Deletion of the zinc-binding motif of CD13/aminopeptidase N molecules
results in loss of epitopes that mediate binding of inhibitory antibodies
RA Ashmun, LH Shapiro and AT Look
Department of Hematology-Oncology, St Jude Children's Research Hospital,
Memphis, TN 38101.
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.
Volume 79,
Issue 12,
pp. 3344-3349,
06/15/1992
Copyright © 1992 by The American Society of Hematology
