Joiner K A, Goldman R, Schmetz M, Berger M, Hammer C H, Frank M M, Leive L
J Immunol. 1984 Jan;132(1):369-75.
The binding of serum C3 to the O-antigen capsule (OAg Cap), lipopolysaccharide (LPS), and outer membrane proteins (OMP) of Escherichia coli 0111B4 was examined. Bacteria were intrinsically labeled with [3H] or [14C]galactose (*gal) in the OAg Cap and LPS moieties or with [14C]leucine (*leu) to label proteins. Organisms were then incubated in serum containing differentially labeled C3, the above fractions were separated, and the proportion of each binding to a column containing anti-C3 was measured. The OAg Cap fraction bound 72 to 82% of the C3, which bound to E. coli 0111B4 during incubation in absorbed 10% pooled normal human serum (10% PNHS) or absorbed 40% C8-deficient serum (C8D). This distribution did not change when the organism was presensitized with immune IgG before serum incubation. A total of 2.93% +/- 0.48 of OAg Cap and 0.52% +/- 0.16 of LPS *gal bound specifically to Sepharose-containing antibodies to C3 (A:C3-Seph) after incubation in 10% PNHS; these values increased to 10.1% +/- 4.5 and 1.8% +/- 0.3, respectively, when C3 deposition was increased fourfold by incubation in 40% C8D. When encapsulated E. coli 0111B4 was incubated in 10% PNHS containing biotinylated C3, specific attachment of OAg Cap *gal to avidin-Sepharose was demonstrated in 1% sodium dodecyl sulfate (SDS), and complete release of bound *gal but not C3 occurred with 1 M NH2OH. When a mutant of E. coli 0111B4 lacking OAg Cap was incubated in 40% C8D, the outer membrane (OM) bound 85% of C3. Five percent of OM *gal from the unencapsulated organism bound to A:C3-Seph in 0.05% SDS, indicating that the fraction of LPS molecules with bound C3 increased threefold in the absence of OAg Cap. OAg Cap does not contain protein, and no net specific binding of *leu from OAg Cap fractions to A:C3 was detectable; 2.4 to 3.6% of OM *leu bound to A:C3-Seph. Immunoprecipitation of 82.9% of OAg Cap *gal with antisera that were directed to E. coli 0111B4 was associated with co-precipitation of 69.5% of C3 in the capsular fraction. Therefore, the majority of C3 bound to E. coli 0111B4 was covalently attached to OAg Cap and LPS. As corroboration of experiments with whole bacteria, purified OAg Cap and purified LPS consumed C3 when incubated in serum in the fluid phase. These results are the first to evaluate the acceptor site for C3 deposition on a Gram-negative organism incubated in serum, and show that LPS, OAg Cap, and OMP are all major acceptor sites for C3 in nonimmune serum.
检测了血清C3与大肠杆菌O111B4的O抗原荚膜（OAg Cap）、脂多糖（LPS）及外膜蛋白（OMP）的结合情况。细菌在OAg Cap和LPS部分用[3H]或[14C]半乳糖（*gal）进行内在标记，或用[14C]亮氨酸（*leu）标记蛋白质。然后将细菌在含有不同标记C3的血清中孵育，分离上述各部分，并测定每一部分与含抗C3柱的结合比例。在吸收的10%混合正常人血清（10% PNHS）或吸收的40% C8缺陷血清（C8D）中孵育期间，OAg Cap部分结合了72%至82%与大肠杆菌O111B4结合的C3。当在血清孵育前用免疫IgG对细菌进行预致敏时，这种分布没有改变。在10% PNHS中孵育后，共有2.93%±0.48的OAg Cap和0.52%±0.16的LPS *gal特异性结合到含抗C3抗体的琼脂糖珠（A:C3-Seph）上；当在40% C8D中孵育使C3沉积增加四倍时，这些值分别增加到10.1%±4.5和1.8%±0.3。当包被的大肠杆菌O111B4在含生物素化C3的10% PNHS中孵育时，在1%十二烷基硫酸钠（SDS）中证明了OAg Cap gal与抗生物素蛋白琼脂糖珠的特异性结合，并且用1 M NH2OH可使结合的gal完全释放，但C3不释放。当缺乏OAg Cap的大肠杆菌O111B4突变体在40% C8D中孵育时，外膜（OM）结合了85%的C3。来自未包被细菌的OM gal中有5%在0.05% SDS中与A:C3-Seph结合，这表明在没有OAg Cap的情况下，结合C3的LPS分子比例增加了三倍。OAg Cap不含蛋白质，未检测到OAg Cap部分的leu与A:C3的净特异性结合；2.4%至3.6%的OM *leu与A:C3-Seph结合。用针对大肠杆菌O111B4的抗血清对82.9%的OAg Cap *gal进行免疫沉淀时，荚膜部分中69.5%的C3也被共沉淀。因此，与大肠杆菌O111B4结合的大部分C3共价连接到OAg Cap和LPS上。作为对全菌实验的佐证，纯化的OAg Cap和纯化的LPS在液相血清中孵育时会消耗C3。这些结果首次评估了血清中孵育的革兰氏阴性菌上C3沉积的受体位点，并表明LPS、OAg Cap和OMP都是非免疫血清中C3的主要受体位点。
