Characterization of the anti-DnaJ monoclonal antibodies and their use to compare immunological properties of DnaJ and its human homologue HDJ-1.
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.
Krzewski
Konrad
K
Department of Biochemistry, University of Gdańsk, Poland.
Kunikowska
Danuta
D
Wysocki
Jan
J
Kotlarz
Agnieszka
A
Thompkins
Philip
P
Ashraf
William
W
Lindsey
Nigel
N
Picksley
Steven
S
Głośnicka
Renata
R
Lipińska
Barbara
B
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
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