Cross-reactivity of the anti-La monoclonal antibody SW5 with early endosome antigen 2.
Coimmunoprecipitation studies with SW5, a frequently used and specific mouse monoclonal antibody (mAb) directed against the human La autoantigen, led to the identification of a functionally unrelated 80 000 MW protein, designated early endosome antigen 2 (EEA2). EEA2 appeared to be directly targeted by mAb SW5. Because an RNA-binding domain, a structural element of La containing the SW5-epitope, was not discernable in the primary structure of EEA2, the SW5-epitope on EEA2 was determined. Coiled-coil region 3 of EEA2 appeared to contain the epitope recognized by SW5. The SW5 epitope regions of La and EEA2 share a limited sequence homology and probably share a higher degree of structural similarity at the tertiary level. Most likely, the most critical determinants for recognition by SW5 reside in elements adopting alpha-helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross-reactivity of mAbs with structurally, but not functionally, similar proteins.
Fouraux
Michael A
MA
Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, the Netherlands.
van der Heijden
Annemarie
A
van Venrooij
Walther J
WJ
Pruijn
Ger J M
GJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western; D003429:Cross Reactions; D011992:Endosomes / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D015531:Radioimmunoprecipitation Assay; D011994:Recombinant Proteins / Q000276:immunology; D012261:Ribonucleoproteins / Q000276:immunology*; D000097585:SS-B Antigen
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; C464460:early endosome antigen 2, human