Insights into the Mammalian Neuromuscular Junction Using the Torpedo Californica Electric Organ as a Model Tissue
Open AccessThe neuromuscular junction (NMJ) is a highly specialized sub-region of the myofiber membrane with a limited repertoire of NMJ proteins characterized. We hypothesized that many additional NMJ protein components remain to be discovered, and the existing limited repertoire inhibits molecular understanding of the NMJ. The miniscule size and low abundance of the NMJ in muscle presents technical limitations in studying its proteome. To overcome this, we used the Torpedo californica electric organ as a model. We hypothesized that profiling the proteins of the electric organ will reveal novel synaptic proteins that were overlooked in the mammalian NMJ. We conducted a non-targeted proteomics study to investigate the molecular constituents of the electric organ and assessed its concordance with the mammalian NMJ. We showed that the electric organ is a repository of candidate NMJ proteins and selected Eps homology domain containing protein 1 (EHD1) for additional characterization. We found that EHD1 localized to the primary synaptic cleft of the NMJ but that the function of EHD1 is not critical to maintaining the NMJ architecture. However, EHD4 expression is increased in EHD1-/- skeletal muscle and likely compensates in function for the loss of EHD1 such that double knockout mouse models are necessary to assess the function of EHD1 at the NMJ. Many synaptic proteins are post-translationally modified by the addition of carbohydrates. We hypothesized that a proteomic approach targeting N-linked glycoproteins expressed in the electric organ could lead to novel biochemical insights regarding the structure of the NMJ. We identified the SITS-binding protein (SP105) as the most abundant N-glycosylated protein in the electric organ. Using a glycoproteomics approach, we identified five of the eight asparagine residues within consensus sequence N-X-(S/T) were glycosylated. Furthermore, glycans were of variable form, from high mannose to complex tri-antennary structures. The studies in this dissertation provide new knowledge concerning NMJ protein repertoire that will aid in presenting a more complete NMJ proteome to guide future studies on NMJ structure and function in health and disease. In addition, this work shows the conservation and diversity of N-linked glycan post-translation modifications of a highly abundant electrocyte protein, SP105.
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