ABSTRACT
Oculopharyngeal muscular dystrophy is caused by small alanine expansions in polyadenylate binding protein nuclear 1 (PABPN1) protein resulting in its intranuclear accumulation in skeletal muscle. 3F5 llama antibody specifically interferes with the PABPN1 aggregation process in vitro and in vivo. To understand the structural basis for its epitope recognition we mapped the binding interface of 3F5 with PABPN1 and provide a structural model of the 3F5-PABPN1 complex. We show that 3F5 complementarity determining regions create a cavity in which PABPN1 alpha-helix domain resides by involving critical residues previously implicated in the aggregation process. These results may increase our understanding of the PABPN1 aggregation mechanism and the therapeutic potential of 3F5.
Subject(s)
Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/immunology , Muscular Dystrophy, Oculopharyngeal/immunology , Poly(A)-Binding Protein II/immunology , Poly(A)-Binding Protein II/metabolism , Humans , Kinetics , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Poly(A)-Binding Protein II/chemistry , Protein Binding , Protein Conformation , Spectrometry, FluorescenceABSTRACT
Oculo-pharyngeal muscular dystrophy (OPMD) is characterised by progressive eyelid drooping (ptosis) and difficulties with swallowing (dysphagia). In order to determine the role of growth factors, cytokines and chemokines in the physiopathology of muscle disease we have compared the level of expression of 174 factors in both the affected (cricopharyngeal) and non-affected (sternocleidomastoid) muscles of 8 OPMD patients by means of antibody arrays. Despite an important inter-individual variability the expression of sixty factors was found to be persistently different between affected and non-affected muscles. Many of the differentially expressed factors in our study are known to be involved in the formation of fibrosis in both the liver and the lung, indicating the possibility that treatments such as those used in hepatic fibrosis may have a beneficial effect in OPMD patients.
Subject(s)
Cytokines/metabolism , Fibrosis/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophy, Oculopharyngeal/metabolism , Aged , Aged, 80 and over , Biomarkers/analysis , Biomarkers/metabolism , Chemokines/analysis , Chemokines/metabolism , Computational Biology/methods , Cytokines/analysis , Female , Fibrosis/immunology , Fibrosis/physiopathology , Humans , Immunohistochemistry , Intercellular Signaling Peptides and Proteins/analysis , Male , Middle Aged , Muscle, Skeletal/immunology , Muscle, Skeletal/physiopathology , Muscular Dystrophy, Oculopharyngeal/immunology , Muscular Dystrophy, Oculopharyngeal/physiopathology , Pilot Projects , Protein Array Analysis/methods , Proteomics/methods , SoftwareABSTRACT
Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.
