Shohet J M, Pemberton P, Carroll M C
Department of Pathology, Harvard Medical School, Boston, Massachusetts.
J Biol Chem. 1993 Mar 15;268(8):5866-71.
Activation of the alternative pathway of complement by immune complexes involves the covalent attachment of the third component (C3) to the IgG heavy chain. In order to localize the site/sites of attachment, adducts of human C3.IgG were digested in situ with endoproteinase Lys-C and Staphylococcus aureus V8 protease, and the fragments were analyzed. The dimeric peptide containing the covalent bond, identified by alkylation of the free thiol group (Cys1010) with iodo[14C]acetamide, was isolated by high-performance liquid chromatography fractionation. A double sequence with NH2 termini corresponding to position 134 of IgG heavy chain and position 1002 of the C3 alpha' chain was found by analysis with automated Edman degradation. The intact dimeric peptide had a mass of 3453 Da and was composed of IgG and C3 fragments with predicted sizes of 23 and 12 residues, respectively. The IgG peptide includes a cluster of six potential acceptor sites for ester bond formation. Thus, it appears that C3 binding is limited to a single region within the CH1 domain of the IgG1 heavy chain.
免疫复合物激活补体替代途径涉及第三成分（C3）与IgG重链的共价连接。为了定位连接位点，用人C3.IgG加合物原位用内肽酶Lys-C和金黄色葡萄球菌V8蛋白酶消化，并对片段进行分析。通过用碘[14C]乙酰胺对游离巯基（Cys1010）进行烷基化鉴定出含有共价键的二聚体肽，通过高效液相色谱分级分离进行分离。通过自动Edman降解分析发现了一个双序列，其NH2末端对应于IgG重链的第134位和C3α'链的第1002位。完整的二聚体肽质量为3453 Da，由预测大小分别为23和12个残基的IgG和C3片段组成。IgG肽包括六个潜在的酯键形成受体位点簇。因此，似乎C3结合仅限于IgG1重链CH1结构域内的单个区域。
