Anti‐integrase abzymes from the sera of HIV‐infected patients specifically hydrolyze integrase but nonspecifically cleave short oligopeptides

Abstract

In contrast to canonical proteases, total immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies (Abs) from HIV‐infected patients hydrolyze effectively only HIV integrase (IN), reverse transcriptase (RT), human casein, and serum albumin. Anti‐IN IgG and IgM isolated by chromatography on IN‐Sepharose hydrolyze specifically only IN but not many other tested proteins. Total Abs from HIV‐infected patients hydrolyze not only globular proteins but also different specific and nonspecific tri‐, tetra‐, and 20‐ to 25‐mer oligopeptides (OPs) with a higher rate than anti‐IN Abs isolated using IN‐Sepharose. A similar situation was observed for IgG from patients with multiple sclerosis and HIV‐infected patients, which after purification on myelin basic protein (MBP)–Sepharose and RT‐Sepharose specifically hydrolyze only MBP and RT, respectively. The active sites of all anti‐protein abzymes are localized on their light chains, whereas the heavy chain is responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide the specificity of protein hydrolysis. The affinity of anti‐IN and anti‐MBP abzymes for intact IN and MBP is approximately 102‐ to 105‐fold higher than for short and long specific and nonspecific OPs. The data suggest that all OPs interact mainly with the light chain of different Abs, which possesses a lower affinity for substrates, and therefore, depending on the OP sequences, their hydrolysis may be less specific or completely nonspecific. The data indicate that the relative activity of Abs not fractionated on specific protein sorbents in the hydrolysis of specific and nonspecific OPs can correspond to an average proteolytic activity of light chains of polyclonal Abs directed against many different proteins. Copyright © 2012 John Wiley & Sons, Ltd.

Multiple Sites of the Cleavage of 21- and 25-Mer Encephalytogenic Oligopeptides Corresponding to Human Myelin Basic Protein (MBP) by Specific Anti-MBP Antibodies from Patients with Systemic Lupus Erythematosus

“…The relative rate of the MCA formation was approximately 4-5- (sh-OP2) and 20-25-fold (sh-OX3) higher in the presence of ms-IgG mix than that for sle-IgG mix . Similar situation was observed for longer nonspecific 20-mer oligopeptides in-OP1 and in-OP2 (Fig 1B) corresponding to viral integrase, that, as revealed by a MALDI analysis, contain several sites of IN cleavage in the case of anti-IN IgGs from HIV-infected patients [33], [34]. Nonspecific in-OP1 and in-OP2 oligopeptides corresponding to HIV integrase were slightly hydrolyzed nonspecifically after 24 of the incubation, but there was no detectable difference in the fluorescence intensities of the spots after the incubation of these OPs without (lanes 2) and with sle-IgG mix (lanes 3) (Fig.…”

“…Polyclonal IgG preparations from patients with rheumatoid arthritis also displayed Pro-Phe-Arg-MCA-hydrolyzing activity [32]. Anti-integrase IgGs and IgMs from HIV-infected patients hydrolyze not only globular HIV-1 integrase but also different specific and nonspecific tri- and tetraoligopeptides [33], [34]. Therefore, first we have analyzed the efficiency of hydrolysis of nonspecific tri- and tetrapeptides using sle-IgG mix and ms-IgG mix purified by affinity chromatography on MBP-Sepharose.…”

Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus

“…Polyclonal IgG preparations from patients with rheumatoid arthritis also displayed Pro-PheArg-MCA-hydrolyzing activity [32]. Anti-integrase IgGs and IgMs from HIV-infected patients hydrolyze not only globular HIV-1 integrase but also different specific and nonspecific tri-and tetraoligopeptides [33,34]. Therefore, first we have analyzed the efficiency of hydrolysis of nonspecific tri-and tetrapeptides using sle-IgG mix and ms-IgG mix purified by affinity chromatography on MBP-Sepharose.…”

“…All sites corresponding to major and moderate products of the cleavage are shown by long and short arrows respectively, while to minor ones by diamonds (panels C and F). doi:10.1371/journal.pone.0051600.g006 KIIGQVRDQAE (in-OP2) [33,34]. All mentioned OPs contained fluorescent residue 6-O-(Carboxymethyl)fluorescein ethyl ester (X) on its N-terminus.…”

Constant Domain-regulated Antibody Catalysis

“…Polyclonal antibody studies indicated that the average catalytic activity of IgMs far exceeds that of IgGs (15,19,35). IgGs develop from IgMs by a switch of C-domain gene usage, usually accompanied by immunogen-driven adaptive accumulation of V-domain mutations.…”