Monoclonal antibody AA4, which inhibits binding of IgE to high affinity receptors on rat basophilic leukemia cells, binds to novel alpha-galactosyl derivatives of ganglioside GD1b.
Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.
Guo
N H
NH
Laboratory of Structural Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Her
G R
GR
Reinhold
V N
VN
Brennan
M J
MJ
Siraganian
R P
RP
Ginsburg
V
V
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000502:physiology*; D000918:Antibody Specificity; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000276:immunology / Q000302:isolation & purification; D001491:Basophils / Q000378:metabolism*; D001667:Binding, Competitive; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D005690:Galactose / Q000031:analogs & derivatives*; D005732:Gangliosides / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D007073:Immunoglobulin E / Q000378:metabolism*; D007938:Leukemia / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011920:Rats, Inbred WF; D011961:Receptors, Fc / Q000378:metabolism*; D017455:Receptors, IgE; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000944:Antigens, Differentiation, B-Lymphocyte; D000951:Antigens, Neoplasm; D005732:Gangliosides; D011961:Receptors, Fc; D017455:Receptors, IgE; C033851:ganglioside, GD1b; D007073:Immunoglobulin E; D005690:Galactose