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Human Colonic Sulfomucin Identified by a Specific Monoclonal Antibody¹

The Departments of Tumor Biology [T. I., D. M. W., L. G. H.], Pathology [K. R. C.], and General Surgery [D. M. O.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

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 µg/ml, depending on different preparations. Various glycosaminoglycans or sulfatides did not show inhibitory activity. Sulfomucin reactivity with mAb 91.9H, as determined by solid-phasebinding 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.

¹ This work was supported by NIH Grants RO1 CA39319 and CA50231 and by Texas Advanced Technology Program 1549.

² To whom requests for reprints should be addressed, at Division of Chemical Toxicology and Immunohistochemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan.

Received 4/11/91. Accepted 8/ 6/91.

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