Abstract
The LGI1 gene has been shown to predispose to epilepsy and influence cell invasion in glioma cells. To identify proteins that interact with LGI1 and gain a better understanding of its function, we have used co-immunoprecipitation (co-IP) of a secreted green fluorescent protein-tagged LGI1 protein combined with mass spectrometry to identify interacting partners from lysates prepared from human subcortical white matter. Proteins were recovered from polyacrylamide gels and analyzed using liquid chromatography coupled to tandem mass spectrometry. This analysis identified a range of proteins, but in particular synaptotagmin, synaptophysin, and syntaxin 1A. Each of these proteins is found associated with synaptic vesicles. These interactions were confirmed independently by co-IP and Western blotting and implicate LGI1 in synapse biology in neurons. Other vesicle-related proteins that were recovered by co-IP include clathrin heavy chain 1, syntaxin binding protein 1, and a disintegrin and metalloprotease 23. These observations support a role for LGI1 in synapse vesicle function in neurons.
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Acknowledgments
We are grateful to Rama-Dey Rao and Kyoung-Soo Choi for assistance with the proteomics analysis. This work was supported by grant CA from the National Institutes of Health. The authors have no conflicts of interest.
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Supplemental Figures S1–3
Chromatograms, MS and MS/MS Spectra of representative peptides from: (Figure S1) synaptotagmin, m/z 792.91, [M+2H]+2, TLNPVFNEQFTFK; (Figure S2) synaptophysin, m/z 502.88, [M+3H]+3, LHQVYFDAPTCR; (Figure S3) Adam23, m/z 618.84, [M+2H]+2, SVVNLVDSIYK. The procedures for processing the immunoprecipitated sample by 1D SDS-PAGE, in-gel trypsinization of the protein bands, LC–MS/MS, and database search for peptide and protein identification are described in the text. The base peak intensities (BPI) and extracted ion current (EIC) chromatograms are shown in (A) and (B), respectively, and the inserts in (B) show the MS spectra of the peptides. The MS/MS spectra and de novo sequencing indicating the b and y ions that were identified are shown in (C). The MASCOT scores and peptide mass tolerances (shown in parentheses) are also reported. (PDF 713 kb)
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Kunapuli, P., Jang, GF., Kazim, L. et al. Mass Spectrometry Identifies LGI1-Interacting Proteins that Are Involved in Synaptic Vesicle Function in the Human Brain. J Mol Neurosci 39, 137–143 (2009). https://doi.org/10.1007/s12031-009-9202-y
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DOI: https://doi.org/10.1007/s12031-009-9202-y