Lambris J D, Dobson N J, Ross G D
Proc Natl Acad Sci U S A. 1981 Mar;78(3):1828-32. doi: 10.1073/pnas.78.3.1828.
A glycoprotein binding complement component C3d was isolated from media used for culture of Raji human 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 molecular weight of 72,000, and several different bound carbohydrates. Several lines of evidence indicated that this medium-derived C3d-binding protein originated from membrane complement receptor type two (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 sulfate gel electrophoresis of the radiolabeled and solubilized lymphocyte antigens reactive with the anti-C3d-binding protein sera revealed a single-chain cell-surface protein of molecular weight 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.
从用于培养Raji人淋巴母细胞的培养基中分离出一种结合补体成分C3d的糖蛋白。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和气相/液相色谱分析表明，C3d结合糖蛋白由一条具有广泛链内二硫键的单多肽链组成，分子量为72,000，以及几种不同的结合碳水化合物。多条证据表明，这种源自培养基的C3d结合蛋白起源于二型膜补体受体（CR2，C3d受体），可能在膜周转过程中脱落。C3d结合蛋白与包被有C3d的绵羊红细胞（EC3d）结合，但不与包被有C3b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抑制了与EC3d的淋巴细胞玫瑰花结形成，但不抑制与EC3b的形成。对与抗C3d结合蛋白血清反应的放射性标记和可溶淋巴细胞抗原进行十二烷基硫酸钠凝胶电泳分析，发现一种分子量为72,000的单链细胞表面蛋白，它显然与分离出的C3d结合蛋白相同。用F(ab')2抗C3d结合蛋白（抗CR2）通过直接免疫荧光法和平行检测淋巴细胞与EC3d的玫瑰花结形成来检测CR2，结果表明这两种检测方法具有相同的特异性和几乎相同的灵敏度。在这两种系统中，CR2的表达仅限于B细胞，在T细胞、单核细胞或中性粒细胞上无法检测到。
