All the antibodies were diluted in 1% BSA/PBS; this was followed by washes with PBS

All the antibodies were diluted in 1% BSA/PBS; this was followed by washes with PBS. of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair conversation of LGI1 with the ADAM22 and ADAM23 receptors around the cell CLEC4M surface. These results support the presence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE. Author Summary Temporal lobe epilepsy is the most common form of focal epilepsy. It is frequently DB07268 associated with structural brain abnormalities, but genetic forms caused by mutations in major genes have also been described. Autosomal dominant lateral temporal epilepsy (ADLTE) is usually a familial condition characterized by focal seizures with prominent auditory symptoms. ADLTE-causing mutations are found in the gene in about 30% of affected families. encodes a protein, LGI1, that is secreted by neurons. Most mutations suppress protein secretion, thereby preventing protein function in the extracellular environment. In this paper, we examine the effects of four mutations and show that they do not inhibit secretion of the LGI1 protein but impair its conversation with the neuronal receptors ADAM22 and ADAM23. In agreement with these findings, a three- dimensional model of the protein predicts that these mutations have no impact on LGI1 structure but instead may affect amino acids that are critical for interactions with ADAM receptors. Our results provide novel evidence for an extracellular mechanism through which mutant LGI1 proteins cause ADLTE and strengthen the importance of LGI1-ADAM22/23 protein complex in the mechanisms underlying ADLTE. Introduction Mutations in the leucine-rich, glioma-inactivated 1 (mutations are found in about 30% of families with this syndrome [7]. To date, more than 30 ADLTE-causing mutations have been detected throughout the protein-coding region of is mainly expressed in neurons [1,10,11] and shows no similarity to known ion channels. The predicted structure of the LGI1 protein comprises a signal peptide, four leucine-rich repeats (LRRs) [12], and seven repeats named EPTP [13] or EAR [14] likely forming a beta-propeller structural domain name [15]. Both LRR and beta-propeller domains mediate protein-protein interactions [15,16]. The LGI1 protein is usually secreted [10,17,18], and most ADLTE-causing mutations inhibit protein secretion [10,17,19C21], consistent with a loss-of-function effect of mutations. We recently reported the first disease-causing mutation (R407C) with no inhibitory effect on LGI1 secretion DB07268 [22]. LGI1 has been implicated in various functions, some of which are mediated by interactions with two ADAM (A Disintegrin And Metalloprotease domain name) receptors. LGI1 has been shown to bind to the postsynaptic receptor ADAM22 and this ligand-receptor complex participates in the control of synaptic strength at excitatory synapses [23]. It also binds to ADAM23 to stimulate neurite outgrowth both and [24] and may act as a trans-synaptic protein connecting the pre-synaptic ADAM23 with the post-synaptic ADAM22 receptors [25]. Though different in nature, each of these functions may potentially be related to epilepsy if impaired by mutations of that prevent or disturb interactions with ADAM22 and ADAM23 receptors. Recent work has shown that serum LGI1 autoantibodies from patients with limbic encephalitis (LE), which is usually characterized by cognitive dysfunction and seizures [26, 27], prevent conversation of LGI1 with ADAM22 [28]. It has also been shown that some ADLTE-related mutations allowing secretion of LGI1 impair its binding to ADAM22 but not to ADAM23 [29]. In this paper we show that secretion-positive DB07268 LGI1 mutations impair extracellular binding to both ADAM22 and ADAM23 receptors, providing further.