Molecular and cellular analysis of peptidase D (prolidase) deficiency.

肽酶D缺乏症是一种罕见的常染色体隐性遗传病，以各种皮肤损害、智力低下等症状为特征。肽酶D（Peptidase D，EC 3.4.13.9）是一类水解酰亚胺肽并从底物肽中释放羧基端脯氨酸的寡肽酶。我们已经从人血液中分离出这种酶，并克隆了人肽酶D的cDNA。然后我们确定了该酶的一级结构、基因结构和基因定位，并对该患者进行了基因分析。在一个有两个姐妹篇的日本家族中，我们从培养的细胞中发现了192 bp的mRNA偏转。我们从患者中克隆了覆盖缺失突变的基因片段，并测定了核苷酸序列。结果表明，患者的基因中约759 bp的序列缺失。在比利时的病例中（最初来自Meddle East），我们发现了一个点突变（核苷酸826处的A至G），导致氨基酸取代（Asp-Asn）。对突变体cDNA的表达分析表明，失活的cDNA由cDNA合成。另一名来自中东的患者也发现了同样的突变。此外，我们还测定了内含子-外显子连接点周围的核苷酸序列。这些研究使我们有可能扩增所有外显子用于该疾病的基因分析。

Peptidase D (prolidase) deficiency is a rare autosomal recessive disorder characterized by various skin lesions, mental retardation, and other symptom. Peptidase D (EC 3.4.13.9) is a kind of oligopeptidases which hydrolysis imidopeptides and releases carboxyl terminal proline from the substrate peptides. We have isolated the enzyme from human blood and cloned the CDNA for human peptidase D. Then we have determined the primary structure of the enzyme, the structure of the gene and the localization of the gene.We carried out the gene analysis of the patient with the disease. In a Japanese family with two sisters with the-disease, we found 192base pairs (bp) deflection in MRNA from cultured cells. We cloned the gene fragment which covered the deletion mutation from the patients and determined the nucleotide sequences. It was demonstrated that approximately 759 bp sequence was deleted in the gene from the patients. In the case from Belgium (originally from Meddle East), we found a point mutation (A to G at nucleotide 826) which resulted in an amino acid substitution (Asp-Asn). Expression analysis of the mutant CDNA revealed that the inactive synthesized from the CDNA. The same mutation was found in the other patient who came from Middle East. Thus the point mutation might be originated from the Middle East.In addition we determined the nucleotide sequences surrounding the intron-exon junctions. These studies make us possible to amplify the all exons for gene analysis of this disease.