Autoantigenicity of DFS70 is restricted to the conformational epitope of C-terminal alpha-helical domain

Abstract

Autoantibodies against DFS70 (Dense Fine Speckles 70) are found in 30% of Japanese atopic dermatitis patients, and less frequently in patients with other diseases. We have recently reported that they are also seen in 11% of hospital workers, but in only ∼2% of patients with systemic rheumatic disease. In this study, in order to investigate the possible pathological role of anti-DFS70 antibodies, fine epitope mapping was carried out using 93 anti-DFS70 autoantibody-positive sera. Immunoblotting using overlapping peptides failed to reveal major linear epitopes. Western blotting using various truncated proteins showed a strikingly uniform epitope distribution on a suspected tertiary structure expressed by DFS70^349–435. Some sera showed reactivity only in an immunoprecipitation assay using an in vitro translated DFS70. Circular dichroism analysis revealed that DFS^349–435 contains an approximately 40% alpha-helical conformation, while an overlapping, non-antigenic peptide is composed of random coiled structures. The skewed single major epitope enabled us to establish a highly quantitative ELISA for the epitope region. Antibody titers showed no significant differences between the diseased group and healthy individuals. We propose that anti-DFS70 antibody may be a natural autoantibody, which might modify or reflect the inflammatory process of various disorders.

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.