title: Characterization of the anti-DnaJ monoclonal antibodies and their use to compare immunological properties of DnaJ and its human homologue HDJ-1.
abstract: Escherichia coli DnaJ (Hsp40) is suspected to participate in rheumatoid arthritis (RA) pathogenesis in humans by an autoimmune process. In this work a set of 6 anti-DnaJ monoclonal antibodies (mAbs) was raised and localization of the epitopes recognized by the mAbs was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) experiments showed that the mAbs efficiently bound only native antigen. Using DnaJ mutant proteins with deletions of specified domains and ELISA, we found that AC11 mAb reacted with the best conserved in evolution N-terminal J domain, whereas BB3, EE11, CC5, CC8, and DC7 bound to the C-terminal part after residue 200. Mapping performed with the use of a random peptide library displayed by filamentous phage indicated that (1) AC11 mAb bound to a region between residues 33-48, including D-34 which belongs to the HPD triad, present in all DnaJ homologues, (2) BB3 recognized residues localized in the 204-224 region, (3) EE11 recognized the 291-309 region, (4) CC5--the region 326-359, and (5) CC8--the 346-366 region. All these mAbs, as well as the polyclonal antibodies against the N- or C-terminal domain, bound efficiently to HDJ-1, human Hsp40. These results show the presence of a significant immunological similarity between bacterial DnaJ and human HDJ-1, which is not restricted to the evolutionarily conserved parts of the proteins, and suggest that HDJ-1 could be a possible target of immune response triggered by DnaJ.
authors.affiliation: Department of Biochemistry, University of Gdańsk, Poland.
authors.forename: Konrad
authors.initials: K
authors.lastname: Krzewski
authors.forename: Danuta
authors.initials: D
authors.lastname: Kunikowska
authors.forename: Jan
authors.initials: J
authors.lastname: Wysocki
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kotlarz
authors.forename: Philip
authors.initials: P
authors.lastname: Thompkins
authors.forename: William
authors.initials: W
authors.lastname: Ashraf
authors.forename: Nigel
authors.initials: N
authors.lastname: Lindsey
authors.forename: Steven
authors.initials: S
authors.lastname: Picksley
authors.forename: Renata
authors.initials: R
authors.lastname: Głośnicka
authors.forename: Barbara
authors.initials: B
authors.lastname: Lipińska
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005260:Female; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary
chemical_list: D000911:Antibodies, Monoclonal; C494647:DNAJB1 protein, human; C494635:DnaJ protein, E coli; D000939:Epitopes; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins