Autoantigenicity of DFS70 is restricted to the conformational epitope of C-terminal alpha-helical domain
Introduction
Autoantibodies against Dense Fine Speckles 70 (DFS70), also identical to lens epithelium derived growth factor (LEDGF) and transcription coactivator p75, were identified in 30% of Japanese atopic dermatitis (AD) patients, and less frequently in patients with asthma and interstitial cystitis [1]. We have recently reported that autoantibodies to DFS70 were seen in 11% of 597 Japanese hospital workers, who represented 54% of the antinuclear antibody-positive population, but only 1.5% of patients with systemic rheumatic disease had this autoantibody [2]. On the other hand, LEDGF, which is considered to be a secreted protein translocating into the nucleus, was initially cloned as an autoantigen of cytotoxic antibodies found in sera from patients with age-related cataracts [3] and has been found to link with various cellular processes [4]. It was shown to be a DNA-binding transcription coactivator [5], promote cell survival and enhance stress resistance in various cell lines, and induce the disassembly of gap junction of lens epithelial cells via activation of protein kinase C gamma [4], [6]. LEDGF interacts with HIV-1 integrase and mediates the chromosomal targeting of integrase [7]. The mRNA of p75 was shown to be expressed ubiquitously in a variety of tissues but was most abundant in thymus [5]. Alternative splicing of LEDGF produces a second protein product, p52, from the same gene; 325 of their N-terminal amino acid residues are identical, whereas C-terminal 205 and eight residues are different in LEDGF and p52 [4]. LEDGF and p52 show distinct nuclear distributions and cellular functions, which is considered to be due to the unique C-terminal 205 residue fragment of LEDGF/p75 [5], [7], [8], [9].
Since autoimmunity to DFS70 appears in patients with various diseases and even in healthy individuals, our interest is whether these autoantibodies are clinically relevant; whether the autoantibodies mediate any pathological processes in these patients, or alternatively, whether the appearance of anti-DFS70 antibodies is a reflection of any immunological events occurring in these patients. To find a clue to these questions, we conducted fine epitope mapping of 93 anti-DFS70-positive sera from patients with various clinical manifestations as well as those from healthy individuals.
Section snippets
Serum samples
Fifty-nine sera out of 670 sera in the serum bank of the Department of Dermatology, Nagoya University Hospital, were judged to be positive for anti-DFS70 antibody by indirect immunofluorescence assay (IIF) and western blotting assay using HeLa nuclear extract [2], and were selected for further study. The gender and age for all donors are shown in Table 1, and the clinical diagnosis of the patients are shown in Table 2. Thirty-four healthy human sera containing anti-DFS70 antibody were obtained
The vast majority of anti-DFS70 positive sera did not recognize the linear continuous epitopes of DFS70
The profiles of the sera used in this study are described in Table 1, Table 2. In order to determine the fine epitope specificity of these autoantibodies, they were screened by a synthetic peptide array (Fig. 1). Series of peptides of 12 amino acid residues were synthesized with overlapping by 11 amino acids, and blotted in nitrocellulose membranes. Because a previous report [1] had shown that the autoepitope of DFS70 lies in the C-terminal 205 residues that are not shared with its splicing
Discussion
Anti-DFS70 antibodies have been recently recognized to represent a major ANA subset in the non-rheumatologic population [2], [20]. The clinical impact of DFS70 antibodies is marked by the fact that they are one of the most prevalent autoantibodies in AD reported so far [1], and that they are found in more than one half of ANA-positive healthy individuals [2]. However, detailed examinations of these autoantibodies and comparative studies regarding the clinical features of donors have not been
Acknowledgement
The authors thank Dr. Tan at Scripps Research Institute for the pET-DFS plasmid and advice, and Drs. Toshikazu Usuda and Masanari Kodera at Chukyo Hospital for supplying sera from healthy volunteers. This work was supported in part by grants 13670879 and 15591174 from the Ministry of Education, Science, Sports and Culture of Japan (YM).
References (34)
- et al.
Autoantibodies to DFS 70 kd/transcription coactivator p75 in atopic dermatitis and other conditions
J Allergy Clin Immunol
(2000) - et al.
LEDGF, a survival factor, activates stress-related genes
Prog Retin Eye Res
(2002) - et al.
LEDGF activation of PKC gamma and gap junction disassembly in lens epithelial cells
Exp Eye Res
(2003) - et al.
LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells
J Biol Chem
(2003) - et al.
A novel transcriptional coactivator, p52, functionally interacts with the essential splicing factor ASF/SF2
Mol Cell
(1998) - et al.
An epidemiologic study of fatigue with relevance for the chronic fatigue syndrome
J Psychiatr Res
(1997) - et al.
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa
Anal Biochem
(1987) - et al.
Synthetic compound peptide simulating antigenicity of conformation-dependent autoepitope
J Biol Chem
(1994) - et al.
A charged segment mainly composed of basic amino acids forms an autoepitope of CENP-A
Clin Immunol Immunopathol
(1996) - et al.
LEDGF/p75: a novel nuclear autoantigen at the crossroads of cell survival and apoptosis
Autoimmun Rev
(2003)
Advances in B-cell epitope analysis of autoantigens in connective tissue diseases
Clin Immunol
Technical problems concerning the use of immunoblots for the detection of antinuclear antibodies
J Immunol Methods
Humoral immune response directed against LEDGF in patients with VKH
Immunol Lett
Self-reactive antibodies (natural autoantibodies) in healthy individuals
J Immunol Methods
Cluster analysis of human autoantibody reactivities in health and in type 1 diabetes mellitus: a bio-informatic approach to immune complexity
J Autoimmun
Autoantibodies to factor VIII
Autoimmun Rev
The PWWP domain: a potential protein–protein interaction domain in nuclear proteins influencing differentiation?
FEBS Lett
Cited by (50)
Clinical value of anti-DFS70 antibodies in a cohort of patients undergoing routine antinuclear antibodies testing
2020, Journal of Immunological MethodsA better definition of the anti-DFS70 antibody screening by IIF methods
2018, Journal of Immunological MethodsAnti-DFS70 among HIV-positive individuals – A prospective study
2018, Best Practice and Research: Clinical RheumatologyAnti-DFS70 antibodies detected by immunoblot methods: A reliable tool to confirm the dense fine speckles ANA pattern
2016, Journal of Immunological Methods