Proteolytic cleavage sites of native AE2 anion exchanger in gastric mucosal membranes.
The AE2 anion exchanger in pig and rabbit gastric mucosal membranes was subjected to limited proteolysis with trypsin, chymotrypsin, and papain, and to enzymatic N-deglycosylation. A monoclonal antibody to the AE2 C-terminal peptide was raised, characterized, and used to purify pig AE2 and its C-terminal cleavage products. Five distinct proteolytic cleavage sites within the AE2 transmembrane domain were defined by amino 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 glycopeptides and was unaffected by deglycosylation, whereas the smaller rabbit AE2 C-terminal tryptic peptide lacked oligosaccharide, consistent with the respective amino acid sequences. The third consensus N-glycosylation site in pig Z-loop was heterogeneously glycosylated. Rapid papain cleavage in the Z-loop and slower cleavage in loop 7-8 produced C-terminal peptide products which were not N-glycosylated. Chymotryptic cleavage of the rabbit AE2 Z-loop required prior deglycosylation. Chymotryptic cleavage in the pig AE2 Z-loop produced C-terminal glycopeptides. Prior deglycosylation of pig AE2 unmasked novel, ionic strength-sensitive chymotryptic cleavage sites in the adjacent exofacial loop 7-8. These results provide experimental confirmation for some aspects of AE2 topography previously predicted from primary structure alone.
Zolotarev
A S
AS
Molecular Medicine Unit, Beth Israel Hospital, Boston, Massachusetts 02215, USA.
Chernova
M N
MN
Yannoukakos
D
D
Alper
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D017920:Antiporters; D002918:Chymotrypsin / Q000378:metabolism; D018076:DNA, Complementary; D005753:Gastric Mucosa / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011817:Rabbits; D064606:SLC4A Proteins; D017386:Sequence Homology, Amino Acid; D013552:Swine
D027321:Anion Transport Proteins; D000838:Anions; D000911:Antibodies, Monoclonal; D017920:Antiporters; D018076:DNA, Complementary; D008565:Membrane Proteins; D064606:SLC4A Proteins; D002918:Chymotrypsin