PROTEOLYTIC CLEAVAGE SITES OF NATIVE AE2 ANION-EXCHANGER IN GASTRIC-MUCOSAL MEMBRANES

The AE2 anion exchanger in pig and rabbit gastric mucosal membranes wa s subjected to limited proteolysis with trypsin, chymotrypsin, and pap ain, and to enzymatic N-deglycosylation. A monoclonal antibody to the AE2 C-terminal peptide was raised, characterized, and used to purify p ig AE2 and its C-terminal cleavage products. Five distinct proteolytic cleavage sites within the AE2 transmembrane domain were defined by am ino acid sequencing. The amino acid sequence of pig AE2 in the region encompassing the N-glycosylated Z-loop was also determined by RT-PCR. Tryptic cleavage of pig AE2 in the Z-loop produced C-terminal glycopep tides and was unaffected by deglycosylation, whereas the smaller rabbi t AE2 C-terminal tryptic peptide lacked oligosaccharide, consistent wi th the respective amino acid sequences. The third consensus N-glycosyl ation site in pig Z-loop was heterogeneously glycosylated. Rapid papai n cleavage in the Z-loop and slower cleavage in loop 7-8 produced C-te rminal peptide products which were not N-glycosylated. Chymotryptic cl eavage of the rabbit AE2 Z-loop required prior deglycosylation. Chymot ryptic cleavage in the pig AE2 Z-loop produced C-terminal glycopeptide s. Prior deglycosylation of pig AE2 unmasked novel, ionic strength-sen sitive chymotryptic cleavage sites in the adjacent exofacial loop 7-8. These results provide experimental confirmation for some aspects of A E2 topography previously predicted from primary structure alone.