5. Five ways to obtain catalytic antibodies Immunization by transition state analog of reaction Production of antiidiotypic antibody Reactive immunization Induction of autoimmune disease Screening of phage- display libraries Belogurov et al, BioEssays 2009
10. -streptavidin molecule -BSA molecule -Biotin group -Phosphonate group Control well Experimental well Phage pull after reaction with Bt-X phosphonate Nonspecific sorbsion Nonspecific and specific sorbsion Trypsin elution Phage amplification Next rounds of selection Bt-X Reactive (covalent) selection
11. A.17 Catalytic Antibody Biotin-X-phosphonate Griffin.1 scFv library + Screening for biotin-X binding Conversion into the full-size human antibody crystal Reshetnyak et al, JACS 2007
12. A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base
13. A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base Comparison of active site cavities of natural and created de novo biocatalysts. Chemically selected reactibody A.17 possess deepest substrate binding niche. Cross-section views of the active center of esterolytic antibodies 49G7, TEPC15, aldolase antibody 33F12, choline esterases AChE and BChE and antibody A.17 complexed with their ligands.
14. The pre-existing primitive active site of the A.17 antibody stereo-selectively interacts with P(R)-isomer of the phosphonate molecule Tyr59 Tyr53 Tyr33 Tyr34 Trp109 Tyr37 Trp92 Trp48 Phe100 OP compound Tyr 37 OH P(R) Tyr 37 P(S) SN2 Tyr59 Tyr53 Tyr33 Tyr34 Trp109 Asn105 Ala107 Phe100 Trp92 Trp48 Cl - 2.5 Å 3.3 Å 3.2 Å Ser51 2.8 Å Tyr37
15. A.17 antibody hydrolyzes organophosphorus pesticide – paraoxon by multi-step covalent catalysis. Covalent intermediate Amount of released p-nitrophenol in case of the reaction with paraoxon is evidently higher than concentration of active sites The reaction rate increased linearly with hydroxylamine concentration. It allows to define that dephosphorylation is rate limiting step k 2 = 1.1 ± 0.1 x10 -1 min -1 k 3 = 1.6 ± 0.2 x10 -2 min -1 k 4 = 1.26 ± 0.09 x10 -3 min -1 Tyr Tyr + Tyr Tyr Tyr Tyr + +
16. The reaction mechanism of paraoxon hydrolysis by A.17 antibody proceeds through the phosphotyrosine covalent intermediate. 1578.5 m/z=30 1578.5 m/z=30 1583.0 m/z=30 Δ m=135 ( ) (unmodified Fab A.17) (phosphonylated Fab A.17) (unmodified Fab A.17)
19. CDRH3 DNA Possible catalytic residues Schuster et al, Science, 1992, Gololobov et al, PNAS, 1995; Gololobov et al. Mol Immunol. 1997. Structural Similarity Between BV04-01 and MRL-4 anti-DNA Autoantibodies: DNA-binding and DNA-cleaving Activities are Germline-Encoded
20. Do Myelin-Directed Antibodies Predict Multiple Sclerosis? N EJM, 2003 The B-Cell – Old Player, New Position on the Team NEJM, 2008 Multiple sclerosis B-cells as one of the key players in the MS Environmental hypothesis of MS induction. EBV virus involvement.
21. Environmental hypothesis of MS induction. EBV virus involvement. Antibodies selected from MS Phage-display library are crossreactive towards both, Myelin Basic Protein and EBV latent membrane protein 1.
22. Abzymatic Site-specific MBP hydrolysis The cleavage sites are localized inside the encephalitogenic epitopes MBP Musse et al, PNAS 2006 Ponomarenko et al, PNAS 2006
23. Specific B-Cells depletion in MS Toxin Autoreactive B-Cell B-Cell Receptor (BCR), fragments of myelin basic protein (MBP) BCR MBP CD 4 CD 25High TOLERANCE
25. Specific B-Cells depletion in MS Fc-based immunotoxins are shown to be the best in the presented set due to the excellent specific/unspecific cytotoxicity ratio
26. MDS score versus days post disease induction (surrounding pictures). Peptides were applied at days 7-11 (120 μg/rat per day) after disease induction by nasal route. Maximum clinical score in each group of rats, median and 95% confidential interval (in the middle). NS - not significant Belogurov et. al Autoimmunity Administration of MBP peptides to DA rats with EAE
38. Der Mensch als Industriepalast (Man as Industrial Palace) Stuttgart, 1926. Chromolithograph. National Library of Medicine. Fritz Kahn (1888-1968) Kahn’s modernist visualization of the digestive and respiratory system as "industrial palace," really a chemical plant
39. M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Authors: Alexandre G. Gabibov , Alexey A. Belogurov Jr., Ivan V. Smirnov, Inna N. Kurkova, Ekaterina Kuzina, Alexey Kononikhin, Alexey Stepanov, Natalie A. Ponomarenko, Andrey V. Reshetnyak
40. COLLABORATION RUSSIA Prof. Eugene Nikolaev Institute of Biochemical Physics RAS Prof. Alexey Boyko Moscow MS Center Prof. Dmitry Knorre , Prof. Olga Fedorova, Dr. Nikita Kuznetsov Dr. Dmitry Genkin Dr. Dobroslav Melamed USA Prof. Al Tramontano , UC Davis, Medical school National Institute of Allergy and Infectious Diseases National Institutes of Health Dr. Herbert C. Morse III ; Ciphergen Biosystems, Inc FRANCE Prof. Daniel Thomas, Alain Friboulet, Drs. Dominidue Pillet, Marjorie Paon, Berangere Avalle . The University of Technology, Compiegne Prof. Patrick Masson , Drs Eugénie Carletti, Florian Nachon Département de Toxicologie – CRSSA Institut de Recherche Biomédicale des Armées
Editor's Notes
5
основе действия ФОТ лежит необратимое связывание токсина с ацетилхолинэстеразой, что приводит к накоплению ацетилхолина в холинергических синапсах и коллапсу периферической и центральной нервной системы. Различные данные указывают на роль бутирилхолинэстеразы, в предотвращении холинергического кризиса путем связывания и выведения ФОТ из организма. Поэтому БуХэ представляется преспективным средством андотной профилактики и лечения отравлений ФОТ. На сегодняшний день для получения одной дозы БуХЭ необходимо 400 литров человеческой крови. Существующие системы экспресии БуХэ не являются достаточно эффективными
MS is well-known chronic neurodegenerative disease. Long period of time it was postulated that the main aggressors in MS are autoreactive T-Cells specific to major component of myelin membrane – myelin basic protein. However nowadays there is no doubt that B-cells and autoantibodies also have their own important position in attackers team of neurodegenerative process are thought to be one of the key-players in MS.
При рассеянном склерозе одна из причин демиелинизации – это атака цитотоксических лимфоцитов на олигодендроциты в центральной нервной системе. Нами было показано, что подобная атака может быть связана с презентацией на поверхности олигодендроцитов фрагментов основного белка миелина, генерируемых особым энзиматическим комплексом – иммунопротеасомой. Данный переход в свою очередь связан с переходом протеасома – иммунопротеасома, индуцируемым интерфероном. Ингибирование иммунопротеасомы позволит предотвратить некорректную презентацию и тем самым защитить олигодендроциты от цитотоксических лимфоцитов.
