The contributions of N-linked glycosylation to hair cell mechanoelectrical transduction-linked proteins and auditory function - PROJECT SUMMARY/ABSTRACT Cochlear hair cells are highly specialized, sound-responsive cells that contain actin-rich stereocilia at their apical surface. The tips of stereocilia are endowed with mechanically-sensitive ion channels that open in response to sound wave-induced deflection, leading to ion flux and hair cell depolarization, in a process known as mechanoelectrical transduction (MET). MET leads to neurotransmitter release and transmission of a neural signal to the central auditory pathway. The MET channel complex is a unique heteromeric protein complex consisting of multiple protein subunits, each with discrete contributions to channel structure and function. Recent work has shown that TMC1 is the primary pore-forming subunit of the MET channel in mature cochlear hair cells. However, very little is known about the mechanisms that regulate TMC1 processing, trafficking, degradation, or assembly with other MET channel subunits. Post-translational modifications, including N-linked glycosylation, have major impacts on protein trafficking, stability, complex assembly, and function. Mutations in key molecules involved in N-glycosylation biosynthetic processing, such as the glucosyltransferase Alg10b, cause hearing loss and cochlear hair cell death in mice. However, it is unclear how mutations in N-linked glycosylation processing machinery lead to hearing loss. Our preliminary data using a PNGase F assay shows that, consistent with computational predictions, the MET complex proteins TMC1, TMC2, and PCDH15 are all N-glycosylated. To examine the importance of N-linked glycosylation sites on TMC1, we generated a mutant form of TMC1 that abolished three conserved N-linked glycosylation sites. Using coimmunoprecipitation and in vivo AAV-mediated expression of mutant constructs in Tmc1-/- mice, we show that inhibiting N-linked glycosylation on TMC1 impairs protein-protein interactions and prevents proper localization to stereocilia. We found that Alg10b mRNA is expressed in neonatal and adult cochlear hair cells and that TMC1 interacts with ALG10B in vitro. Based on previous research and our preliminary data, we hypothesize that N-linked glycosylation is essential for proper localization of TMC1 to stereocilia, and that errors in N-linked glycosylation caused by mutations in the glucosyltransferase Alg10b affect the localization and function of critical auditory glycoproteins, including TMC1, leading to hearing loss. To test our hypothesis, we will dissect the specific contributions of N-linked glycosylation sites on TMC1 to protein-protein interactions, localization to stereocilia and auditory function. To gain insights into the critical role of N-glycosylation biosynthetic machinery on auditory function, we will evaluate the roles of ALG10B on cochlear hair cell structure, auditory function, and the critical N-glycosylated MET-linked proteins: TMC1, TMC2, and PCDH15. We will use complementary experiments in heterologous cells and mouse models to explore effects on complex assembly, protein trafficking, and auditory function. This work will characterize the functional importance of N-linked glycosylation on MET-linked proteins and auditory function, and will provide insights into the pathophysiology of hearing loss due to deficits in N-linked glycosylation.
