Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1.
Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1.
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DOI：
10.1210/mend.11.13.0035
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发表时间：
1997-12
影响因子：
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通讯作者：
G. Fu;D. Lin;Martin Y. H. Zhang;D. Bikle;C. Shackleton;W. Miller;A. Portale
中科院分区：
文献类型：
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作者：
G. Fu;D. Lin;Martin Y. H. Zhang;D. Bikle;C. Shackleton;W. Miller;A. Portale
第二类固醇激素1,25-二羟基维生素D [1,25(OH)2D]在正常骨生长、钙代谢和组织分化中起着至关重要的作用。生物合成1,25(OH)2D的关键步骤是肾脏中25-羟基维生素D （25- ohd）的1 α -羟基化。由于其在肾脏中的表达非常低，我们从人角质形成细胞中克隆并测序了25-OHD-1 α -羟化酶（P450c1 α）的cDNA，其中1 α -羟化酶活性和mRNA表达可以诱导得更高。P450c1 α mRNA在人肾、脑和睾丸中的表达水平低得多。转染克隆的P450c1 α cDNA的哺乳动物细胞表现出强大的1 α -羟化酶活性。用高效液相色谱和气相色谱-质谱联用技术对转染细胞合成的1,25(OH)2D3产物进行了鉴定。编码P450c1 α的基因定位于12号染色体，1 α -羟化酶缺乏综合征，维生素d依赖性佝偻病1型（VDDR-1）已经定位。人类成人和新生儿角质形成细胞的原代培养物表现出丰富的1 α -羟化酶活性，而来自vdr -1患者的原代培养物则缺乏可检测到的活性。来自vdr -1患者的角质细胞P450c1 α cDNA在密码子211或密码子231处含有缺失/移码突变，表明该患者是两个零突变的复合杂合子。这些发现奠定了VDDR-1的分子遗传学基础，为其在角质形成细胞中的研究提供了新的手段，并提供了维生素D生物活化关键酶的序列。
The secosteroid hormone, 1,25-dihydroxyvitamin D [1,25(OH)2D], plays a crucial role in normal bone growth, calcium metabolism, and tissue differentiation. The key step in the biosynthesis of 1,25(OH)2D is its 1 alpha-hydroxylation from 25-hydroxyvitamin D (25-OHD) in the kidney. Because its expression in the kidney is very low, we cloned and sequenced cDNA for 25-OHD-1 alpha-hydroxylase (P450c1 alpha) from human keratinocytes, in which 1 alpha-hydroxylase activity and mRNA expression can be induced to be much greater. P450c1 alpha mRNA was expressed at much lower levels in human kidney, brain, and testis. Mammalian cells transfected with the cloned P450c1 alpha cDNA exhibit robust 1 alpha-hydroxylase activity. The identity of the 1,25(OH)2D3 product synthesized in transfected cells was confirmed by HPLC and gas chromatography-mass spectrometry. The gene encoding P450c1 alpha was localized to chromosome 12, where the 1 alpha-hydroxylase deficiency syndrome, vitamin D-dependent rickets type 1 (VDDR-1), has been localized. Primary cultures of human adult and neonatal keratinocytes exhibit abundant 1 alpha-hydroxylase activity, whereas those from a patient with VDDR-1 lacked detectable activity. Keratinocyte P450c1 alpha cDNA from the patient with VDDR-1 contained deletion/frameshift mutations either at codon 211 or at codon 231, indicating that the patient was a compound heterozygote for two null mutations. These findings establish the molecular genetic basis of VDDR-1, establish a novel means for its study in keratinocytes, and provide the sequence of the key enzyme in the biological activation of vitamin D.