title: Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells.
abstract: Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, United Kingdom.
authors.forename: H C
authors.initials: HC
authors.lastname: Morton
authors.forename: J D
authors.initials: JD
authors.lastname: Atkin
authors.forename: R J
authors.initials: RJ
authors.lastname: Owens
authors.forename: J M
authors.initials: JM
authors.lastname: Woof
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002846:Chromatography, Affinity; D015922:Complement C1q / Q000378:metabolism; D006224:Cricetinae; D005822:Genetic Vectors; D006801:Humans; D007070:Immunoglobulin A / Q000145:classification / Q000302:isolation & purification* / Q000502:physiology; D010447:Peptide Hydrolases / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012697:Serine Endopeptidases; D014162:Transfection
chemical_list: D007070:Immunoglobulin A; D011993:Recombinant Fusion Proteins; D015922:Complement C1q; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase