The main objective of this research was to increase our knowledge of the pathological mechanisms underlying LGMD 2B, to better characterize each member of the dysferlin complex and, more... Show moreThe main objective of this research was to increase our knowledge of the pathological mechanisms underlying LGMD 2B, to better characterize each member of the dysferlin complex and, more importantly, to try to identify potential modifier genes that might modulate the clinical course in patients. In chapter 2, by diverse selection methodologies on different truncated forms of recombinant dysferlin, VHH antibody fragments with specificity for two different dysferlin domains were selected from a non-immune phage display library. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation. Using these antibody fragments, we found that CAPN3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.In order to gain functional insight into the molecular mechanisms of dysferlin, we have searched for yet unknown proteins that interact with dysferlin in skeletal muscle. By co-immunoprecipitation coupled to mass spectrometry, we demonstrated that dysferlin interacts with AHNAK. Furthermore, the binding domains in dysferlin and AHNAK were mapped and the AHNAK expression in normal skeletal muscle dysferlinopathy was assessed by immonofluorescence analysis (Chapter 3).Chapter 4 describes the proteolysis of AHNAK by CAPN3. Direct interaction of AHNAK and CAPN3 was studied by GST-pull down assay and specific AHNAK domains that were cleaved by CAPN3 were identified in a cell model. The biological relevance of this cleavage was also studied at cellular level.Finally, in chapter 5 the selection strategies of antibody phage display and the biomedical application of VHH will be discussed. This chapter will mainly focus on the role of dysferlin complex in LGMD 2B and future perspectives will be discussed in this chapter. Show less
