Immunoprecipitation of membrane proteins from rat basophilic leukemia cells by the antiganglioside monoclonal antibody AA4.
In previous studies, mAb AA4 inhibited IgE binding, induced rapid morphologic changes, and blocked histamine release in rat basophilic leukemia (RBL-2H3) cells. It bound to two novel derivatives of ganglioside GD1b (Ag I and Ag II) that appear to be present only in rat mast cells. The present study demonstrates the importance of gangliosides Ag I and Ag II for binding of mAb AA4 to intact cells. We also investigated the presence of gangliosides Ag I and Ag II and proteins immunoprecipitated with mAb AA4 in the parental and four variant cell lines. In comparison with the parental RBL-2H3, two variant cell lines had very low (0.5% and 2.0%) and two others had intermediate levels (9% and 18%) of 125I-AA4 binding. mAb AA4 inhibited 125I-IgE binding to the parental RBL-2H3 cells and to only one variant with intermediate amounts of gangliosides Ag I and Ag II. Therefore, there are variations in the proximity of these gangliosides to the high affinity IgE receptor (Fc epsilon RI) among different cell lines. mAb AA4 immunoprecipitated proteins of 50 to 60, 120, and 135 kDa from 125I-surface labeled cells. These were different from the subunits of Fc epsilon RI. The amount of gangliosides Ag I and Ag II in cell extracts correlated with the number of mAb AA4 binding sites on the cell surface and with the quantity of proteins precipitated from the different cell lines. Thus, these membrane proteins appear to be associated with gangliosides Ag I and Ag II. The binding of mAb AA4 to the surface gangliosides could induce intracellular changes through transmembrane signaling by these proteins.
Stephan
V
V
Clinical Immunology Section, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
Guo
N
N
Ginsburg
V
V
Siraganian
R P
RP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology; D005732:Gangliosides / Q000032:analysis / Q000276:immunology*; D007073:Immunoglobulin E / Q000378:metabolism; D007457:Iodine Radioisotopes; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D051379:Mice; D009439:Neuraminidase / Q000494:pharmacology; D011233:Precipitin Tests; D051381:Rats; D011961:Receptors, Fc / Q000276:immunology; D017455:Receptors, IgE
D000911:Antibodies, Monoclonal; D000944:Antigens, Differentiation, B-Lymphocyte; D005732:Gangliosides; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011961:Receptors, Fc; D017455:Receptors, IgE; D007073:Immunoglobulin E; D009439:Neuraminidase