Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells.
ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D (PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesin-1 in human granulocytes, we generated specific polyclonal and monoclonal antibodies (mAbs). We also overexpressed GFP-ARNO and GFP-cytohesin-1 in RBL-2H3 cells to characterize the specificity and the ability of cytohesin-1 mAbs to immunoprecipitate cytohesin-1. Among the hybridomas secreting cytohesin-1 mAbs, only the clones 2E11, 1E4, 3C8, 6F5, 4C7, 7A3 and 8F7 were found to be specific for cytohesin-1. Furthermore, mAb 2E11 immunoprecipitated GFP-cytohesin-1 but not GFP-ARNO under native conditions. In contrast, mAbs 5D8, 4C3, 2G8, 6G11, 4C3, 6D4, 7B4 and 6F8 detected both cytohesin-1 and ARNO as monitored by immunoblotting. Although mAb 6G11 detected both proteins, this antibody immunoprecipitated GFP-ARNO but not GFP-cytohesin-1 under native conditions. Another antibody, mAb 10A12, also selectively immunoprecipitated GFP-ARNO under native conditions, but the epitope recognized by this mAb is unlikely to be linear as no signal was obtained by immunoblotting. Immunoprecipitation with a cytohesin-1 polyclonal antibody and blotting with cytohesin-1 specific mAbs revealed that cytohesin-1 is highly expressed in neutrophils. Cytohesin-1 can be detected in HL-60 cells but the endogenous protein levels were low in undifferentiated cells. Using the specific cytohesin-1 mAb 2E11 we observed a marked increase in levels of cytohesin-1 expression during dibutyryl-cyclic AMP-induced granulocytic differentiation of HL-60 cells. These data suggest that cytohesin-1, which may have important functions in neutrophil physiology, can be useful as a potential marker for granulocytic differentiation.
Garceau
V
V
Centre de Recherche en Rhumatologie et Immunologie, MRC Group on the Molecular Mechanisms of Inflammation, Centre de Recherche du CHUL, Ste-Foy, G1V 4G2, Québec, Canada.
Houle
M G
MG
Chouinard
F
F
Gagnon
S
S
Harbour
D
D
Naccache
P H
PH
Bourgoin
S G
SG
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015815:Cell Adhesion Molecules / Q000276:immunology*; D002454:Cell Differentiation / Q000276:immunology; D006098:Granulocytes / Q000276:immunology*; D020662:Guanine Nucleotide Exchange Factors; D018922:HL-60 Cells; D006801:Humans; D009504:Neutrophils / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D020662:Guanine Nucleotide Exchange Factors; C100723:cytohesin-1