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.
Copyright © 2013 John Wiley & Sons, Ltd.