Mutation analysis of five new patients affected by prolidase deficiency: the lack of enzyme activity causes necrosis-like cell death in cultured fibroblasts
Mutation analysis of five new patients affected by prolidase deficiency: the lack of enzyme activity causes necrosis-like cell death in cultured fibroblasts
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DOI：
10.1007/s00439-002-0792-5
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发表时间：
2002-10-01
期刊：
影响因子：
5.3
通讯作者：
Cetta, G
中科院分区：
文献类型：
--
作者：
Forlino, A;Lupi, A;Cetta, G
氨酰基脯氨酸二肽酶是一种广泛分布的二肽酶，参与内源性和膳食蛋白质的后期降解，在胶原蛋白降解中特别重要。水解C-末端含有脯氨酸或羟脯氨酸的二肽。脯氨酰二肽酶基因突变导致脯氨酰二肽酶缺乏症（PD），这是一种常染色体隐性遗传疾病，主要表现为皮肤损害、智力低下和反复感染。在这项工作中，我们报告了5例PD患者的分子缺陷的鉴定。PCR扩增的基因组DNA的直接测序显示在两个同胞中的纯合G>A颠换导致G448 R取代。在第三个先证者中检测到引起外显子II跳跃的杂合IVS 11 +1G>C转换和无效等位基因。在两个不相关的患者中，鉴定了纯合IVS 7 -1G>A颠换，并显示其引起多种选择性剪接转录物。所有突变导致氨酰基脯氨酸二肽酶活性丧失。来自这些PD患者的长期培养的成纤维细胞用于开发允许受影响细胞的研究的体外模型。光学和电子显微镜显示，PD细胞比对照组更圆和分支，胞质空泡化增加，质膜中断，线粒体肿胀，线粒体基质和嵴修饰。JC-1标记显示线粒体膜电位降低。通过毛细管电泳分析检测到Gly-Pro二肽的显著细胞内积累。我们的研究结果提供了第一个证据，即氨酰基脯氨酸二肽酶活性的缺乏会导致坏死样细胞死亡的激活，这可能是PD中典型皮肤病变的原因。
Prolidase, a ubiquitously distributed dipeptidase, is involved in the latter stage of degradation of endogenous and dietary proteins and is particularly important in collagen catabolism. It hydrolyzes dipeptides containing proline or hydroxyproline at the C-terminal position. Mutations in the gene encoding for prolidase cause prolidase deficiency (PD), an autosomal recessive disorder mainly characterized by skin lesions, mental retardation and recurrent infectious. In this work we reported the identification of the molecular defect in five PD patients. Direct sequencing of PCR amplified genomic DNA showed a homozygous G>A transversion in two siblings leading to a G448R substitution. A heterozygous IVS11+1G>C transition causing the skipping of exon I I and a null allele were detected in a third proband. In two unrelated patients, a homozygous IVS7-1G>A transversion was identified and shown to cause multiple alternative spliced transcripts. All the mutations result in loss of prolidase activity. Long-term cultured fibroblasts from these PD patients were used to develop an in vitro model that allowed investigation of the affected cells. Light and electron microscopy revealed that PD cells were more round and branched out than controls with increased cytosolic vacuolization, interruptions of the plasma membrane, mitochondria swelling, mitochondrial matrix and cristae modifications. JC-1 labeling showed decreased mitochondrial membrane potential. A significant intracellular accumulation of the Gly-Pro dipeptide was detected by capillary electrophoresis analysis. Our results provide the first evidence that absence of prolidase activity causes the activation of a necrosis-like cellular death, which could be responsible for the typical skin lesions in PD.