TY - JOUR
T1 - Mapping and molecular characterization of novel monoclonal antibodies to conformational epitopes on NH2 and COOH termini of mammalian tryptophanyl-tRNA synthetase reveal link of the epitopes to aggregation and Alzheimer's disease
AU - Paley, Elena L.
AU - Smelyanski, Larisa
AU - Malinovskii, Vladimir
AU - Subbarayan, Pochi R.
AU - Berdichevsky, Yevgeny
AU - Posternak, Natalia
AU - Gershoni, Jonathan M.
AU - Sokolova, Olga
AU - Denisova, Galina
N1 - Funding Information:
This work was supported in parts by Fellowship from the Howard Hughes Medical Institute (O.S.), Grants from the Israel Ministries of Health and Science (E.L.P.), Fellowship from the Gileadi Program of the Israel Inter-University Commission (E.L.P.) and Grant from the Israel Science Foundation (G.D.). We thank Dr. Valery Filonenko for providing mAb F25, Dr. Niko Grigorieff for help with electron microscopy and Dr. Olga Volpert for support. The mAbs 6C10 and 9D7 used in this study were produced at the Expert BioMed, Inc.
PY - 2007/1
Y1 - 2007/1
N2 - Tryptophanyl-tRNA synthetase (TrpRS) is an interferon-induced phosphoprotein with autoantigenic and cytokine activities detected in addition to its canonical function in tRNA aminoacylation. The availability of monoclonal antibodies (mAbs) specific for TrpRS is important for development of tools for TrpRS monitoring. A molecular characterization of two mAbs raised in mice, using purified, enzymatically active bovine TrpRS as the inoculating antigen, is presented in this report. These IgG1 antibodies are specific for bovine, human and rabbit but not E. coli TrpRS. Immunoreactivity and specificity of mAbs were verified with purified recombinant hTrpRS expressed in E. coli and TrpRS-derived synthetic peptides. One of the mAbs, 9D7 is able to disaggregate fibrils formed by Ser32-Tyr50 TrpRS-peptide. Epitope mapping revealed that disaggregation ability correlates with binding of 9D7 to this peptide in ELISA and immunocytochemistry. This epitope covers a significant part of N-terminal extension that suggested to be proteolytically deleted in vivo from the full-length TrpRS whereas remaining COOH-fragment possesses a cytokine activity. For epitope mapping of mAb 6C10, the affinity selected phage-displayed peptides were used as a database for prediction of conformational discontinuous epitopes within hTrpRS crystal structure. Using computer algorithm, this epitope is attributed to COOH-terminal residues Asp409-Met425. In immunoblotting, the 6C10 mAb reacts preferably with (i) oligomer than monomer, and (ii) bound than free TrpRS forms. The hTrpRS expression was shown to correlate with growth rates of neuroblastoma and pancreatic cancer cells. Immunohistochemically both mAbs revealed extracellular plaque-like aggregates in hippocampus of Alzheimer's disease brain.
AB - Tryptophanyl-tRNA synthetase (TrpRS) is an interferon-induced phosphoprotein with autoantigenic and cytokine activities detected in addition to its canonical function in tRNA aminoacylation. The availability of monoclonal antibodies (mAbs) specific for TrpRS is important for development of tools for TrpRS monitoring. A molecular characterization of two mAbs raised in mice, using purified, enzymatically active bovine TrpRS as the inoculating antigen, is presented in this report. These IgG1 antibodies are specific for bovine, human and rabbit but not E. coli TrpRS. Immunoreactivity and specificity of mAbs were verified with purified recombinant hTrpRS expressed in E. coli and TrpRS-derived synthetic peptides. One of the mAbs, 9D7 is able to disaggregate fibrils formed by Ser32-Tyr50 TrpRS-peptide. Epitope mapping revealed that disaggregation ability correlates with binding of 9D7 to this peptide in ELISA and immunocytochemistry. This epitope covers a significant part of N-terminal extension that suggested to be proteolytically deleted in vivo from the full-length TrpRS whereas remaining COOH-fragment possesses a cytokine activity. For epitope mapping of mAb 6C10, the affinity selected phage-displayed peptides were used as a database for prediction of conformational discontinuous epitopes within hTrpRS crystal structure. Using computer algorithm, this epitope is attributed to COOH-terminal residues Asp409-Met425. In immunoblotting, the 6C10 mAb reacts preferably with (i) oligomer than monomer, and (ii) bound than free TrpRS forms. The hTrpRS expression was shown to correlate with growth rates of neuroblastoma and pancreatic cancer cells. Immunohistochemically both mAbs revealed extracellular plaque-like aggregates in hippocampus of Alzheimer's disease brain.
KW - Aggregation/disaggregation
KW - Alzheimer's disease
KW - Aminoacyl-tRNA synthetases
KW - Epitope mapping
KW - Monoclonal antibodies
KW - Neuroblastoma
KW - Pancreatic, cervical and kidney cancer cells
UR - http://www.scopus.com/inward/record.url?scp=33748632378&partnerID=8YFLogxK
U2 - 10.1016/j.molimm.2006.02.006
DO - 10.1016/j.molimm.2006.02.006
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AN - SCOPUS:33748632378
SN - 0161-5890
VL - 44
SP - 541
EP - 557
JO - Molecular Immunology
JF - Molecular Immunology
IS - 4
ER -