Discrimination of MUC1 mucins from other sialyl-Le(a)-carrying glycoproteins produced by colon carcinoma cells using a novel monoclonal antibody

Abstract

Earlier studies (Baeckström et al., J. Biol. Chem., 266: 21537-21547, 1991) have revealed that the colon carcinoma cell line COLO 205 produces two different proteins carrying the carcinoma-associated sialyl-Le(a) carbohydrate epitope. One is the MUC1 mucin apoprotein, and the other protein is smaller and has not been characterized in detail. This paper describes a comparison of COLO 205 with three other colon carcinoma cell lines, aided by the use of a novel MUC1-reactive monoclonal antibody, Ma552. Ma552 reacted with H-CanAg, the MUC1 mucin purified from COLO 205, the binding increasing greatly upon partial deglycosylation of H-CanAg with trifluoromethanesulfonic acid. This pattern of reactivity was in contrast with that of the MUC1-reactive monoclonal antibody HMFG-2, which did not recognize H-CanAg without prior deglycosylation. The nature of the epitope of Ma552 was further investigated using a synthetic peptide corresponding to the tandem-repeat sequence of the MUC1 protein. When the peptide was used as an inhibitor of antibody binding to immobilized, partially deglycosylated H-CanAg mucin, Ma552 was inhibited, as was HMFG-2. Using short, immobilized synthetic peptides identical to parts of the MUC1 tandem repeat, the reactivity of Ma552 was mapped to the hexapeptide TRPAPG. Ma552 and C50, a monoclonal antibody reactive with sialyl-Le(a), were used in immunofluorometric assays to analyze gel filtration fractions of extracts and spent media from the colorectal carcinoma cell lines COLO 205, SW1116, LoVo, and LS 174T. Using C50 in a homologous assay, all sialyl-Le(a)-carrying glycoproteins were detected. Among these, mucins based on the MUC1 apoprotein were identified using Ma552 and C50 in a combination assay. The results showed that sialyl-Le(a) was present on more than one glycoprotein not only in COLO 205 but also in SW1116 and LoVo. The Ma552/Eu-C50 assay revealed the presence of sialyl-Le(a)-carrying MUC1 in COLO 205 as expected, as well as in SW1116. The presence of MUC1 in Ma552-reactive fractions was confirmed by deglycosylation followed by assaying with the monoclonal antibody HMFG-2. Furthermore, Northern blots revealed the presence of MUC1 mRNA only in the two Ma552-positive cell lines.