Comparative levels of CALLA/neutral endopeptidase on normal granulocytes, leukemic cells, and transfected COS-1 cells.
We discovered that the common acute lymphoblastic leukemia antigen, CALLA (CD10), was identical to human neutral endopeptidase 3.4.24.11 (NEP), a Zn-binding glycoprotein with an extracellular active site capable of hydrolyzing several biologically active peptides. In this study we compare the expression of CALLA/NEP in terms of antigenic density and enzymatic activity at the cell surface and of messenger RNA (mRNA) levels on granulocytes, leukemic cells, and CALLA-transfected COS-1 cells. Mature granulocytes, the only readily available source of normal human CALLA, express relatively low but constant levels of antigen, NEP activity (3.5 pmol/min/10(6) cells), and mRNA. The two major CALLA-mRNA species of 6.5 kb and 3.8 kb, observed to date in a variety of cells and tissues, were also found in four independent granulocyte preparations. With leukemia cell lines, a correlation was established between the density of CALLA antigen and the level of enzymatic activity (3.4 to 21.0 pmol/min/10(6) cells). This paper constitutes the first report of NEP activity on blast cells derived from patients with non-T acute lymphoblastic leukemia (ALL); the levels of activity were variable (1.5 to 35.9 pmol/min/10(6) cells for six cases) but correlated with the level of CALLA assessed by flow cytometry. Heterogeneous levels of expression of the CALLA-mRNA species were also observed in non-T ALL cases that correlated with the level of CALLA expression at the surface of these cells. Very high levels of NEP activity were achieved by transfecting COS-1 cells with pSV-CALLA; 20% of the transfected cells were CALLA+ and expressed 550 pmol/min/10(6) cells. Extracts prepared from COS-1 cells transfected with pSV-CALLA (carrying human NEP cDNA) and pSVENK19 (carrying rabbit NEP-cDNA), respectively, gave Michaelis constant (Km) values of 50 mumol/L and similar inhibition curves with thiorphan. Thus the recombinant proteins encoded by these two genes have similar enzymatic properties, confirming the high degree of their structural relatedness. The expression of high levels of CALLA/NEP on COS-1 cells should allow the use of this system to test the effects of specific mutations on activity and might lead to the understanding of the role of CALLA in the onset and/or progression of leukemia.
Tran-Paterson
R
R
Division of Immunology, Hospital for Sick Children, Toronto, Canada.
Boileau
G
G
Giguère
V
V
Letarte
M
M
D000818:Animals; D000943:Antigens, Differentiation / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology / Q000648:ultrastructure; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D015870:Gene Expression; D006098:Granulocytes / Q000032:analysis* / Q000201:enzymology / Q000276:immunology; D006801:Humans; D007938:Leukemia / Q000097:blood / Q000276:immunology / Q000473:pathology*; D007942:Leukemia, Experimental / Q000097:blood / Q000276:immunology / Q000473:pathology*; D015260:Neprilysin; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000276:immunology / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology; D014158:Transcription, Genetic; D014162:Transfection
D000943:Antigens, Differentiation; D000951:Antigens, Neoplasm; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010450:Endopeptidases; D015260:Neprilysin