Ghebrehiwet B, Lu P D, Zhang W, Keilbaugh S A, Leigh L E, Eggleton P, Reid K B, Peerschke E I
Department of Medicine, State University of New York, Stony Brook 11794, USA.
J Immunol. 1997 Aug 1;159(3):1429-36.
Two types of widely coexpressed, highly acidic, cell membrane binding proteins that display preferential domain specificity for C1q have been described: a 60-kDa calreticulin homologue, designated cC1q-R, that binds to the collagen-like "stalk" and a 33-kDa glycoprotein with affinity for the globular "heads" (gC1q-R). Although the two molecules are known to be coexpressed on all cell types examined to date and often coelute during purification, there is no direct evidence showing that they associate with each other either on the membrane or when examined in a purified system. In this report we present the first evidence that 1) biotinylated cC1q-R binds to recombinant as well as native gC1q-R, as assessed by solid phase ELISA; 2) binding sites for cC1q-R are located within N-terminal residues 76 through 93 of the mature form of gC1q-R and within residues 204 through 218; 3) this interaction is inhibited by two mAbs, 60.11 and 46.23, that recognize primarily epitopes within the N terminus of gC1q-R corresponding to residues 74 through 96 and by mAb 74.5.2 that recognizes epitopes within residues 204 through 218; and 4) biotinylated cC1q-R binds to microtiter-fixed Raji and K562 cells, and this interaction is inhibited by mAb 60.11. Furthermore, coimmunoprecipitation analysis of Raji cell membranes with anti-gC1q-R mAbs showed the presence of cC1q-R in addition to gC1q-R. Taken together, the evidence suggests that cC1q-R is able to form a complex with gC1q-R and may associate with gC1q-R on the cell surface.
已描述了两种广泛共表达、高度酸性的细胞膜结合蛋白，它们对C1q显示出优先的结构域特异性：一种60 kDa的钙网蛋白同源物，命名为cC1q-R，它与胶原样“柄”结合；另一种33 kDa的糖蛋白，对球状“头部”（gC1q-R）具有亲和力。尽管已知这两种分子在迄今为止检测的所有细胞类型上均共表达，并且在纯化过程中经常一起洗脱，但没有直接证据表明它们在膜上或在纯化系统中检测时相互关联。在本报告中，我们提供了首个证据表明：1）通过固相ELISA评估，生物素化的cC1q-R与重组以及天然的gC1q-R结合；2）cC1q-R的结合位点位于gC1q-R成熟形式的N端残基76至93以及残基204至218内；3）这种相互作用被两种单克隆抗体60.11和46.23抑制，这两种抗体主要识别gC1q-R N端对应于残基第74至96位的表位，以及被识别残基204至218内表位的单克隆抗体74.5.2抑制；4）生物素化的cC1q-R与微量滴定板固定的Raji细胞和K56细胞结合，并且这种相互作用被单克隆抗体60.11抑制。此外，用抗gC1q-R单克隆抗体对Raji细胞膜进行共免疫沉淀分析显示，除了gC1q-R外还存在cC1q-R。综上所述，证据表明cC1q-R能够与gC1q-R形成复合物，并且可能在细胞表面与gC1q-R相关联。
