Neuroscience Letters 374 (2005) 203–206 Identification of the epitope of a monoclonal antibody to DJ-1 David W. Miller ∗ , Carmen R. Wilson, Mona A. Kaleem, Jeff Blackinton, Mark R. Cookson Laboratory of Neurogenetics, National Institute on Aging, Bldg 35, Rm 1A-1002, 35 Convent Drive, Bethesda, MD 20892, USA Received 15 September 2004; received in revised form 15 October 2004; accepted 21 October 2004 Abstract Mutations in DJ-1 can cause early onset parkinsonism. Various antibodies have been generated to detect this protein, one of which is a commonly used monoclonal antibody (clone 3E8). Since results of in situ examinations of DJ-1 expression with this antibody have differed from analyses with species-specific antibodies (e.g. rat), it would be useful to know the epitope for this antibody. Using GFP-tagged deletion constructs of human DJ-1, we have localized the epitope region for this antibody to within residues 56–78 of human DJ-1. Mapping this region to the published three-dimensional structure of DJ-1 indicates that this is a solvent-accessible surface epitope. Immunonegativity of E64D mutant DJ-1 with the monoclonal antibody suggests that glutamate 64 of human DJ-1 contributes to the epitope recognized by this antibody. Moreover, the loss of immunoreactivity due to such a small substitution demonstrates the remarkable sensitivity of the monoclonal antibody 3E8 to DJ-1. Published by Elsevier Ireland Ltd. Keywords: DJ-1; Early onset parkinsonism; Epitope map; PARK7; Parkinson’s disease Mutations in DJ-1 are associated with early onset parkinson- ism in humans [2]. The function of the protein is still unclear, although chaperone, protease, and transcriptional regulation activities have been implicated [4]. It is clear that DJ-1 re- sponds to oxidative stress and protects cells against a number of toxic insults [3,15]. As all the mutations identified to date are recessive and presumably loss of function, it is reason- able to hypothesize that mutations in DJ-1 reduce the ability of cells to withstand pro-cell death insults including oxidative paradigms. Detailed neuropathology of the parkinsonian syndrome linked to DJ-1 mutations is not yet available, but PET stud- ies in these cases implicate presynaptic neuronal loss in the nigrostriatal system [5,7]. This suggests that loss of DJ-1 in neurons is associated directly or indirectly with neuronal cell loss. However, the majority of DJ-1 protein is detected in glia rather than neurons in human brain [1,11]. Most of these stud- ies to date have used a commercially available monoclonal antibody (clone 3E8) that was raised against full-length DJ-1 [10], but whose epitope is not known. Neuronal labeling has ∗ Corresponding author. Tel.: +1 301 451 3831; fax: +1 301 480 2830. E-mail address: millerda@mail.nih.gov (D.W. Miller). been reported using anti-peptide antibodies in rodent tissue [12,14], which may represent either a difference in epitope availability between neurons and glia or a species difference between rodents and humans. The mRNA for DJ-1 is present in neurons of mouse brain [13]. Because of the differences in apparent expression in neurons and glia between different antibodies and species, we felt it would be important to map the epitope of monoclonal antibody 3E8 to DJ-1. We generated a series of expression constructs that had GFP fused to fragments of human DJ-1. PCR of the DJ-1 cDNA sequence was carried out using primers that yielded various fragments of DJ-1 cDNA (primer sequences avail- able upon request). These fragments were then ligated into pcDNA3.1/NT-GFP-TOPO vector according to manufac- turer’s specifications (Invitrogen, Carlsbad, CA, USA). Con- structs were sequenced using the BigDye Terminator Kit v3 on an ABI3100 Sequencer. The resulting fusion proteins en- coded the following N-terminal amino acid sequences of DJ- 1: 6–27, 6–79, 6–94, 6–147, 6–174 and 6–189. Another set of fusion proteins encoded the following C-terminal amino acid sequences of DJ-1: 26–189, 56–189, 78–189, 102–189, 150–189 and 179–189. HEK293 cells were transiently trans- fected with these constructs using FuGENE (Roche Applied 0304-3940/$ – see front matter Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2004.10.088