ABSTRACT
ZF5 was originally cloned as a transcriptional repressor on the mouse c-myc promoter. It contains the Kruppel-type zinc fingers and a conserved POZ domain, which is found in a growing number of zinc finger proteins and mediate protein-protein interactions. Autoantibodies against transcription factors are sometimes found in sera from patients with high levels of anti-nuclear antibody (ANA). Using Western blotting with ZF5 and sera of autoimmune disease, we detected one serum, named M6 serum, which contains the antibody against a transcriptional repressor ZF5. The confirmed epitope was specific to ZF5 and was not reactive to the other analogous factors: BCL-6, ZID, and Sp1. This epitope also has a molecular mimicry of the viral proteins. From these results, we predict that viral protein which mimics host ZF5 antigen triggers self-reactive T cell clones and induces the autoantibody in M6 serum after the destruction of host tissues.
Subject(s)
Autoantibodies/immunology , DNA-Binding Proteins/immunology , Repressor Proteins/immunology , Transcription Factors/immunology , Amino Acid Sequence , Antibody Specificity , Antigen-Antibody Reactions , Autoantigens/immunology , DNA-Binding Proteins/blood , Epitopes/blood , Epitopes/immunology , Epitopes/isolation & purification , Humans , Immune Sera/metabolism , Kruppel-Like Transcription Factors , Molecular Sequence Data , Repressor Proteins/bloodABSTRACT
Murine ZF5 is a transcription factor with five zinc finger motifs that represses the c-myc gene by binding to two GC-rich elements at the promoter region. Because of its ubiquitous expression in a variety of tissues, elucidation of biological functions and cellular target genes of ZF5 is of great interest. As the first step of identifying cellular target genes, we have attempted to determine the consensus binding motif for ZF5. We succeeded in isolating 19 oligonucleotide duplex DNAs to which ZF5 binds and determined the binding sequences with DNase I footprinting analysis. From these sequences, we deduced the consensus binding motif for ZF5 to be GSGCGCGR. In addition, we have analyzed the DNA-binding domain of ZF5 by testing a series of deletion mutants. It turned out that the zinc fingers 3 and 4 of the five finger motifs play a critical role in DNA binding.
Subject(s)
Consensus Sequence , DNA-Binding Proteins/chemistry , DNA/chemistry , Peptide Fragments/chemistry , Repressor Proteins/chemistry , Zinc Fingers , Animals , Binding Sites , DNA/metabolism , DNA-Binding Proteins/metabolism , Mice , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Heteroduplexes/metabolism , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Peptide Fragments/metabolism , Repressor Proteins/metabolismABSTRACT
We isolated genomic DNA containing the entire sequence of ZF5, which was originally identified by its ability to repress the mouse c-myc promoter and which was characterized as one of the POZ (Poxvirus and zinc finger) proteins. The POZ motif is a protein-protein interaction interface found at the N-terminal region of zinc finger proteins. Sequence analysis demonstrated that the ATG translation initiation codon was separately located from the remainder of the coding sequence. Using both RNase protection and primer extension assay, a single major transcription start site was determined. Promoter analysis by transient transfection assay suggested positive autoregulation by ZF5 itself. The ZF5 N-terminal region, including the POZ domain, was required for this regulation. Sp1 also activated the ZF5 promoter and this activity was repressed by addition of ZF5. ZF5 expression was stronger in mouse ovary, lung and brain than in other organs.
Subject(s)
DNA-Binding Proteins/genetics , Repressor Proteins/genetics , Zinc Fingers/genetics , Animals , DNA-Binding Proteins/biosynthesis , Exons , Female , Gene Expression , Genomic Library , Mice , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Repressor Proteins/biosynthesis , Tissue Distribution , Transcription, Genetic , TransfectionABSTRACT
BACKGROUND: A high molecular weight allergen, M-177 (177 kDa) was isolated from Dermatophagoides farinae using a specific antibody raised to an allergenic clone Mag 3, which was obtained by immunoscreening a mite cDNA library. The potent IgE reactivity of M-177 is comparable with that of Der f 2. OBJECTIVE: The aim of this study was to analyse the molecular characteristics and the allergenic activity of M-177 in stored mite extracts. METHODS: Antigens were analysed by immunoblotting and enzyme-linked immunosorbent assay (ELISA; inhibition). Allergenic activity was estimated from IgE reactivity and the results of a histamine release assay. RESULTS: The intact M-177 molecule was present in high concentrations in fresh extract obtained from purified mite bodies, but was only detected in small amounts in stored extracts. Instead of the intact molecule, anti-Mag 3 antibody detected various cross-reactive antigens in the stored preparations. Studies of a stored liquid extract showed that these cross-reactive antigens were produced by the degradation of M-177, and that this change was suppressed by the addition of protease inhibitors. Interestingly, the allergenic activity of the fragmented M-177 (sM-177) isolated from the stored extracts was greater than that of the intact antigen. Specific IgE reacted with sM-177 in 84.2% of 38 sera samples from patients allergic to mites, while 65.8% were positive for M-177-specific IgE. Similarly, the histamine release test showed that sM-177 had greater allergenic activity in vitro. ELISA inhibition indicated that the increased allergenic activity resulted from alteration of the antigenicity with the degradation of M-177. CONCLUSIONS: M-177 is a protease-sensitive allergen. The breakdown products of M-177 provoked higher allergenic activity than the intact allergen.
