Deletion of the zinc-binding motif of CD13/aminopeptidase N molecules results in loss of epitopes that mediate binding of inhibitory antibodies.
The myeloid cell-surface glycoprotein CD13/aminopeptidase N (APN; EC 3.4.11.2) contains a pentapeptide (HExxH) in its extracellular domain that is characteristic of many zinc-dependent metalloproteinases. This region contains residues important for zinc binding and constitutes part of the catalytic domain of several metalloproteases. We deleted an internal fragment of 117 base pairs (bp) from the human CD13/APN cDNA, resulting in an in-frame deletion that included the sequences coding for this pentapeptide motif. The mutant cDNA was subcloned into a retroviral expression vector, and polypeptides encoded by the altered cDNA were expressed in transfected murine NIH-3T3 fibroblasts. The mutant CD13/APN molecules lacked enzymatic activity, and their intracellular processing to the cell surface was retarded by comparison with normal CD13/APN polypeptides. The mutant molecules also lacked epitopes required for binding of four of 19 CD13-specific monoclonal antibodies (MoAbs) tested in flow cytometric assays. Each of the four MoAbs also inhibited the enzymatic activity of wild-type APN molecules, suggesting that these antibodies may inhibit aminopeptidase activity by interfering with the enzyme's zinc-coordinating properties. Cells engineered to express mutant CD13/APN polypeptides at the cell surface provide a tool for defining the physiologic role of this enzyme on normal and malignant myeloid cells and marrow stromal cells.
Ashmun
R A
RA
Department of Hematology-Oncology, St Jude Children's Research Hospital, Memphis, TN 38101.
Shapiro
L H
LH
Look
A T
AT
D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015703:Antigens, CD / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D018826:CD13 Antigens; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D007163:Immunosorbent Techniques; D051379:Mice; D008970:Molecular Weight; D016296:Mutagenesis; D014162:Transfection; D015032:Zinc / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000939:Epitopes; D004247:DNA; D000626:Aminopeptidases; D018826:CD13 Antigens; D015032:Zinc