Micro-sequencing strategies for the human A33 antigen, a novel surface glycoprotein of human gastrointestinal epithelium.

Monoclonal antibody (mAb) A33, which recognizes a M(r) approximately 43,000 differentiation antigen (A33) expressed in normal human colonic and small bowel epithelium as well as in 95% of colon cancers, shows specific targeting of colon cancer in humans and is currently being evaluated for clinical use. Here, we describe strategies for the purification and structural analysis of the A33 antigen from the human colorectal carcinoma cell lines LIM1215 and SW1222. Edman degradation of the intact protein and nine peptides, derived by proteolytic digestion of the A33 antigen with Asp-N endoproteinase, thermolysin, trypsin and pepsin followed by micropreparative reversed-phase high-performance liquid chromatography, allowed the unambiguous sequence assignment of 153 amino acid residues; these data reveal one N-glycosylation sequeon in Asp-N endoproteinase peptide D4, and a disulfide linkage between peptides D1 and D4. This amino acid sequence information has facilitated the cloning and subsequent sequencing of a cDNA for the A33 antigen which demonstrates that it is a novel human cell surface molecule of the immunoglobulin superfamily.