ISOLATION OF LYMPHOCYTE MEMBRANE COMPLEMENT RECEPTOR TYPE-2 (THE C3D RECEPTOR) AND PREPARATION OF RECEPTOR-SPECIFIC ANTIBODY
ISOLATION OF LYMPHOCYTE MEMBRANE COMPLEMENT RECEPTOR TYPE-2 (THE C3D RECEPTOR) AND PREPARATION OF RECEPTOR-SPECIFIC ANTIBODY
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DOI：
10.1073/pnas.78.3.1828
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
ROSS, GD
中科院分区：
文献类型：
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作者：
LAMBRIS, JD;DOBSON, NJ;ROSS, GD
从用于培养 Raji 人[伯基特淋巴瘤]淋巴母细胞的培养基中分离出糖蛋白结合补体成分 C3d。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和气/液色谱分析表明，C3d结合糖蛋白由一条具有广泛链内二硫键的多肽链组成，分子量为72,000，并结合了几种不同的碳水化合物。这种源自培养基的 C3d 结合蛋白显然源自 2 型膜补体受体（CR2，C3d 受体），可能在膜周转期间脱落。 C3d 结合蛋白与 C3d 包被的绵羊红细胞 (EC3d) 结合，但不与 C3b 包被的绵羊红细胞 (EC3b) 结合。用纯化的 C3d 结合糖蛋白免疫制备的抗血清可抑制 EC3d 淋巴细胞玫瑰花结的形成，但不抑制 EC3b。通过十二烷基钠电泳对与抗 C3d 结合蛋白血清反应的放射性标记和溶解的淋巴细胞抗原进行分析，发现了 MW 72,000 的单链细胞表面蛋白，其显然与分离的 C3d 结合蛋白相同。通过使用 F(ab'')2 抗 C3d 结合蛋白（抗 CR2）直接免疫荧光和 EC3d 玫瑰花结形成对淋巴细胞进行平行测定，表明两种测定具有相同的特异性和几乎相同的灵敏度。在这两个系统中，CR2 表达仅限于 B 细胞，在 T 细胞、单核细胞或中性粒细胞上检测不到。
A glycoprotein binding complement component C3d was isolated from media used for culture of Raji human [Burkitt''s lymphoma] lymphoblastoid cells. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gas/liquid chromatography indicated that the C3d-binding glycoprotein consisted of a single polypeptide chain with extensive intrachain disulfide bonds, a MW of 72,000 and several different bound carbohydrates. This medium-derived C3d-binding protein apparently originated from membrane complement receptor type 2 (CR2, the C3d receptor), presumably shed during membrane turnover. The C3d-binding protein bound to sheep erythrocytes coated with C3d (EC3d) but not to sheep erythrocytes coated with C3b (EC3b). Antisera, prepared by immunization with the purified C3d-binding glycoprotein, inhibited lymphocyte rosette formation with EC3d but not with EC3b. Analysis by sodium dodecyl electrophoresis of the radiolabeled and solubilized lymphocyte antigens reactive with the anti-C3d-binding protein sera revealed a single-chain cell-surface protein of MW 72,000 that was apparently identical to the isolated C3d-binding protein. Parallel assay of lymphocytes for CR2 by direct immunofluorescence with F(ab'')2 anti-C3d-binding protein (anti-CR2) and rosette formation with EC3d indicated that both assays had the same specificity and nearly the same sensitivity. With both systems CR2 expression was limited to B cells and was undetectable on T cells, monocytes or neutrophils.