水的运输对于保持无血管晶状体的透明度至关重要,并且已知晶状体表达来自水通道蛋白(AQP)家族的至少五个明显不同的水通道。在这项研究中,我们报告了第六晶状体AQP的鉴定,AQP3是一种水甘油,除了水还输送甘油和H2O2。使用RT-PCR和蛋白质印迹在转录水平和蛋白质水平上鉴定AQP3,分别,在老鼠身上,rat,牛和人的晶状体,表明其表达在哺乳动物晶状体中是保守的。Western印迹显示晶状体中的AQP3在所有晶状体物种中以25kDa非糖基化和37kDa糖基化的单体形式存在。为了确定在晶状体中表达AQP3的区域,使用上皮重复进行蛋白质印迹,从小鼠晶状体分离的外皮质和内皮质/核心部分。在所有晶状体区域都发现了AQP3,在上皮中发现非糖基化AQP3的最高信号。而在内部皮质/核心区,AQP3信号不仅较低,而且主要来自AQP3的糖基化形式。用AQP3抗体对晶状体切片进行免疫标记,证实在成年小鼠的所有区域都发现了AQP3,还揭示了AQP3的亚细胞分布随纤维细胞分化而变化。在外皮质的上皮和外周纤维细胞中,AQP3标记主要与细胞质中的膜囊泡相关,但在晶状体的较深区域,AQP3标记与位于内皮质和晶状体核心的成纤维细胞的质膜有关。为了确定AQP3亚细胞分布的成人模式是如何建立的,在胚胎和出生后的晶状体上进行AQP3的免疫标记。AQP3的表达在胚胎第11天首次在已经开始拉长并填充晶状体囊泡内腔的原代纤维细胞的膜中检测到,而在E16后期,原代纤维细胞中的AQP3标记已转移到主要的细胞质位置。在随后的P3和P6的晶状体生长的出生后(P)阶段,AQP3标记在晶状体的所有区域都保持细胞质,直到P15,AQP3的定位模式才变为成人分布,其中在外部皮质中检测到细胞质标记,在内部皮质和晶状体核心中检测到膜定位。AQP3标记模式与先前获得的AQP0和AQP5的比较表明,亚细胞分布与AQP5比AQP0更相似,但仍存在显着差异,表明AQP3在维持晶状体透明度方面可能具有独特的作用。
Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP,
AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens.
AQP3 was found in all lens regions, with the highest signal of non-glycosylated
AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of
AQP3. Immunolabelling of lens sections with
AQP3 antibodies confirmed that
AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for
AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.