Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Lavrentiev Ave. 8, Novosibirsk 630090, Russia.
J Mol Recognit. 2013 Mar;26(3):121-35. doi: 10.1002/jmr.2253.
HIV-infected patients possess anti-integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN-Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti-IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG-mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse-phase chromatography, matrix-assisted laser desorption/ionization spectrometry, and thin-layer chromatography to analyze the cleavage products of two 20-mer OPs corresponding to these AGDs. Both OPs contained 9-10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti-IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti-IN Abzs for intact integrase was ~1000-fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN.
HIV 感染患者拥有抗整合酶（IN）IgG 和 IgM，经 IN-Sepharose 色谱分离后，与典型的蛋白酶不同，这些抗体特异性地水解 IN，但不水解许多其他测试蛋白。完整球状 IN 的水解首先导致蛋白的许多长片段形成，而其与抗-IN 抗体的长时间孵育，特别是在具有高蛋白水解活性的 Abz（Abzs）的情况下，会导致短肽和极短寡肽（OPs）的形成。为了鉴定与整合酶已知 AGD 相对应的 IgG 介导的蛋白水解所有位点，我们使用反相色谱、基质辅助激光解吸/电离光谱和薄层色谱组合来分析对应于这些 AGD 的两个 20 肽 OPs 的切割产物。两个 OPs 都包含 9-10 个主要聚集的大、中、小切割位点。完整 IN 中 AGD 的主要表面切割位点和分析的肽的部分或深水解位点不重合。抗-IN Abzs 的活性位点定位于其轻链上，而重链负责蛋白底物的亲和力。完整球状蛋白与 Abzs 的轻链和重链的相互作用提供了 IN 水解的高度特异性。抗-IN Abzs 对完整整合酶的亲和力比 OPs 高约 1000 倍。数据表明，两个 OPs 主要与 Abzs 总 IgG 池的不同单克隆 Abzs 的轻链相互作用，该 Abzs 对底物的亲和力较低；因此，根据寡肽序列，其水解可能不如完整球状 IN 的切割特异性和明显不同。
