Qu Z X, Odin J, Glass J D, Unkeless J C
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
J Exp Med. 1988 Mar 1;167(3):1195-210. doi: 10.1084/jem.167.3.1195.
We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.
我们通过从FcγRβ1 cDNA克隆中缺失编码跨膜和细胞质结构域的序列，分离出一种重组分泌型FcγRβ分子，并将截短的cDNA插入真核表达载体pcEXV-3中。为了表达和扩增截短的FcγRβ分子的产量，我们将截短的cDNA质粒与二氢叶酸还原酶缺陷型CHO细胞系以及二氢叶酸还原酶小基因一起转染，并使用甲氨蝶呤扩增基因产物。所得细胞系每24小时分泌2 - 3微克/毫升的截短FcγRβ，可通过IgG-琼脂糖亲和层析轻松纯化。截短的FcγRβ在SDS-PAGE上的分子量为31 - 33,000，且进行了糖基化修饰。N-糖苷酶F切割使分子量降至19,000，与截短产物176个氨基酸残基的大小一致。该蛋白中有两个二硫键。DNP20BSA与抗DNP单克隆抗体形成的免疫复合物的结合显示，IgG1聚集体的结合优于IgG2b和IgG2a聚集体。与之前纯化的FcγR与免疫复合物包被珠子结合的实验相反，免疫复合物在更酸性的pH值下结合稍好。我们惊讶地观察到截短的FcγRβ不与抗FcγR单克隆抗体6B7C反应。先前的工作表明，6B7C在免疫印迹上与FcγR反应，不与静止B细胞和腹膜巨噬细胞表面结合，但与巨噬细胞系和LPS刺激的B细胞结合。我们通过单克隆抗体6B7C与肽缀合物的结合表明，6B7C表位位于完整FcγRβ的169 - 183位残基内，该区域恰好在质膜外。微克量截短的FcγRβ的可得性应有助于进一步分析这些受体的结构和功能。
