Section on Molecular Structure and Functional Genomics, National Eye Institute, National Institutes of Health, Bethesda, MD, USA.
Genetic Engineering Facility, National Eye Institute, National Institutes of Health, Bethesda, MD, USA.
Exp Eye Res. 2018 Apr;169:111-121. doi: 10.1016/j.exer.2018.02.001. Epub 2018 Feb 7.
KLPH/lctl belongs to the Klotho family of proteins. Expressed sequence tag analyses unexpectedly revealed that KLPH is highly expressed in the eye lens while northern blots showed that expression is much higher in the eye than in other tissues. In situ hybridization in mouse localized mRNA to the lens, particularly in the equatorial epithelium. Immunofluorescence detected KLPH in lens epithelial cells with highest levels in the germinative/differentiation zone. The gene for KLPH in mouse was deleted by homologous recombination. Littermate knockout (KO) and wild type (WT) mice were compared in a wide panel of pathology examinations and were all grossly normal, showing no systemic effects of the deletion. However, the lens, while superficially normal at young ages, had focusing defects and exhibited age-related cortical cataract by slit lamp examination. Whole-lens imaging showed that KO mice had disorganized lens sutures, forming a loose double-y or x instead of the tight y formation of WT. RNA-seq profiles for KO and WT littermates confirmed the absence of KLPH mRNA in KO lens and also showed complete absence of transcripts for Clic5, a protein associated with cilium/basal body related auditory defects in a mouse model. Immunofluorescence of lens epithelial flat mounts showed that Clic5 localized to cilia/centrosomes. Mice mutant for Clic5 (jitterbug) also had defective sutures. These results suggest that KLPH is required for lens-specific expression of Clic5 and that Clic5 has an important role in the machinery that controls lens fiber cell extension and organization.
KLPH/lctl 属于 Klotho 蛋白家族。表达序列标签分析出人意料地显示，KLPH 在眼睛晶状体中高度表达，而 northern blot 显示其在眼睛中的表达远高于其他组织。在小鼠的原位杂交中，mRNA 定位于晶状体，特别是在赤道上皮细胞。免疫荧光检测到 KLPH 在晶状体上皮细胞中表达，在生殖/分化区表达水平最高。用同源重组的方法敲除了小鼠 KLPH 的基因。敲除（KO）和野生型（WT）同窝仔鼠在广泛的病理学检查中进行了比较，均表现为大体正常，无缺失的系统影响。然而，晶状体在年轻时虽然表面正常，但存在聚焦缺陷，并通过裂隙灯检查显示出与年龄相关的皮质性白内障。全晶状体成像显示 KO 小鼠的晶状体缝线排列紊乱，形成疏松的双 Y 或 X 形，而不是 WT 的紧密 Y 形。KO 和 WT 同窝仔鼠的 RNA-seq 图谱证实了 KO 晶状体中 KLPH mRNA 的缺失，也显示了与纤毛/基体相关的听觉缺陷相关的蛋白 Clic5 的转录本完全缺失，该蛋白在一个小鼠模型中。晶状体上皮平铺免疫荧光显示 Clic5 定位于纤毛/中心体。Clic5 突变（jitterbug）的小鼠也有缝线缺陷。这些结果表明 KLPH 是晶状体特异性表达 Clic5 所必需的，Clic5 在控制晶状体纤维细胞延伸和组织的机制中起着重要作用。
