Water permeability of C-terminally truncated aquaporin 0 (AQP0 1-243) observed in the aging human lens
Water permeability of C-terminally truncated aquaporin 0 (AQP0 1-243) observed in the aging human lens
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DOI：
10.1167/iovs.02-1317
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发表时间：
2003-11-01
影响因子：
4.4
通讯作者：
Schey, KL
中科院分区：
文献类型：
--
作者：
Ball, LE;Little, M;Schey, KL
目的。首先评估水通道蛋白 0 (AQP0) 翻译后截短产物在正常人晶状体解剖切片中的分布，并确定主链裂解对 AQP0 水渗透性的影响。方法。一个 27 年前的镜头被同心地分割成六个部分。从每个切片中分离膜蛋白并用溴化氰裂解，并通过反相（RP）-HPLC-质谱（MS）分离和分析肽。通过质谱法确定翻译后 AQP0 C 末端截短的位点。截短形式的 AQP0 在非洲爪蟾卵母细胞系统中表达，并在卵母细胞渗透膨胀测定中评估截短对 AQP0 水渗透性的影响。 结果。 C 末端内许多位点的截短程度随着纤维细胞年龄的增加而增加，并且检查了残基 234、238 和 243 之后的截短对 AQP0 水渗透性的影响。与全长蛋白 AQP0 1-263 相比，残基 243 后的截短导致渗透性降低约 15%。然而，表面蛋白表达的分析表明，通透性降低是由于蛋白质运输到卵母细胞表面效率较低的结果，而不是对水运输的直接影响，并且全长 AQP0 和 1-243 AQP0 的通透性无法区分。此外，AQP0 的 C 端截短为 1-234 和 1-238，完全损害了进入质膜的运输，从而妨碍了渗透性的测量。结论。这些数据提供证据表明，C 端丢失 20 个氨基酸可能不会直接影响 AQP0 运输水的能力。
PURPOSE. To first assess the distribution of posttranslationally truncated products of aquaporin 0 (AQP0) in dissected sections of a normal human lens and to determine the effect of backbone cleavage on the water permeability of AQP0.METHODS. A 27-year-old lens was concentrically dissected into six sections. Membrane protein was isolated from each section and cleaved with cyanogen bromide, and the peptides were separated and analyzed by reverse-phase (RP)-HPLC-mass spectrometry (MS). The sites of posttranslational AQP0 C-terminal truncation were determined by mass spectrometry. Truncated forms of AQP0 were expressed in a Xenopus laevis oocyte system, and the effect of truncation on AQP0 water permeability was assessed in an oocyte osmotic swelling assay.RESULTS. The extent of truncation at many sites within the C terminus increased with fiber cell age, and the effects of truncations after residues 234, 238, and 243 on AQP0 water permeability were examined. Truncation after residue 243 resulted in an approximate 15% decrease in permeability compared with the full-length protein, AQP0 1-263. However, rather than a direct effect on water transport, analysis of surface protein expression indicated that the decrease in permeability was a result of less efficient protein trafficking to the oocyte surface and that the permeabilities of full-length and 1-243 AQP0 were indistinguishable. Further, C-terminal truncation of AQP0 to 1-234 and 1-238, completely impaired trafficking into the plasma membrane, precluding the measurement of permeability.CONCLUSIONS. These data provide evidence that loss of 20 amino acids from the C terminus may not directly affect the ability of AQP0 to transport water.