Maliszewski C R, VandenBos T, Shen L, Schoenborn M A, Kubagawa H, Beckmann M P, Monteiro R C
Department of Immunology, Immunex Corporation, Seattle, Washington 98177.
J Leukoc Biol. 1993 Mar;53(3):223-32. doi: 10.1002/jlb.53.3.223.
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.
我们之前描述了一种免疫球蛋白A（FcαR）Fc区的人髓样细胞表面受体的克隆。在本研究中，通过从全长FcαR cDNA中去除跨膜和细胞质编码区并连接到哺乳动物表达载体中，产生了一种可溶性FcαR（solFcαR）。用solFcαR质粒转染的COS-7细胞分泌一种蛋白，该蛋白可抑制免疫球蛋白A（IgA）和抗FcαR单克隆抗体（mAb）与FcαR+ U937细胞的结合。此外，solFcαR特异性结合抗FcαR mAb免疫亲和柱并可从该柱上洗脱，保留生物活性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，重组全长FcαR在约40-60 kd的分子量范围内迁移，与天然存在的髓样细胞表面FcαR的报道大小和异质性一致。solFcαR在SDS-PAGE上以较小的条带（37-55 kd）迁移，经N-糖苷酶处理后减少为23和25 kd的两条带，表明FcαR是一种高度糖基化的蛋白。生化数据，再加上流式细胞术研究表明重组FcαR与五种不同的抗FcαR mAb结合，清楚地证明克隆的FcαR直接对应于人单核细胞、中性粒细胞和髓样细胞系上表达的主要FcαR种类。可溶性受体蛋白的产生将有助于研究FcαR在IgA介导的免疫调节、效应功能和疾病中的作用。
