ABSTRACT
The identification of cross-reacting antigens is an important process in the characterization of antibodies. We describe here the application of mRNA-display technology with a cDNA library for the isolation of cross-reacting antigens using a monoclonal antibody against human tumor protein p53 (hTP53) as a model. A mixed cDNA library constructed from mRNAs prepared from several human tissues and cell-lines was used for the mRNA-display. After several rounds of panning, five annotated polypeptides, topoisomeraseII-binding protein 1 (TOBP1), RAS protein activator like 2 isoform 1 (RASAL2), endosome-associated FYVE-domain protein (ZFYVE16), and utrophin (UTRN) as well as hTP53, were identified as cross-reactive antigen candidates. All of them had a consensus motif, -S-D-L-( )-K-L-, which was included in the known epitope of the antibody. They were synthesized in vitro, and their binding was compared by conducting a pull-down assay. In cross-activity to the antibody, they ranked as follows: ZYVE16 congruent with hTP53 congruent with TOBP1 > UTRN > RASAL2.
Subject(s)
Antigens/immunology , Gene Expression Profiling/methods , Gene Library , Tumor Suppressor Protein p53/immunology , Amino Acid Motifs , Amino Acid Sequence , Antibodies/immunology , Antigens/chemistry , Antigens/genetics , Antigens/isolation & purification , Carrier Proteins/chemistry , Carrier Proteins/immunology , Chromatography, Affinity , Cloning, Molecular , Cross Reactions , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/immunology , Epitopes/genetics , Epitopes/immunology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/immunology , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/immunology , Peptides , Polymerase Chain Reaction , Sequence Analysis, DNA , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Tumor Suppressor Protein p53/chemistry , Utrophin/chemistry , Utrophin/immunology , ras GTPase-Activating Proteins/chemistry , ras GTPase-Activating Proteins/immunologyABSTRACT
Helper-dependent adenovirus vector (AdV)-mediated full-length dystrophin expression leads to significant mitigation of the dystrophic phenotype of the mdx mouse. However, dystrophin, as a neoantigen, elicits antibody formation. As an alternative approach, we evaluated gene transfer of full-length murine utrophin, a functional homologue of dystrophin that is normally present only at the neuromuscular junction. A single injection in the tibialis anterior (TA) muscle of the helper-dependent adenovirus vector encoding utrophin provided very good transduction, with 58% of fibers demonstrating sarcolemmal utrophin expression in the neonates, and 35% utrophin-positive (Utr(+)) fibers in adults. The presence of utrophin prevented extensive necrosis in the neonates, halted further necrosis in the adults, and led to restoration of sarcolemmal expression of dystrophin-associated proteins up to 1 year after injection. Marked physiological improvement was observed in both neonates and adults. Neither increased humoral responses nor cellular immune responses were evident. However, there was a time-related decline of the initial high utrophin expression. Although viral DNA persisted in animals that were injected in the neonatal stage, viral DNA levels decreased in muscles of adult mice. These results demonstrate that although utrophin gene transfer leads to amelioration of the dystrophic phenotype, the effects are not sustained upon loss of utrophin expression.
