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

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 462-469 ◽  
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.

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

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 462-469 ◽  
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.

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

Blood ◽  
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

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.

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

Blood ◽  
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.

scholarly journals CD84 Leukocyte Antigen Is a New Member of the Ig Superfamily

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2398-2405 ◽  
Author(s):  
Miguel Angel de la Fuente ◽  
Pilar Pizcueta ◽  
Marga Nadal ◽  
Jaime Bosch ◽  
Pablo Engel
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Surface Glycoprotein ◽  
Single Chain ◽  
Cell Surface Glycoprotein ◽  
Leukocyte Antigen ◽  
Acid Protein ◽  
Ig Superfamily ◽  
Cloned Cdna ◽  
Human B Cell

Abstract cDNA isolated from a human B-cell line Raji library was analyzed and shown to encode the full-length cDNA sequence of a novel cell-surface glycoprotein, initially termed HLy9-β. The predicted mature 307-amino acid protein was composed of two extracellular Ig-like domains, a hydrophobic transmembrane region, and an 83-amino acid cytoplasmic domain. The extracellular Ig-like domains presented structural and sequence homology with a group of members of the Ig superfamily that included CD2, CD48, CD58, and Ly9. Northern blot analysis showed that the expression of HLy9-β was predominantly restricted to hematopoietic tissues. Chromosome localization studies mapped the HLy9-β gene to chromosome 1q24, where other members of this Ig superfamily (CD48 and HumLy9) have been mapped. CD84 monoclonal antibodies (MoAbs) were shown to react with cells transfected with the cloned cDNA. These MoAbs were further used to show that CD84 is expressed as a single chain cell-surface glycoprotein of Mr 64,000 to 82,000, which was highly glycosylated. CD84 had a unique pattern of expression, being found predominantly on lymphocytes and monocytes. Thus, the glycoprotein HLy9-β is recognized by MoAbs previously clustered as CD84 and represents a newly identified member of the Ig superfamily that may play a significant role in leukocyte activation.

scholarly journals CD84 Leukocyte Antigen Is a New Member of the Ig Superfamily

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2398-2405 ◽  
Author(s):  
Miguel Angel de la Fuente ◽  
Pilar Pizcueta ◽  
Marga Nadal ◽  
Jaime Bosch ◽  
Pablo Engel
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Surface Glycoprotein ◽  
Single Chain ◽  
Cell Surface Glycoprotein ◽  
Leukocyte Antigen ◽  
Acid Protein ◽  
Ig Superfamily ◽  
Cloned Cdna ◽  
Human B Cell

cDNA isolated from a human B-cell line Raji library was analyzed and shown to encode the full-length cDNA sequence of a novel cell-surface glycoprotein, initially termed HLy9-β. The predicted mature 307-amino acid protein was composed of two extracellular Ig-like domains, a hydrophobic transmembrane region, and an 83-amino acid cytoplasmic domain. The extracellular Ig-like domains presented structural and sequence homology with a group of members of the Ig superfamily that included CD2, CD48, CD58, and Ly9. Northern blot analysis showed that the expression of HLy9-β was predominantly restricted to hematopoietic tissues. Chromosome localization studies mapped the HLy9-β gene to chromosome 1q24, where other members of this Ig superfamily (CD48 and HumLy9) have been mapped. CD84 monoclonal antibodies (MoAbs) were shown to react with cells transfected with the cloned cDNA. These MoAbs were further used to show that CD84 is expressed as a single chain cell-surface glycoprotein of Mr 64,000 to 82,000, which was highly glycosylated. CD84 had a unique pattern of expression, being found predominantly on lymphocytes and monocytes. Thus, the glycoprotein HLy9-β is recognized by MoAbs previously clustered as CD84 and represents a newly identified member of the Ig superfamily that may play a significant role in leukocyte activation.

scholarly journals Human Herpesvirus 8 K14 Protein Mimics CD200 in Down-Regulating Macrophage Activation through CD200 Receptor

2004 ◽  
Vol 78 (14) ◽  
pp. 7667-7676 ◽  
Author(s):  
Mildred Foster-Cuevas ◽  
Gavin J. Wright ◽  
Michael J. Puklavec ◽  
Marion H. Brown ◽  
A. Neil Barclay
Keyword(s):  
Cell Surface ◽  
Macrophage Activation ◽  
Myeloid Cells ◽  
Human Herpesvirus ◽  
Lytic Cycle ◽  
Surface Glycoprotein ◽  
Human Herpesvirus 8 ◽  
Cell Contact ◽  
Cell Surface Glycoprotein ◽  
Herpesvirus 8

ABSTRACT Many viral proteins limit host immune defenses, and their genes often originate from their hosts. CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) that is implicated in locally preventing macrophage activation. Distant, but recognizable, homologues of CD200 have been identified in many herpesviruses and poxviruses. Here, we show that the product of the K14 open reading frame from human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) interacts with human CD200R and is expressed at the surfaces of infected cells solely during the lytic cycle. Despite sharing only 40% primary sequence identity, K14 and CD200 interacted with CD200R with an almost identical and low affinity (KD = 0.5 μM), in contrast to other characterized viral homologue interactions. Cells expressing CD200 or K14 on the cell surface were able to inhibit secretion by activated macrophages of proinflammatory cytokines such as tumor necrosis factor alpha, an effect that could be specifically relieved by addition of monoclonal antibodies and soluble monomeric CD200 protein. We conclude that CD200 delivers local down-modulatory signals to myeloid cells through direct cell-cell contact and that the K14 viral homologue closely mimics this.

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