Research Article Received: 12 August 2013,

Revised: 22 September 2013,

Accepted: 26 September 2013,

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jmr.2329

Why specific anti-integrase antibodies from HIVinfected patients can efficiently hydrolyze 21mer oligopeptide corresponding to antigenic determinant of human myelin basic protein Elena S. Odintsovaa, Pavel S. Dmitrenokb, Anna M. Timofeevaa, Valentina N. Bunevaa,c and Georgy A. Nevinskya,c* Human immunodeficiency virus-infected patients possess anti-integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti-myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti-MBP and anti-IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti-IN abzymes efficiently hydrolyze a 21-mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti-MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG-mediated and IgM-mediated proteolysis of OP21 by anti-IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti-IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti-IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non-cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease-like abzymes against different proteins, anti-IN abzymes possess serine, thiol, acidic, and metal-dependent protease activities. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: human blood antibodies; HIV infected patients; catalytic antibodies; hydrolysis of human myelin basic protein peptide

INTRODUCTION

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Human immunodeficiency virus-1 is the etiologic agent of an extremely dangerous human disease, AIDS (Fauci et al., 2008 and refs therein). The replication cycle of HIV-1 involves reverse transcription of the RNA genome into DNA prior to integration into the host cell genome (Katz and Skalka, 1994). The reverse transcription is carried out by a retroviral encoded RNAdependent DNA polymerase (reverse transcriptase (RT)) (Skalka and Goff, 1993; Litvak, 1996). Replication of retroviruses depends on the integration of a double-stranded DNA copy of the retroviral genome into the host cell nuclear genome (Katz and Skalka, 1994). The integration step is catalyzed by the retroviral enzyme integrase (IN) (Asante-Appiah and Skalka, 1999). Therefore, IN and RT are two important targets of anti-HIV drugs and suppression of their action is very important for suppressing HIV reproduction. The sera of HIV-infected patients contains Abs to many different viral proteins including RT and IN as well as human proteins (Zandman-Goddard and Shoenfeld, 2002; Fauci et al., 2008; Nevinsky, 2011 and references therein). Multiple sclerosis (MS) is a chronic autoimmune (AI) demyelinating disease of the central nervous system leading to the manifestation of different nervous and psychiatric disturbances (O’Connor et al., 2001). Its etiology remains unclear, and the most

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widely accepted theory of MS pathogenesis assigns the main role in the destruction of myelin to the inflammation related to AI reactions (O’Connor et al., 2001).

* Correspondence to: Georgy A. Nevinsky, Laboratory of repair enzymes, Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Avenue, Novosibirsk 630090, Russia. E-mail: [email protected] a E. S. Odintsova, A. M. Timofeeva, V. N. Buneva, G. A. Nevinsky Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Lavrentiev Ave. 8, Novosibirsk 630090, Russia b P. S. Dmitrenok Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, Vladivostok 690022, Russia c V. N. Buneva, G. A. Nevinsky Novosibirsk State University, Pirogova Ave. 10, Novosibirsk 630090, Russia Abbreviations: Ab, antibody; HIV-1, human immunodeficiency virus type 1; IN, HIV-1 integrase; MCA, 4-methylcoumaryl-7-amine; MBP, human myelin basic protein; OP, oligopeptide; RPC, reverse-phase chromatography; SDS, polyacrylamide gel electrophoresis; IgM and IgG, polyclonal IgG and IgM antibodies; IgGmix and IgMmix, mixtures of different individual antibodies from the sera of HIV-infected patients; X, fluorescent residue 6-O-(Carboxymethyl) fluorescein ethyl ester

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ANTI-INTEGRASE ANTIBODIES HYDROLYSE HUMAN MYELIN BASIC PROTEIN

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patients with MS (Polosukhina et al., 2004, 2005, 2006; Legostaeva et al., 2010) and the specific sites of the neural antigen cleaved by abzymes have been established (Ponomarenko et al., 2006). Recently, it was shown that electrophoretically and immunologically homogeneous IgGs (approximately 86% of patients with SLE) purified using several affinity resins including Sepharose with immobilized MBP (MBP-Sepharose) specifically hydrolyze only MBP but not many other tested proteins (Bezuglova et al., 2011). Several rigid criteria were applied to show that the MBP-hydrolyzing activity is an intrinsic property of MS and SLE abzymes but not from healthy donors (Polosukhina et al., 2004, 2005, 2006; Ponomarenko et al., 2006; Legostaeva et al., 2010; Bezuglova et al., 2011, 2012a, 2012b). It was shown that anti-MBP abzymes from the sera of patients with SLE hydrolyze MBP at the same four immunodominant sites of the protein (Ponomarenko et al., 2006; Bezuglova et al., 2011, 2012a, 2012b). In MS and SLE, anti-MBP abzymes with protease activity can attack MBP of the myelin-proteolipid sheath of axons. The established MS drug Copaxone appears to be a specific inhibitor of MBP-hydrolyzing activity of the abzymes (Ponomarenko et al., 2006). Consequently, MBP-hydrolyzing abzymes may play an important negative role in MS and SLE pathogenesis. The first abzymes observed in AIDS were polyclonal IgGs hydrolyzing DNA (Odintsova et al., 2006a). Recently, it was shown that IgGs and IgMs from HIV-infected patients after their purification on sorbents bearing immobilized HIV-1 RT, viral IN, human serum albumin, and casein hydrolyzed only the cognate protein but not many other proteins (Odintsova et al., 2005, 2006b, 2011, 2012, 2013; Baranova et al., 2009, 2010). The sites of IN cleavage by abzymes from HIV-infected patients determined by matrix-assisted laser desorption/ ionization (MALDI mass spectrometry were localized mainly within seven known immunodominant regions of IN (Odintsova et al., 2012). A prolonged incubation of IN with AIDS IgGs and IgMs having high catalytic activity usually produces many OP of different lengths including short ones. To identify all sites of IgG-mediated proteolysis corresponding to known antigenic determinants (AGDs) of IN, we have used a combination of RPC, MALDI spectrometry, and thin layer chromatography (TLC) to analyze the cleavage products of two 20-mer OP corresponding to these AGDs. Both OP contained 9–10 mainly clustered major, medium, and minor sites of cleavage (Odintsova et al., 2013). This suggests that within the pools of IgGs and IgMs of HIV-infected patients, only specific anti-IN Abs are able to hydrolyze intact globular molecules of viral integrase. It was shown that Abs against MBP from the sera of patients with MS and SLE hydrolyze short nonspecific OPs extremely slow, whereas anti-IN abzymes of HIV-infected patients cleavage them with high rate (Odintsova et al., 2012). This phenomenon may be a consequence of different reasons. Taking this into account, it was interesting to study the Abz-dependent hydrolysis of MBP specific sequence by anti-IN abzymes in more detail. In this paper, we have analyzed for the first time site-specific degradation of 21-mer OP (OP21) corresponding to AGD of MBP using a combination of MALDI mass spectrometry, RPC, TLC, and affinity chromatography. In addition, we have compared the hydrolysis of globular intact human MBP and HIV IN, as well as OPs corresponding to AGDs of MBP and IN by anti-MBP and anti-IN abzymes.

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The polyetiologic and polysyndromic character of AI polyetiologic diffuses disease systemic lupus erythematosus (SLE) leads to highly variable manifestations of this disease in terms of many biochemical, immunological, and clinical indices (Hhachn, 1996). We find it interesting that SLE and MS demonstrated some similarity in the development of the same medical, biochemical, and immunological indexes. For example, neuropsychiatric involvement occurs in about 50% of patients with SLE and carries a poor prognosis (reviewed in O’Connor et al., 2001). SLE predominantly affects the central nervous system and within its cerebral complications, it has a particular propensity—perhaps more than any other systemic inflammatory disease—to cause psychiatric disorders (O’Connor et al., 2001). The distinctive production of diverse auto-Abs seems to be related to defective clearance of apoptotic cells. Ab-mediated neural cell injury and rheological disturbances represent the two principal suggested mechanisms of tissue injury (O’Connor et al., 2001). Interplay between these processes, underlying genetic factors, their modification by hormones, complicated by a number of secondary factors, may explain the wide spectrum of features encountered in MS and SLE diseases. Some indicators of disease common to SLE and MS were observed (O’Connor et al., 2001). Several recent findings imply an important role of B cells and auto-Abs against myelin autoantigens including MBP in the pathogenesis of MS (Archelos et al., 2000; O’Connor et al., 2001; Hemmer et al., 2002). It was recently shown that titers of Abs against MBP in patients with SLE 4.2-fold higher than in healthy individuals but 2.1-fold lower than in patients with MS (Bezuglova et al., 2011). In addition, anti-MBP abzymes hydrolyzing MBP were revealed in the sera of patients with SLE and MS (see succeeding text). Catalytically active artificial Abs or abzymes against transition chemical states of different reactions were studied intensively (reviewed in (Keinan, 2005). Natural abzymes hydrolyzing DNA, RNA, polysaccharides, OPs, and proteins are described from the sera of patients with several AI (SLE, Hashimoto’s thyroiditis, polyarthritis, MS, asthma, rheumatoid arthritis, etc.) and viral diseases with a pronounced immune system disturbance (viral hepatitis, AIDS, and tick-borne encephalitis) (reviewed in [14–20]). Healthy humans and patients with many diseases with insignificant AI reactions usually lack abzymes or develop Abs with very low catalytic activities, with these activities being often on a borderline of the sensitivity of detection methods (Nevinsky and Buneva, 2002, 2003, 2005, 2010, 2012; Nevinsky et al., 2002; Nevinsky, 2010). Abzymes may play a significant positive and/or negative role in broadening Ab properties, forming specific pathogenic patterns and clinical settings in different AI conditions (Nevinsky and Buneva, 2002, 2003, 2005, 2010, 2012; Nevinsky et al., 2002; Nevinsky, 2010). It was shown that SLE and MS IgGs and/or IgMs effectively hydrolyzed DNA, RNA, and polysaccharides (Shuster et al., 1992; Baranovskii et al., 1998, 2001; Savel’ev et al., 1999, 2004; Saveliev et al., 2003, Andrievskaya et al., 2000, 2002). Because DNase abzymes of patients with MS (Nevinsky and Buneva, 2003) similar to (Kozyr et al., 1998) patients with SLE are cytotoxic and induce cell apoptosis, they can play an important role in SLE and MS pathogenesis. It has been recently shown that MBP-hydrolyzing activity is an intrinsic property of IgGs, IgMs, and IgAs from the sera of

E. S. ODINTSOVA ET AL.

MATERIALS AND METHODS Chemicals, donors, and patients All chemicals were from Sigma or Pharmacia. Homogeneous HIV-1 IN was obtained as in (Caumont et al., 1999). IN-Sepharose was prepared using BrCN-activated Sepharose according to the standard manufacturer’s protocol. Recently, we have obtained blood sera from 10 healthy volunteers and 24 HIV-infected patients (18–40 years old; men and women) including 16 at the stage of pre-AIDS and 8 at the stage of generalized lymphadenopathy according to the classification of the Center of Disease Control and Prevention and screened them for proteolytic abzymes specifically hydrolyzing HIV integrase (Baranova et al., 2009, 2010). In this work, we have used several previously described catalytically active IgGs and IgMs from HIV-infected patients to study the degradation of MBP and its specific OPs. The blood sampling protocol conformed to the local human ethics committee guidelines (Ethics committee of Novosibirsk State Medical University, Novosibirsk, Russia; Institutional ethics committee specifically approved this study) including written consent of patients and healthy donors to present their blood for scientific purposes in accordance with Helsinki ethics committee guidelines. I confirm that patients actually gave their written consent. Antibody purification

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Electrophoretically homogeneous IgGs and IgMs were obtained from healthy donors and HIV-infected patients by sequential affinity chromatography of the serum proteins on Protein A-Sepharose and gel filtration on a Superdex 200 HR 10/30 column as in (Baranova et al., 2009, 2010). IgMs were separated from IgAs and IgGs by FPLC gel filtration of the total Ab fraction on a Superdex 200 HR 10/30 column (GE Healthcare, New York, NY, USA) equilibrated with 50 mM Tris–HCl (pH 7.5) containing 0.3 M NaCl as described previously (Baranova et al., 2009, 2010) The type of Abs (IgA, IgG, or IgM) in the fractions during different chromatographies was determined by type-specific Western blotting (Baranova et al., 2009, 2010). The fractions corresponding to the central parts of the IgGs and IgMs peaks were concentrated and used in further purification and catalytic activity assays. In order to protect the Ab preparations from bacterial contamination, they were filtered through a Millex filter (pore size 0.2 μm). To exclude artifacts due to possible traces of contaminating enzymes, the IgGs and IgMs from HIVinfected patients as well as from healthy donors were analyzed using SDS-PAGE assay of proteolytic activity. Specific hydrolysis of IN only by Abs from HIV-infected patients was shown similar to (Baranova et al., 2009, 2010). To analyze the ‘average’ catalytic heterogeneity of proteolytic polyclonal Abs, we have prepared a mixture of equal amounts of electrophoretically homogeneous IgGs (IgGmix) and IgMs (IgMmix) with different relatively high activities from the sera of seven HIV-infected patients. In some control experiments, a mixture of equal amounts of IgG (hd-IgGmix) and IgM (hd-IgMmix) preparations from the sera of seven healthy donors was used. The IgGmix and IgMmix (from HIV-infected patients) were chromatographed on an IN-Sepharose column (1 ml) equilibrated with 50 mM Tris–HCl (pH 7.5) containing 0.1 M NaCl. After loading, the column was washed with the same buffer to zero optical density, and IgGs or IgMs were eluted first with a gradient of NaCl (0.2–2.0 M), then with 3 M NaCl

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(Abs(salt)) and finally with 40 mM glycine-HCl (pH 2.6) (Abs(acid)). All Ab fractions were dialyzed against 50 mM Tris–HCl (pH 7.5) and concentrated using Centricon-50 spin concentrators. After each purification step, protein concentrations in the final fractions were measured using a standard Bradford assay calibrated with bovine serum albumin.

Ab proteolytic activity assay The analysis of the hydrolysis of OP was carried out. The mixtures (10–20 μl) containing 50 mM Tris–HCl (pH 7.5), 30 mM NaCl, 0.5–2.0 mM specific OPs, and 0.01–0.03 mg/ml IgGmix or IgMmix separated on antigen IN-modified Sepharose (or 0.2 mg/ml hd-IgGmix or hd-IgMmix) were incubated for 1–24 hr at 37 °C. Non-specific X-OP21 (X-YLASASTMDHARHGFLPRRHR) corresponding to one of the four known IgG-dependent specific cleavage sites of human MBP (Ponomarenko et al., 2006; Bezuglova et al., 2012a, 2012b) was used. This OP contained a fluorescent residue 6-O-(carboxymethyl)fluorescein ethyl ester (X) on its N-end. As controls, we have used OPs corresponding to specific cleavage sites of the two known AGDs of HIV-1 integrase, which were identified in the case of abzymes from HIV-infected patients (Odintsova et al., 2011, 2012, 2013). The products of X-OP21 hydrolysis (0.5–2 μl of the reaction mixture) were analyzed by TLC on Kieselgel 60 F254 plates using the system acetic acid–n-butanol– H2O (1:4:5). The plates were dried and photographed. To quantify the intensities of the fluorescent spots after TLC, the control OPs incubated without Abs were used. The progress of reaction was followed by the decrease in the fluorescence of non-hydrolyzed X-OP21 (%) within the linear region of the time course. The sum of fluorescence intensities of a non-hydrolyzed OP and all accumulated fragments of its cleavage was taken for 100%. In some experiments, the cleavage products of specific X-OP21 were first separated by RPC on Nucleosil C-18 column (4.6 × 250 mm) using 0.05% trifluoroacetic acid and gradient of acetonitrile concentration (0–80%). The relative amount of various cleavage products was calculated by the fluorescence. Excitation was performed at 320 nm and fluorescence emission detected at 490 nm. The fractions corresponding to different peaks were collected, evaporated to minimal volume, and products of the hydrolysis were analyzed by TLC (see previously mentioned statement) and by MALDI spectrometry (see later text).

MALDI-TOF analysis Ab-dependent hydrolysis of oligopeptides In all cases, the products of X-OP21 hydrolysis were analyzed by MALDI-TOF mass spectrometry using a Reflex III system (Bruker, Germany) equipped with a 337-nm nitrogen laser (VSL-337 ND, Laser Science, Newton, MA, USA), 3 ns pulse duration. Saturated solution of cyano-4-hydroxycinnamic acid in a mixture of 0.1% acetonitrile and trifluoroacetic acid (1:2) was used as the matrix. To a 1 μl of the reaction mixture containing hydrolyzed OPs before or after their separation by RPC or TLC, 1 μl of 0.2% trifluoroacetic acid and 2 μl of the matrix were added, and 1 μl of the final mixture was spotted on the MALDI plate, air-dried, and used for the analysis. Calibration of the MALDI spectra was performed using the protein and OP standards I and II (Bruker Daltonic, Germany) in the external and internal calibration mode.

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ANTI-INTEGRASE ANTIBODIES HYDROLYSE HUMAN MYELIN BASIC PROTEIN Homology analysis The lack of appreciable homology of the complete protein sequence of X-OP25 and HIV integrase was verified using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Statistical analysis The results are reported as mean ± S.E. of at least three independent experiments. Errors in the values were within 8–12%.

RESULTS Abzyme characterization In this work, electrophoretically and immunologically homogeneous IgGs and IgM were purified from the sera HIV-infected

patients and characterized similar to (Baranova et al., 2009, 2010; Odintsova et al., 2012, 2013). To analyze an ‘average’ situation, we have prepared a mixture of equal amounts of homogeneous IgGs (IgGmix) and IgMs (IgMmix) from the sera of seven HIV-infected patients and seven healthy donors (hd-IgGmix and hd-IgMmix). Then, IgGmix and IgMmix fractions having affinity for IN were separated by affinity chromatography on Sepharose bearing immobilized IN as in (Baranova et al., 2009, 2010; Odintsova et al., 2013). The fractions of IgGmix and IgMmix eluted from IN-Sepharose with 3 M NaCl (Abs(salt)) and acidic buffer, pH 2.6 (Ab(acid)) were used in this study. To exclude possible artifacts due to traces of contaminating canonical proteases, the purified IgGmix and IgMmix preparations, eluted with salt and acidic buffer, were separated by SDS-PAGE under non-reducing and reducing conditions, respectively, and their proteolytic activities were detected after the extraction of

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Figure 1. Profile of RPC of anti-IN IgG(salt)mix-dependent products of X-OP21 deep hydrolysis (A) and analysis of the products corresponding to different peaks after RPC by TLC (B) or mass spectrometry (C–E): (—), relative fluorescence (A). Numbers of lines in panel B coincide with numbers of peaks on panel A; lane C1 corresponds to the TLC of the final reaction mixture; C2 and C3 correspond to X-OP21 incubated in the absence of Abs and this OP before incubation, respectively, whereas lane C4 shows the position of free fluorescent label X. Lanes C5 and C6 correspond to X-OP21 incubated with hd-IgGmix and hd-IgMmix. Panel C demonstrates the MALDI spectra of the products corresponding to OPs of peak 2 (panel A) and initial non-hydrolyzed X-OP21. Panel D shows signals of the products eluted in two badly separated peaks 5 and 6. Panel E corresponds to MALDI specters of the final reaction mixture of X-OP21 hydrolysis. See Materials and Methods for other details.

E. S. ODINTSOVA ET AL. proteins from excised gel slices as in (Baranova et al., 2009, 2010). IN-hydrolyzing activity of Abs from HIV-infected patients was detected only in the zones corresponding to IgGmix (150 kDa) in the non-reducing gel and to separated light chains of IgMmix (intact IgMs, 970 kDa, cannot enter the gel) in the reducing gel. This, together with the absence of any other band of the activity or protein and the absence of activity in the hd-IgGmix and hd-IgMmix preparations, provided direct evidence that all AIDS IgGmix and IgMmix preparations purified on IN-Sepharose are not contaminated with canonical proteases. In addition, similar to (Baranova, et al., 2009; Odintsova et al., 2011), it was shown that, in contrast to canonical proteases, IgGmix and IgMmix preparations

purified on IN-Sepharose specifically hydrolyzed only IN but not many other tested globular proteins including MBP. Ab-dependent hydrolysis of oligopeptides It was shown earlier that IgGs and IgMs from healthy donors hydrolyze intact IN and OPs corresponding to its AGDs (Baranova et al., 2009, 2010; Odintsova et al., 2011, 2012, 2013). We have confirmed here that hd-IgGmix and hd-IgMmix of healthy donors cannot (for example, Figure 1(B), lanes C5 and C6), whereas INAbs from HIV-infected patients hydrolyze X-OP21 corresponding to AGD of human MBP (Figure 1, lane C1). In this article, to analyze in more detail the hydrolysis of X-OP21 by anti-IN

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Figure 2. Profile of RPC of anti-IN IgG(acid)mix-dependent products of X-OP21 deep hydrolysis (A) and analysis of X-OP products of the hydrolysis corresponding to different peaks after RPC by TLC (B) or mass spectrometry (C–E): (—), relative fluorescence (A). Numbers of lines in panel B coincide with numbers of peaks on panel A; lane C1 corresponds to the TLC of the final reaction mixture; C2 and C3 correspond to X-OP21 incubated in the absence of Abs and this OP before incubation, respectively, whereas lane C4 shows the position of free fluorescent label X. Panel C demonstrates the MALDI spectra of initial non-hydrolyzed X-OP21 (m/z = 2884.1) and products corresponding to peak 5 (panel A); Panel D corresponds to products of peaks 2/3 and 7, whereas panel E to the final reaction mixture of X-OP21 hydrolysis. See Materials and Methods for other details.

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ANTI-INTEGRASE ANTIBODIES HYDROLYSE HUMAN MYELIN BASIC PROTEIN Table 1. The data of RPC, TLC, and MALDI analysis of molecular masses of fluorescent oligopeptides forming after incubation of X-OP21 with anti-IN IgG(salt)mix (Figure 1) Number of AA

0 1 2 3 4 5 6 7 8 9 21

Sites of cleavage of X-OP21 (OPs found by mass spectrometry in the reaction mixture and peaks after RPC) Xa X–Y X–YL X–YLA X–YLAS X–YLASA X–YLASAS X–YLASAST X–YLASASTM X–YLASASTMD X–YLASASTMDHARHGFLPRRHR

Mol. mass, Da (ratio mass/charge, H+-form) Calculated

417 580.94 694.1 765.17 852.25 923.33 1010.41 1111.5 1242.7 1356.79 2884.07

Experimental

416.8 581.0 694.3 765.2 852.3 923.4 1010.6 1112.0 1243.0 1357.0 2884.1

Peak number after RPC (Figure 1(A))

Lane number after TLC (Figure 1(B))

1,RMc 1,2 (3),RMd 8,RM 9(7),RM 3,4(2,7),RM 5/6,7(2–4),RM 4(3,7),RM 7(3,4),RM 3,7(4,8),RM 2,7,RM 2,9,RM

1,RMd 1,2,RM 8,RM 8(7),RM 3,4(2,7),RM 5/6,7(2–4),RM 4(3,7),RM 7(3,4),RM 3,7(4,8),RM 2,7,RM 2,9,RM

Relative content after a deep hydrolysis of OP, % (Figure 1)b 4–6 1–2 6–8 4–6 12–14 25–28 14–16 9–12 16–19 5–7 1–3

RPC, reverse-phase chromatography; TLC, thin layer chromatography; MALDI, matrix-assisted laser desorption/ionization; OP, oligopeptide. a Free fluorescent compound; X-OP21 contained fluorescent X-component. b Total amount of the hydrolysis products was taken for 100%; for each value, a mean of three repeats of TLC chromatography is used. c The same products of hydrolysis corresponding to each peak after RPC (Figure 2(A)) were revealed not only by MALDI spectrometry, but also by TLC (Figure 1(B)). RM reflects the presence of the fluorescent spots corresponding to the analyzed product in the total reaction mixture (RM) analyzed by TLC. d The numbers of the peaks containing some major products were marked in bold, while the numbers of the peaks containing any minor products are given in the parenthesis.

Table 2. The data of RPC, TLC, and MALDI analysis of molecular masses of fluorescent oligopeptides forming after incubation of X-OP21 with anti-IN IgG(acid)mix (Figure 2) Number of AA

0 1 2 3 4 5 6 7 8 9 21

Sites of cleavage of X-OP21 (OPs found by mass spectrometry in the reaction mixture and peaks after RPC)

Mol. mass, Da (ratio mass/charge, H+-form) Calculated

Xa X–Y X–YL X–YLA X–YLAS X–YLASA X–YLASAS X–YLASAST X–YLASASTM X–YLASASTMD X–YLASASTMDHARHGFLPRRHR

417 580.94 694.1 765.17 852.25 923.33 1010.41 1111.5 1242.7 1356.79 2884.07

Experimental 416.8 581.0 694.3 765.2 852.3 923.4 1010.6 1112.0 1243.0 1357.0 2884.1

Peak number after RPC (Figure 2(A))

Lane number after TLC, (Figure 2(B))

Relative content at a deep hydroly-sis of OP, % (Figure 2)b

1,RMc 1,RM 6,7,RM 7,RM 2/3, 6(5,7), RMd 5/6(4,7),RM 2/3(6,7),RM 6,7,RM 6,7,RM 6,7, RM RM

1,RM 1,RM 6,7,RM 6(7) 2/3(5,6,7) 4,5 (6,7) 2/3(6,7) 6,7,RM 6,7,RM 6,7, RM RM

3–6 1–2 8–12 5–8 22–26 26–30 10–13 1–2 3–4 1–2 1–2

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RPC, reverse-phase chromatography; TLC, thin layer chromatography; MALDI, matrix-assisted laser desorption/ionization; OP, oligopeptide. a Free fluorescent compound; X-OP21 contained fluorescent X-component. b Total amount of the hydrolysis products was taken for 100%; for each value, a mean of three repeats of TLC chromatography is used. c The same products of hydrolysis corresponding to each peak after RPC (Figure 2(A)) were revealed not only by MALDI spectrometry, but also by TLC (Figure 2(B)). RM reflects the presence of the fluorescent spots corresponding to the analyzed product in the total reaction mixture (RM) analyzed by TLC. d The numbers of the peaks containing some major products were marked in bold, while the numbers of the peaks containing any minor products are given in the parenthesis.

E. S. ODINTSOVA ET AL. abzymes, we have used IgG(salt)mix, IgM(salt)mix, IgG(acid)mix, and IgM(acid)mix eluted from IN-Sepharose by 3 M NaCl and acidic buffer. MALDI spectrometry analysis of peptide hydrolysis Figure 2 demonstrates that hydrolysis of specific X-OP21 by anti-IN IgG(salt)mix produces several fluorescent OP. TLC alone (Figure 1(B)) cannot unambiguously determine the sequences of these products, because their TLC mobility depends on many factors including the amino acid content, relative hydrophobicity, and the nature of the terminal amino acids. To identify major sites of IgG-mediated proteolysis of this OP, we have

analyzed products of peptide cleavage by a combination of RPC, TLC, and mass spectrometry. We have analyzed the products of nearly complete X-OP21 hydrolysis after 24 hr of incubation. Nine major and several very small peaks corresponding to fluorescent products of X-OP21 hydrolysis were revealed by RPC (Figure 1(A)). The products of all peaks were analyzed by TLC (Figure 1(B)) and by mass spectrometry (for example, Figure 1(C) and (D)). One can see that all RPC peaks according to TLC contain several product of the hydrolysis. It means that some individual products of X-OP21 hydrolysis can be eluted from the sorbent by different concentrations of acetonitrile. The reaction mixture contains Tris–HCl and trifluoroacetic acid, which components can interact with

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Figure 3. Profile of RPC of anti-IN IgM(salt)mix-dependent products of X-OP21 deep hydrolysis (A) and analysis of the products corresponding to different peaks after RPC by TLC (B) or mass spectrometry (C–E): (—), relative fluorescence (A). Numbers of lines in panel B coincide with numbers of peaks on panel A; lane C1 corresponds to the TLC of the final reaction mixture; C2 and C3 correspond to X-OP21 incubated in the absence of Abs and this OP before incubation, respectively, whereas lane C4 shows the position of free fluorescent label X. Panel C demonstrates the MALDI spectra of the products corresponding to OPs of peak 3 (panel A). Panel D shows signals of the products eluted in peak 6. Panel E corresponds to the MALDI specters of the final reaction mixture of X-OP21 hydrolysis. See Materials and Methods for other details.

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ANTI-INTEGRASE ANTIBODIES HYDROLYSE HUMAN MYELIN BASIC PROTEIN positively and negatively charged amino acid residues of products of the X-OP21 hydrolysis. In addition, one cannot exclude that some of the OP products can form interpeptide complexes. An existence of multiple forms of the X-OP21 products can lead to the elution of the same products from the sorbent by different concentration of acetonitrile. Using the combination of RPC, TLC, and MALDI-TOF analyses of the products of X-OP21 hydrolysis were identified. Standard MALDI-TOF analysis is a semi-quantitative approach yielding information only on the molecular masses of analyzed compounds. In some cases, for an additional identification of products of X-OP21 cleavage, the compounds corresponding to different spots after TLC were analyzed by mass spectrometry. An approximate relative content of X-OPs of different length in the final reaction mixture was estimated taking into account the ratio of the relative fluorescence corresponding to the spots with different mobility after TLC (Figure 1(B)); 5-mer > 8-mer ≥ 6-mer ≥ 4-mer ≥ 7-mer ≥ 2-mer ≥ 9-mer ≥ 3-mer ≈ X ≥ 1- (Table 1). Various fractions of abzymes corresponding to a pool of polyclonal Abs eluted from IN-Sepharose with 3 M NaCl and acidic buffer in principle could have different specificity in the hydrolysis of X-OP21. Therefore, we have additionally analyzed products of the X-OP21 hydrolysis by IgG(acid)mix preparation. Seven major and several very small peaks corresponding to fluorescent products of X-OP21 hydrolysis were revealed by RPC (Figure 2(A)). The relative content of X-OPs of different lengths was estimated from the fluorescence corresponding to the spots with different

mobility after TLC (Figure 2(B)): 5-mer > 4-mer ≥ 6-mer ≥ 2-mer ≥ 3mer ≥ X ≈ 8-mer ≥ 7-mer ≈ 9-mer ≈ 1-mer (Table 2). First, immune system produces IgM and later IgG Abs (Fauci et al., 2008). We could not exclude that IgMs and IgGs may be to some extent different in their substrate specificity. First, we have analyzed X-OP21 hydrolysis by IgM(salt)mix preparation, eluted from IN-Sepharose with 3 M NaCl similar to those for IgG preparations (Figure 3 and Table 3). The relative content of products of X-OP21 hydrolysis was estimated: 5-mer ≥ 8-mer ≥ 6-mer ≥ 7-mer ≥ 2-mer ≥ 3-mer ≈ 4-mer ≈ 9-mer ≈ 12-mer ≥ X ≈ 1-mer (Table 3). Then, we have estimated the products of X-OP21 hydrolysis by IgM(acid)mix, eluted from IN-Sepharose with acidic buffer similar to that for IgM(salt)mix preparation (Figure 4 and Table 4). The relative content of the products of X-OP21 hydrolysis was increased in the following order: 5-mer ≈ 8-mer ≥ 6-mer ≥ 4-mer ≥ 2-mer ≥ 7-mer ≈ X ≥ 9-mer ≈ 12-mer ≈ 1-mer (Table 4).

DISCUSSION It was recently shown that anti-MBP IgGs and IgMs from the sera of patients with MS (Polosukhina et al., 2004, 2005, 2006; Legostaeva et al., 2010) and SLE (Bezuglova et al., 2011; Bezuglova et al., 2012a, 2012b) efficiently hydrolyze only globular MBP, whereas anti-IN IgGs and IgMs from the sera of HIV-infected efficiently hydrolyze only globular IN (Baranova et al., 2009, 2010; Odintsova et al., 2011); these abzymes cannot cleavage several other tested proteins. No activity was found

Table 3. The data of RPC, TLC, and MALDI analysis of molecular masses of fluorescent oligopeptides forming after incubation of X-OP21 with anti-IN IgM(salt)mix (Figure 3) Number of AA

0 1 2 3 4 5 6 7 8 9 12 21

Sites of cleavage of X-OP21 (OPs found by mass spectrometry in the reaction mixture and peaks after RPC) Xa X–Y X–YL X–YLA X–YLAS X–YLASA X–YLASAS X–YLASAST X–YLASASTM X–YLASASTMD X–YLASASTMDHAR X–YLASASTMD-HARHGFLPRRHR

Mol. mass, Da (ratio mass/charge, H+-form) Calculated

417 580.94 694.1 765.17 852.25 923.33 1010.41 1111.5 1242.7 1356.79 1722.7 2884.07

Experimental

416.8 581.0 694.3 765.2 852.3 923.4 1010.6 1112.0 1243.0 1357.0 1723.0 2884.1

Peak number after RPC (Figure 3(A))

Lane number after TLC, (Figure 3(B))

Relative content after a deep hydrolysis of OP, % (Figure 3)d

1,RMc 1,RM 7,RM 6(7),RMd 3,RM 5(4,6),RM 3,RM 1(2,3),RM 2,3,6,RM 5(2,3),RM 2,3,RM 2,3,RM

1,RM 1,RM 7,RM 6(7),RM 3,RM 5(4,6),RM 3,RM 1(2,3),RM 2,3,6,RM 5(2,3),RM 2,3,RM 2,3,RM

1–3b 0.5–1.0 4–6 3–5 3–5 25–30 12–15 8–10 20–22 3–5 2–4 5–6

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RPC, reverse-phase chromatography; TLC, thin layer chromatography; MALDI, matrix-assisted laser desorption/ionization, OP, oligopeptide. a Free fluorescent compound; X-OP21 contained fluorescent X-component. b Total amount of the hydrolysis products was taken for 100%; for each value, a mean of three repeats of TLC chromatography is used. c The same products of hydrolysis corresponding to each peak after RPC (Figure 3(A)) were revealed not only by MALDI spectrometry, but also by TLC (Figure 3(B)). RM reflects the presence of the fluorescent spots corresponding to the analyzed product in the total reaction mixture (RM) analyzed by TLC. d The numbers of the peaks containing some major products were marked in bold, while the numbers of the peaks containing any minor products are given in the parenthesis.

E. S. ODINTSOVA ET AL.

Figure 4. Profile of RPC of anti-IN IgM(acid)mix-dependent products of X-OP21 deep hydrolysis (A) and analysis of the products corresponding to different peaks after RPC by TLC (B) or mass spectrometry (C–E): (—), relative fluorescence (A). Numbers of lines in panel B coincide with numbers of peaks on panel A; lane C1 corresponds to TLC of the final reaction mixture; C2 and C3 correspond to X-OP21 incubated in the absence of Abs and this OP before incubation, respectively, whereas lane C4 shows position of free fluorescent label X. Panel C demonstrates the MALDI spectra of the products corresponding to OPs of peak 3 (panel A). Panel D shows signals of the products eluted in peak 7. Panel E corresponds to the MALDI specters of the final reaction mixture of X-OP21 hydrolysis. See Materials and Methods for other details.

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for IgGs and IgMs fraction of healthy donors in the hydrolysis of MBP or IN (Polosukhina et al., 2004, 2005, 2006; Odintsova et al., 2005, 2006a, 2006b, 2011, 2012, 2013; Legostaeva et al., 2010; Bezuglova et al., 2012a, 2012b). The sites of MBP cleavage by MS and SLE IgGs (Bezuglova et al., 2011, 2012a, 2012b) as well as of IN hydrolysis by IgGs and IgMs of HIV-infected patients (Odintsova et al., 2011) determined by mass spectrometry were localized within, respectively, four and seven known immunodominant regions of MBP and IN. It should be mentioned that for the identification of sites of complete MBP hydrolysis, only the largest peptides generated

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by Ab-dependent hydrolysis of intact globular MBP after short times of the incubation were used (Ponomarenko et al., 2006). At the same time, we have seen that long incubation of MBP with MS or SLE IgGs (48–72 hr), especially with abzymes possessing high proteolytic activity, led to the formation of short and very short fragments (Bezuglova et al., 2011, 2012a, 2012b). Similar situation was observed for anti-IN abzymes in the hydrolysis of viral integrase (Odintsova et al., 2011). It means that the total pools of various Abs can contain different subfractions of anti-protein abzymes, which cannot hydrolyze foreign globular proteins, but are capable to hydrolyze cognate proteins and their fragments in many sites but with different rates.

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ANTI-INTEGRASE ANTIBODIES HYDROLYSE HUMAN MYELIN BASIC PROTEIN Table 4. The data of RPC, TLC, and MALDI analysis of molecular masses of fluorescent oligopeptides forming after incubation of X-OP21 with anti-IN IgM(acid)mix (Figure 4) Number of AA

0 1 2 3 4 5 6 7 8 9 12 21

Sites of cleavage of X-OP21 (OPs found by mass spectrometry in the reaction mixture and peaks after RPC) Xa X–Y X–YL X–YLA X–YLAS X–YLASA X–YLASAS X–YLASAST YLASASTM X–YLASASTMD X–YLASASTMDHAR X–YLASASTMD-HARHGFLPRRHR

Peak number after RPC (Figure 4(A))

Lane number after TLC, (Figure 4(B))

Relative content after a deep hydrolysis of OP, % (Figure 4)b

1,RMc 1,RMd 4/5,6,7,RM 2,RM 2,3,6,RM 4/5(2,6,7),RM 3(2,4/5),RM 2(2,4/5),RM 6(2,3,7),RM 2(4/5),RM 1,2,3,RM 2(1,3,4/5,6),RM

1,RM 1,RM 4/5,6,7,RM 2,RM 2,3,6,RM 4/5(2,6,7),RM 3(2,4/5),RM 2(2,4/5),RM 6(2,3,7),RM 2(4/5),RM 1,2,3,RM 2(1,3,4/5,6),RM

4–5 0.5–1 5–8 1–2 8–10 15–18 10–13 3–5 15–17 2–4 1–3 25–30

Mol. mass, Da (ratio mass/charge, H+-form) Calculated

417 580.94 694.1 765.17 852.25 923.33 1010.41 1111.5 1242.7 1356.79 1722.7 2884.07

Experimental

416.8 581.0 694.3 765.2 852.3 923.4 1010.6 1112.0 1243.0 1357.0 1723.0 2884.1

RPC, reverse-phase chromatography; TLC, thin layer chromatography; MALDI, matrix-assisted laser desorption/ionization, OP, oligopeptide. a Free fluorescent compound; X-OP21 contained fluorescent X-component. b Total amount of the hydrolysis products was taken for 100%; for each value, a mean of three repeats of TLC chromatography is used. c The same products of hydrolysis corresponding to each peak after RPC (Figure 5(A)) were revealed not only by MALDI spectrometry, but also by TLC (Figure 5(B)). RM reflects the presence of the fluorescent spots corresponding to the analyzed product in the total reaction mixture (RM) analyzed by TLC. d The numbers of the peaks containing some major products were marked in bold, while the numbers of the peaks containing any minor products are given in the parenthesis.

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Figure 5 summarizes the data concerning X-OP21 hydrolysis by four different preparations of abzymes used. One can see that IgG(salt)mix and IgG(acid)mix hydrolyze X-OP21 at the same 10 clustered sites of X-OP21 beginning from 0 to 1 (the bond between X and first amino acid, X–Y ) to 9–10 (D–Y) bond (Figure 5). Similar result was obtained for IgM(salt)mix and IgM(acid)mix, but in the case of these abzymes in comparison with IgGs, one additional minor site of cleavage between 12 and 13 (R–H) bond was revealed (Figure 5(A)). Deep hydrolysis of X-OP21 (12–24 hr) leading to the formation of short 1-5-mers in principle may be a result of the degradation of longer 8-12-mer X-OPs. However, after a short time of incubation (1–3 hr) according to MALDI spectrometry, the formation of X-OP12 or longer X-OPs was not observed for IgG preparations, and 12-mer was a very minor product of the OP hydrolysis in the case of IgM abzymes. We find it interesting that the cleavage sites corresponding to the formation of 4-mer, 6-mer, 8-mer, and especially 5-mer X-OPs were major sites of the cleavage in the case of all four abzyme preparations after a short time and after a long time of incubation. At the same time, the bonds between 2 and 3 and 3 and 4 amino acids of X-OP21 may be considered as moderate sites of cleavage; these sites of the hydrolysis were the major ones only for IgG(acid) (Figure 5(A)). Depending of the abzyme preparations used, the cleavage sites corresponding to the formation of free X, 1-mer, 7-mer, and 9-mer X-OPs may be minor, moderate, or major ones; the 7-mer X-OP was a major product only in the case of IgG (salt) and IgM(salt) (Figure 5(A), Tables 1–4). We have compared X-OP21 hydrolysis by anti-IN abzymes from HIV-infected patients and by anti-MBP Abs from patients

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In addition, in several reports, short nonspecific OPs were used as substrates of abzymes specifically hydrolyzing only their cognate globular proteins. Thyroglobulin-directed proteolytic IgGs effectively hydrolyzed not only thyroglobulin but also Pro-Phe-Arg-methylcoumarinamide (MCA) at the Arg–MCA bond with a significantly lower affinity (Paul et al., 1997). IgG preparations from patients with rheumatoid arthritis also displayed Pro-Phe-Arg-MCA-hydrolyzing activity (Kalaga et al., 1995) and, Bence Jones proteins cleavage different MCA-peptides (Paul et al., 1995). It was shown that anti-IN abzymes from HIV-infected patients efficiently hydrolyze different short OP, whereas hydrolysis of the same OPs by anti-MBP Abs is extremely slow; remarkable hydrolysis can be revealed only after 4–6 days of incubation (Bezuglova et al., 2012a, 2012b; Odintsova et al., 2012). In addition, according to the data of TLC analysis, anti-IN abzymes efficiently hydrolyzed different 17–25-mer OPs corresponding AGDs of MBP, whereas anti-MBP Abs could not cleavage 20-mer OPs corresponding AGDs of IN (Odintsova et al., 2012). However, anti-IN abzyme-dependent cleavage sites of 17–25mer OPs corresponding AGDs of MBP were not analyzed. Therefore, we have analyzed in this article for the first time the cleavage sites of X-OP21 and a possible difference in the cleavage sites of X-OP21 in the case of anti-MBP and anti-IN abzymes, as well as a possible homology between complete protein sequences of MBP, IN, and OPs corresponding to their AGDs. In addition, we tried to analyze here for the first time possible reasons in significant difference of short OPs hydrolysis by abzymes against various proteins.

E. S. ODINTSOVA ET AL.

Figure 5. All sites of cleavage of X-OP21 determined using a combination of RPC, TLC, and mass spectrometry corresponding to detectable major, moderate, and minor products of this OP digestion by HIV-1 IgG (salt)mix, IgG(acid)mix, IgA(salt)mix, and IgM(acid)mix are shown by long and short arrows, respectively, whereas minor ones by diamonds (A). Panel B shows the trypsin-dependent cleavage sites (line A), a major site which was found previously in the case of hydrolysis of globular intact MBP by MS IgGs (lane B) (34), and all types of sites corresponding to X-OP21 cleavage by anti-MBP abzymes from SLE (lane C) (36).

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with SLE (Figure 5). We find it interesting that several sites of X-OP21 digestion (from X to the 8th amino acid residue) by anti-MBP abzymes (Figure 5, lane C) are the same as for anti-IN Abs (Figure 5(A)). However, in contrast to anti-IN Abs, anti-MBP abzymes cannot hydrolyze X-OP21 leading to the formation of 2-mer, 3-mer, and 9-mer (Figure 5(B), lane C), whereas 2-mer, 3-mer products are minor, moderate or major ones in the case of anti-IN abzymes (Figure 5(A)). In addition, anti-IN IgGs cannot hydrolyze X-OP21 resulting in the formation of 10-mer, 12-mer, and 13-mer (Figure 5(A)), which correspond to major sites of this OP21 hydrolysis by anti-MBP abzymes (Figure 5, line C). AntiIN IgMs also cannot hydrolyze bonds between 10 and 11 and 13 and 14 amino acid residues, whereas 12R–13H-site of the cleavage is the minor one for IgM abzymes. In the case of MS IgGs, the major site of Ab-dependent cleavage of globular MBP within the sequences corresponding to OP21 was identified earlier (Ponomarenko et al., 2006); it is shown in Figure 5 (lane B). An interesting finding was that one cleavage site of X-OP21 coincides with one of the two possible trypsin-dependent sites of this OP hydrolysis (Figure 5, lane A). Anti-IN Abs in contrast to anti-MBP abzyme in fact cannot effectively hydrolyze X-OP21 in sites corresponding to trypsin-dependent ones and bonds around these sites. In this connection, some previously reported data should be mentioned. Many proteolytic abzymes are serine protease-like

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enzymes, and their activity is most strongly reduced after incubation with specific serine protease inhibitors PMSF and/or AEBSF. These abzymes include IgGs hydrolyzing vasoactive intestinal peptide, thyroglobulin, or protrombin from the sera of AI patients (Paul et al., 1989, 1995, 1997; Kalaga et al., 1995; Thiagarajan et al., 2000), casein-hydrolyzing abzymes from human milk and HIV-infected patients (Odintsova et al., 2005, 2006b) and IgMs hydrolyzing HIV gp120 protein from AIDS patients (Paul et al., 2004). In addition, it was shown that small fractions of IgGs and IgMs from patients with MS possess not only serine-like but also metal-dependent protease activity (Polosukhina et al., 2004, 2005, 2006; Legostaeva et al., 2010). For HIV-1 integrase, a very unspecific situation was observed. In contrast to Abs against mentioned proteins, IN-hydrolyzing IgGs and IgMs from only 20% of the patients were inhibited by specific inhibitors of serine proteases and this activity was moderate or low (Baranova et al., 2009, 2010). The total pools of IgGs and IgMs from 40% to 50% of patients contained Ab with metaldependent protease activity, whereas activity of the abzymes of 20% of patients was suppressed by specific inhibitors of acidic proteases. Iodoacetamide, a specific inhibitor of thiol proteases, usually does not significantly affect the activity of proteolytic abzymes (≤ –7% inhibition) (Baranova et al., 2009, 2010 and references therein). Therefore, it was surprising that the IN-hydrolyzing activity of AIDS abzymes could be suppressed in the case of all 100% preparations with iodoacetamide for 12–98%, which is quite different as compared with all other known abzymes with proteolytic activity. We find it interesting that the inhibition of IgG and IgM abzymes by several specific inhibitors analyzed in parallel experiments was observed. For example, one of the IgM preparations was significantly inhibited in across by four specific inhibitors: 46 ± 5% AEBSF; 88 ± 9% Pepstatin A, 64 ± 4% EDTA, and 91 ± 10%, iodineacetamide (the sum of the effects of these four inhibitors was 289%) (Baranova et al., 2010). Overall, in contrast to the known abzymes, the proteolytic activity of 50–60% of individual IgG and IgM preparations from HIV-infected patients was summarily suppressed by four different specific inhibitors of serine, acidic, metal-dependent, and thiol proteases by more than 100% (153–289%) (Baranova et al., 2009, 2010). Thus, it is possible that the immune system of some HIV-infected patients can produce anti-IN abzymes with a combined structure of the active center, carrying amino acid residues typical for four different types of proteases. The effective digestion of X-OP21 by anti-MBP abzymes at site corresponding to that for trypsin may be due to major trypsinlike proteolytic activity of this Abs. However, trypsin-like proteolytic activity is not a major one in the case of anti-IN abzymes, and they can hydrolyze X-OP21 due to their acidic, metaldependent, and especially thiol-like activities, and all of them can possess different substrate specificities. In addition, possibility of the hydrolysis of nonspecific X-OP21 corresponding to MBP by anti-IN abzymes of HIV infected patients could be connected with a partial homology between nucleotide sequences of OP21 and IN. Therefore, it was interesting to analyze a possible homology of OP21 and the complete protein sequence of IN. According to the literature, HIV IN has seven AGDs corresponding to its residues 5–22 (AGD1), 14–35 (AGD2) (Yi et al., 2000), 58–141 (AGD3), 141–172 (AGD4), 248–264 (AGD5) (Bizub-Bender et al., 1994), 208–228 (AGD6), and 251–271(AGD7) (Nilsen et al., 1996). We find it interesting that several fragments of OP21 have some homology with all IN AGDs (Figure 6). The similarity of the

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ANTI-INTEGRASE ANTIBODIES HYDROLYSE HUMAN MYELIN BASIC PROTEIN

Figure 6. All major, moderate, and minor sites of HIV-1 IgG-dependent and IgM-dependent cleavage of N-terminal part of X-OP21 corresponding to human MBP (the data for IgG(acid)mix were used) and additional site of the OP hydrolysis revealed for IgM preparations determined using a combination of RPC, TLC, and mass spectrometry (shown over OP21 sequence, respectively, by gray long and short arrows and diamonds and IgM-dependent site by pentahedron) (A). Complete sequence of HIV integrase and all sites of its cleavage by IgG and IgM from HIV-infected patients (all major, moderate, and minor sites are shown by black long and short arrows and diamonds, respectively, over the sequence (B); the underlined IN fragments are its seven known AGDs. Homology between the complete protein sequence of HIV-1 integrase and the nonspecific OP21 corresponding to the sequence of human MBP (B); identical amino acids at the aligned positions are marked with an asterisk (*) and non-identical amino acids with highly conserved physicochemical properties are marked with a colon (:). All major, moderate, and minor sites of the OP21 cleavage by anti-IN abzymes corresponding to different fragments of OP21 having homology with different part of IN sequence are shown under OP21 fragments using gray symbols.

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specific toward amino acid sequences of OP substrates as compared with Abs against MBP from patients with MS and SLE. One additional question why abzymes against different proteins may be more specific for globular proteins than to different OPs. It is known that catalytic centers of proteolytic abzymes are usually located on the light chain, whereas the heavy chain is more often responsible for specific antigen recognition and increased antigen affinity for Abs (Nevinsky et al., 2002; Nevinsky and Buneva, 2003, 2005, 2010, 2012; Nevinsky, 2010). Intact globular proteins usually interact with both light and heavy chains of abzymes, thus ensuring the specificity of the target protein recognition and its cleavage. At the same time, short OPs may interact mostly with the light chain, which possesses a 100–1000-fold lower affinity for substrates (Nevinsky 2011; Nevinsky and Buneva, 2012; Nevinsky, 2010). In addition, separated light chains of IgGs, IgMs, and IgAs from the sera of patients with different AI and viral diseases usually are significantly more active than intact Abs in the hydrolysis of DNA, RNA, oligosaccharides, and proteins (Nevinsky and Buneva, 2002, 2003, 2005, 2010, 2012; Nevinsky et al., 2002; Nevinsky, 2010). Because of a lower affinity, the separation of the light chains can lead to a decrease in the lifetime of the existence of the complex, and as a consequence, to an increase in the turnover number and Vmax (kcat) of the reaction (Nevinsky and Buneva, 2002, 2003, 2005, 2010, 2012; Nevinsky et al., 2002;

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nonspecific OP21 with these fragments of IN sequence is not absolute and only some positions fully coincide (marked with an asterisk), whereas several positions show good (marked with a colon) conservation of physicochemical and structural properties. We find it interesting that amino acid residues of X-OP21 and complete IN cleavage sites are clustered, but these clusters coincide only partially (Figure 6(B)). N-terminal short (Y)LASA sequence, longer YLASA(–STM; –STMD) fragments, 17-mer (ASTMDHARHGFLPRRHR) and even 18-mer (ASASTMDHARHGFLPRRH) sequences of X-OP21 have good homology with many different fragments of the IN sequence (Figure 6). In addition, different sequences surrounding (on the right and/or at the left) HAR-tripeptide have also homology with various fragments of complete IN sequence. Many sites of cleavage of complete IN by anti-IN abzymes correspond to the sites of the hydrolysis of X-OP21 by these IN Abs. One can see that maximal coinciding sites of the cleavage correspond to uncharged YLASA (Leu-Ala-Ser-Ala-Ser)- or ASA(Ser-Ala-Ser)- fragments of N-terminal amino acid residues and less to the C-terminal sequence of X-OP21 containing several positively charged amino acids (Figure 6(B)). Thus, one can believe that sequence homology between X-OP21 and complete IN can also be important for the cleavage of MBP OP21 by anti-IN abzymes. In addition, one cannot exclude that some of anti-IN abzymes with serine protease-like and especially with acidic, thiol, and metalloprotease activities may be less

E. S. ODINTSOVA ET AL. Nevinsky, 2010). Taken together, lacking or very weak interactions of short substrates with heavy chains of abzymes together with the increase in the rate of OP cleavage in contrast to globular protein molecules may be the main reason for the decrease in specificity of abzymes processing short OPs. In this respect, it should be mentioned that, using MALDI analysis, we have revealed about 40 sites of IN cleavage localized within seven known antigenic determinates (Figure 6(B)), but each individual Ab preparation from HIV-infected patients demonstrated a specific ratio of IN hydrolysis within different AGDs, and some preparations hydrolyzed IN at only a restricted set of sites corresponding to only some of the AGDs (Odintsova et al., 2011). A similar situation can occur for abzymes against other proteins. Therefore, it is not surprising that four fractions of IgGmix and IgMmix preparations from various HIV-infected patients can demonstrate a significant difference in the hydrolysis of X-OP21. In addition, if a specific site of different monoclonal abzymes (included in the pool of polyclonal Abs) recognizing a globular protein is formed mostly by the heavy chain, the specific interaction of the protein with this site will determine the protein sequence available in the active center on the light chain, which can function as to some extent as a nonspecific protease that cleaves any protein sequence in the case of short OPs. Theoretically, a mammalian immune system can produce many variants of Abs against a single antigen. From our point of view, the pool of monoclonal Abs most probably contains abzymes with completely different contributions of their light

and heavy chains to the recognition of substrates (Nevinsky et al., 2002; Nevinsky and Buneva, 2003, 2005, 2010, 2012; Nevinsky, 2010). Taken together, while anti-IN abzymes are specific in the hydrolysis of IN having globular structure, they can, with a different rate, hydrolyze very different specific or nonspecific OPs. Overall, we have revealed here for the first time the cleavage sites of X-OP21 in the case of anti-IN abzymes and compared them with those for globular IN and MBP. One can suppose that a fast cleavage of X-OP21 corresponding to AGD of human MBP by anti-IN abzymes from HIV-infected patients may be a consequence of combination of several factors: the partial homology between peptide sequences of the OP and several fragments of complete IN, interaction of X-OP21 only with light chains of abzymes with significantly low affinity providing higher rate of the reaction as compared with globular protein, and possibility of a less substrate specificity of anti-IN abzymes possessing four different proteolytic activities.

FINDINGS AND ACKNOWLEDGEMENTS This research was made possible in part by grants from the Presidium of the Russian Academy of Sciences (Molecular and Cellular Biology Program, 6.7; Fundamental Sciences to Medicine, 5.13), Russian Foundation for Basic Research (10-04-00281), and funds from the Siberian Division of the Russian Academy of Sciences.

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Why specific anti-integrase antibodies from HIV-infected patients can efficiently hydrolyze 21-mer oligopeptide corresponding to antigenic determinant of human myelin basic protein.

Human immunodeficiency virus-infected patients possess anti-integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, speci...
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