Abstract：
Hearing, the ability to sense sounds, and the processing of auditory information are important for perception of the world. Mice lacking expression of neuroplastin (Np), a type-1 transmembrane glycoprotein, display deafness, multiple cognitive deficiencies, and reduced expression of plasma membrane calcium (Ca2+) ATPases (PMCAs) in cochlear hair cells and brain neurons. In this study, we transferred the deafness causing missense mutations pitch (C315S) and audio-1 (I122N) into human Np (hNp) constructs and investigated their effects at the molecular and cellular level. Computational molecular dynamics show that loss of the disulfide bridge in hNppitch causes structural destabilization of immunoglobulin-like domain (Ig) III and that the novel asparagine in hNpaudio-1 results in steric constraints and an additional N-glycosylation site in IgII. Additional N-glycosylation of hNpaudio-1 was confirmed by PNGaseF treatment. In comparison to hNpWT, transfection of hNppitch and hNpaudio-1 into HEK293T cells resulted in normal mRNA levels but reduced the Np protein levels and their cell surface expression due to proteasomal/lysosomal degradation. Furthermore, hNppitch and hNpaudio-1 failed to promote exogenous PMCA levels in HEK293T cells. In hippocampal neurons, expression of additional hNppitch or hNpaudio-1 was less efficient than hNpWT to elevate endogenous PMCA levels and to accelerate the restoration of basal Ca2+ levels after electrically-evoked Ca2+ transients. We propose that mutations leading to pathological Np variants, as exemplified here by the deafness causing Np mutants, can affect Np-dependent Ca2+ regulatory mechanisms and may potentially cause intellectual and cognitive deficits in humans.
摘要：
听证会,感知声音的能力，听觉信息的处理对于感知世界很重要。缺乏神经激酶（Np）表达的小鼠，1型跨膜糖蛋白,显示耳聋，多重认知缺陷，耳蜗毛细胞和脑神经元的质膜钙(Ca2+)ATP酶(PMCAs)表达降低。在这项研究中,我们将导致失义突变Pitch（C315S）和audio-1（I122N）的耳聋转移到人Np（hNp）构建体中，并在分子和细胞水平研究了它们的影响。计算分子动力学表明，hNppitch中二硫键的丢失会导致免疫球蛋白样结构域（Ig）III的结构不稳定，hNpaudio-1中的新型天冬酰胺会导致空间约束和IgII中的额外N-糖基化位点。通过PNGaseF处理证实了hNpaudio-1的额外N-糖基化。与hNpWT相比，将hNppitch和hNpaudio-1转染到HEK293T细胞中导致正常的mRNA水平，但由于蛋白酶体/溶酶体降解而降低了Np蛋白水平及其细胞表面表达。此外，hNppitch和hNpaudio-1未能促进HEK293T细胞中的外源PMCA水平。在海马神经元中，在电诱发Ca2瞬变后，其他hNppitch或hNpaudio-1的表达在提高内源性PMCA水平和加速基础Ca2水平恢复方面的效率低于hNpWT。我们认为导致病理性Np变异的突变，这里以引起耳聋的Np突变体为例，可以影响Np依赖的Ca2调节机制，并可能导致人类的智力和认知缺陷。
