Cryptic and regulatory epitopes in CD13/aminopeptidase N.
CD13/Aminopeptidase N (APN) is a ubiquitous cell surface enzyme thought to be involved in downregulation of regulatory peptide signals, in binding viruses, and possibly in tumor invasion. Functional aspects of CD13/APN were probed using two monoclonal antibodies (F23 and MY7) to two distinct epitopes on the protein and using substrates and inhibitors of the enzyme. F23 was capable of completely blocking, and MY7 was capable of partially blocking, the binding of substrate to APN and the enzymatic activity of APN. The number of epitopes on APN was quantitated by flow cytometry and by radiobinding with Scatchard analysis. There were approximately 20,000 F23 epitopes per HL60 cell, whereas there were about 10,000 MY7 epitopes per cell. After binding of F23 or natural peptide substrates, the number of MY7 epitopes increased to become equimolar with F23 epitopes, showing a conformational change in CD13/APN structure that reveals MY7 epitopes. Mild proteinase treatment also revealed the hidden epitopes and equalized epitope numbers. The presence of cryptic epitopes may explain the different effects of these antibodies on substrate binding and enzymatic activity. Competition analysis identified the substrate binding site as involving the zinc binding domain of the enzyme. The substrates blocked F23 epitopes (zinc binding domain of the enzyme) and MY7 epitopes, whereas inhibitors blocked only the F23 epitopes, and downregulated MY7 epitopes. A dimeric structure for CD13/APN, in its native, membrane-bound state was directly demonstrated by cross-linking with Bis (sulfosuccinimidyl) suberate and gel electrophoresis. Despite the conformational changes that increased or decreased MY7 epitopes in CD13/APN after substrate or inhibitor binding, neither substrates nor inhibitors caused alterations in the ratio of homodimers to monomers. This suggests that APN undergoes intramolecular conformational changes rather than gross separation of protomers during its regulation. A model for the conformational regulation of APN is proposed.
Xu
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Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Wellner
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Scheinberg
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D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D018826:CD13 Antigens / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018826:CD13 Antigens