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.

Download Full-text

Deletion of the zinc-binding motif of CD13/aminopeptidase N molecules results in loss of epitopes that mediate binding of inhibitory antibodies

Blood ◽

10.1182/blood.v79.12.3344.3344 ◽

1992 ◽

Vol 79 (12) ◽

pp. 3344-3349

Author(s):

RA Ashmun ◽

LH Shapiro ◽

AT Look

Keyword(s):

Cell Surface ◽

Enzymatic Activity ◽

Catalytic Domain ◽

Myeloid Cell ◽

Aminopeptidase N ◽

Surface Glycoprotein ◽

Zinc Binding ◽

Aminopeptidase Activity ◽

Binding Motif ◽

Cell Surface Glycoprotein

Abstract 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.

Download Full-text

Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells

Blood ◽

10.1182/blood.v75.2.462.462 ◽

1990 ◽

Vol 75 (2) ◽

pp. 462-469 ◽

Cited By ~ 106

Author(s):

RA Ashmun ◽

AT Look

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Myeloid Cells ◽

Myeloid Cell ◽

Aminopeptidase N ◽

Surface Glycoprotein ◽

Aminopeptidase Activity ◽

Peptidase Activity ◽

Cell Surface Glycoprotein ◽

Metalloprotease Inhibitors

Abstract We previously found that the myeloid cell surface glycoprotein CD13 (gp150) is identical to aminopeptidase N (EC 3.4.11.2), a widely distributed membrane-bound, zinc-dependent metalloprotease with an extracellular enzymatic domain that cleaves N-terminal amino acid residues from oligopeptides (J Clin Invest 83:1299, 1989). As a first step toward defining the function of this molecule on myeloid cells, we assessed cell surface-associated N-terminal peptidase activity by sensitive spectrophotometric measurements of the cleavage of p- nitroanilide amino acid derivatives. Aminopeptidase activity detected on the surface of normal and malignant hematopoietic cells coincided with the level of cell surface CD13 expression as measured by flow cytometry. The enzyme was specifically inhibited by the zinc-binding metalloprotease inhibitors, bestatin, 1,10-phenanthroline, or 2.2′- dipyridyl, but was not affected by several inhibitors of other classes of proteases. Aminopeptidase activity was demonstrated for CD13 molecules specifically immunoprecipitated from the surface of CD13- positive cells and was blocked by the metalloprotease inhibitor 1,10- phenanthroline. Moreover, cell surface aminopeptidase activity was partially inhibited when viable cells were incubated with two of a panel of 11 monoclonal antibodies (MoAbs) known to be specific for extracellular epitopes of human CD13. This inhibition was apparent in the absence of detectable downmodulation of CD13 molecules from the cell surface, suggesting that these MoAbs either physically interfere with substrate binding or alter the zinc-coordinating properties of aminopeptidase N molecules. Aminopeptidase N could play an important role in modulating signals generated by peptides at the surface of myeloid cells, either by removing key N-terminal residues from active peptides or by converting inactive peptides to active forms. The inhibitory antibodies used in this study should prove useful in delineating the physiologic roles of CD13/aminopeptidase N on normal and malignant myeloid cells.

Download Full-text

Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells

Blood ◽

10.1182/blood.v75.2.462.bloodjournal752462 ◽

1990 ◽

Vol 75 (2) ◽

pp. 462-469 ◽

Cited By ~ 2

Author(s):

RA Ashmun ◽

AT Look

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Myeloid Cells ◽

Myeloid Cell ◽

Aminopeptidase N ◽

Surface Glycoprotein ◽

Aminopeptidase Activity ◽

Peptidase Activity ◽

Cell Surface Glycoprotein ◽

Metalloprotease Inhibitors

We previously found that the myeloid cell surface glycoprotein CD13 (gp150) is identical to aminopeptidase N (EC 3.4.11.2), a widely distributed membrane-bound, zinc-dependent metalloprotease with an extracellular enzymatic domain that cleaves N-terminal amino acid residues from oligopeptides (J Clin Invest 83:1299, 1989). As a first step toward defining the function of this molecule on myeloid cells, we assessed cell surface-associated N-terminal peptidase activity by sensitive spectrophotometric measurements of the cleavage of p- nitroanilide amino acid derivatives. Aminopeptidase activity detected on the surface of normal and malignant hematopoietic cells coincided with the level of cell surface CD13 expression as measured by flow cytometry. The enzyme was specifically inhibited by the zinc-binding metalloprotease inhibitors, bestatin, 1,10-phenanthroline, or 2.2′- dipyridyl, but was not affected by several inhibitors of other classes of proteases. Aminopeptidase activity was demonstrated for CD13 molecules specifically immunoprecipitated from the surface of CD13- positive cells and was blocked by the metalloprotease inhibitor 1,10- phenanthroline. Moreover, cell surface aminopeptidase activity was partially inhibited when viable cells were incubated with two of a panel of 11 monoclonal antibodies (MoAbs) known to be specific for extracellular epitopes of human CD13. This inhibition was apparent in the absence of detectable downmodulation of CD13 molecules from the cell surface, suggesting that these MoAbs either physically interfere with substrate binding or alter the zinc-coordinating properties of aminopeptidase N molecules. Aminopeptidase N could play an important role in modulating signals generated by peptides at the surface of myeloid cells, either by removing key N-terminal residues from active peptides or by converting inactive peptides to active forms. The inhibitory antibodies used in this study should prove useful in delineating the physiologic roles of CD13/aminopeptidase N on normal and malignant myeloid cells.

Download Full-text