Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency
Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency
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DOI：
10.1074/jbc.m600071200
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发表时间：
2006-11-10
影响因子：
4.8
通讯作者：
Inagaki, Nobuya
中科院分区：
文献类型：
--
作者：
Matsumura, Yoshihiro;Ban, Nobuhiro;Inagaki, Nobuya
三磷酸腺苷结合盒转运体ABCA3主要表达在肺泡II型细胞板层小体的界膜上。最近的研究表明，ABCA3基因的突变会导致新生儿致命的表面活性物质缺乏。在这项研究中，我们在HEK293细胞中研究了迄今为止在致命的表面活性物质缺乏患者中发现的ABCA3突变体的细胞内定位和N-糖基化。绿色荧光蛋白标记的L101P、L982P、L1553P、Q1591P和Ins1518fs/ter1519突变蛋白仍定位于内质网，低聚糖的加工受到损害，而野生型和N568D、G1221S和L1580P突变ABCA3蛋白运输到LAMP3阳性的胞内小泡，并伴随着低聚糖从高甘露糖型向复合型的加工。钒酸诱导的核苷酸捕获和ATP结合分析表明，与野生型ABCA3蛋白相比，N568D、G1221S和L1580P突变株的ATP水解酶活性显著降低，而G1221S突变株的ATP结合活性略有降低。此外，对G1221S和L1580P突变体第11跨膜段的Gly-1221残基和胞质尾部的Leu-1580残基的突变分析，以及核苷酸结合域2的同源模拟，证明了这些残基对ATP水解酶的意义，并提出了G1221S和L1580P突变体中损害ATP水解性的机制。因此，ABCA3基因突变所致的表面活性物质缺乏症可分为两类：细胞内定位异常(I型)和细胞内正常定位，并伴有ABCA3蛋白的ATP结合和/或ATP水解性降低(II型)。这些不同的病理生理可能反映了肺表面活性物质缺乏的严重程度和有效的治疗方法。
The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.