D810E mutation depleted the kinase activity of c-KIT protein and
induced the destruction of the inner ear structure
The DFG motif flipped between the “on” and “off” states in the
activation process of the c-KIT protein(Mol et al., 2004; Mol et al.,
2003). Asp810 was directed towards the positively charged guanidinium
group of Arg815 in the autoinhibited conformation and coordinated the
Mg2+ ion bridging of the α- and β-phosphates in the
active conformation (Fig. 4a). Therefore, we deduced that the
substitution of Asp810 with Glu810 elongated the side chain at this
specific site and exerted a deleterious impact on the kinase activity of
the protein. This prediction was further confirmed by SIFT
(https://sift.bii.a-star.edu.sg/) and PolyPhen-2
(http://genetics.bwh.harvard.edu/pph2/) (Supplemental Data). In
addition, c-KIT has been reported to indirectly regulate the
transcription activity of
microphthalmia-associated
transcription factor (MITF) through the MAP kinase machinery to affect
the survival, proliferation and apoptosis of melanocyte derived
intermediate cells(Hemesath, Price, Takemoto, Badalian, & Fisher, 1998;
Ronnstrand, 2004). RT-PCR revealed the downregulation of MITF-regulated
genes (PAX3 , TYR , DCT , and MC1R ) (Fig. 4b),
implying the reduction and dysfunction of the mutant KIT protein
activity. However, the D810E mutation did not affect the transcription
of KIT , MITF , or SOX10 .
To investigate the pathology of hearing loss, we dissected the cochleae
from wild-type and mutant pigs for microcomputed tomography (micro-CT)
scanning and three-dimensional reconstructions to observe the cochlear
structure and integrity. The three-dimensional structure of the cochlea
showed three semicircular canals perpendicular to each other and three
and one-half turns of the cochlear capsule; any marked difference was
not detected between wild-type and mutant pigs (Fig. 4c). The results of
celloidin embedding and hematoxylin-eosin staining (CE-HE) showed that,
in wild-type pigs, the organ of
Corti, the
Reissner’s membrane (RM),
spiral ganglion (SG), tectorial
membrane (TM), and the
stria vascularis (SV) were arrayed
normally in the cochlear duct (Fig. 4d). However, an extensive collapse
of
Reissner’s
membrane onto the
SV
and the disordered organ of Corti were observed inc-KITc.2430T>A/+ pigs, which
induced the profound hearing loss.