colon acidic mucin at doses of 5-50 micrograms/ml, depending on different preparations. Various glycosaminoglycans or sulfatides did not show inhibitory activity. Sulfomucin reactivity with mAb 91.9H, as determined by solid-phase-binding inhibition and by dot blot assays, was significantly reduced by chemical desulfation of sulfomucins with anhydrous hydrochloric acid, suggesting that sulfate groups served as a portion of the immunochemical determinant for this antibody. Sulfate residues were apparently linked to alkaline-sensitive carbohydrate chains, but alkaline-released carbohydrate chains did not react with mAb 91.9H. Immunohistochemical examinations showed that mAb 91.9H bound normal colonic epithelial cells, which also stained with high-iron diamine, more strongly than it bound colon carcinoma cells.

Full Text

Duke Authors

Cited Authors

  • Irimura, T; Wynn, DM; Hager, LG; Cleary, KR; Ota, DM

Published Date

  • October 15, 1991

Published In

Volume / Issue

  • 51 / 20

Start / End Page

  • 5728 - 5735

PubMed ID

  • 1913691

Pubmed Central ID

  • 1913691

International Standard Serial Number (ISSN)

  • 0008-5472

Language

  • eng

Conference Location

  • United States