Recombinant soluble IgA Fc receptor: generation, biochemical characterization, and functional analysis of the recombinant protein.
We previously described the cloning of a human myeloid cell surface receptor for the Fc region of immunoglobulin A (Fc alpha R). In the present study, a soluble version of the Fc alpha R (solFc alpha R) was generated by removing the transmembrane and cytoplasmic coding regions from full-length Fc alpha R cDNA and ligating into a mammalian expression vector. COS-7 cells transfected with the solFc alpha R plasmid secreted a protein that inhibited both immunoglobulin A (IgA) and anti-Fc alpha R monoclonal antibody (mAb) binding to Fc alpha R+ U937 cells. Furthermore, the solFc alpha R bound specifically to and could be eluted from an anti-Fc alpha R mAb-immunoaffinity column, retaining biological activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the recombinant full-length Fc alpha R migrates over a molecular mass range of approximately 40-60 kd, consistent with the reported size and heterogeneity of the naturally occurring myeloid cell surface Fc alpha R. The solFc alpha R ran on SDS-PAGE as a smaller band (37-55 kd) that reduced to two bands of 23 and 25 kd following N-glycanase treatment, indicating that the Fc alpha R is a heavily glycosylated protein. The biochemical data, coupled with flow cytometry studies showing that the recombinant Fc alpha Rs bind to five different anti-Fc alpha R mAbs, clearly demonstrate that the cloned Fc alpha R corresponds directly to the major Fc alpha R species expressed on human monocytes, neutrophils, and myeloid cell lines. The generation of soluble receptor protein will permit investigations of the role of Fc alpha R in IgA-mediated immunoregulation, effector functions, and disease.
Maliszewski
C R
CR
Department of Immunology, Immunex Corporation, Seattle, Washington 98177.
VandenBos
T
T
Shen
L
L
Schoenborn
M A
MA
Kubagawa
H
H
Beckmann
M P
MP
Monteiro
R C
RC
D015703:Antigens, CD; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D004247:DNA; D006801:Humans; D008969:Molecular Sequence Data; D011961:Receptors, Fc / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility
D015703:Antigens, CD; C036832:Fc(alpha) receptor; D011961:Receptors, Fc; D011994:Recombinant Proteins; D004247:DNA