Factor XII Tenri, a novel cross-reacting material negative factor XII deficiency, occurs through a proteasome-mediated degradation.
Factor XII Tenri, a novel cross-reacting material negative factor XII deficiency, occurs through a proteasome-mediated degradation.
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DOI：
10.1182/blood.v93.12.4300.412k01_4300_4308
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发表时间：
1999-06
期刊：
影响因子：
20.3
通讯作者：
Shinichi Kondo;F. Tokunaga;S. Kawano;Yoichi Oono;S. Kumagai;T. Koide
中科院分区：
文献类型：
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作者：
Shinichi Kondo;F. Tokunaga;S. Kawano;Yoichi Oono;S. Kumagai;T. Koide
筛选具有3%抗原和因子XII活性水平的纯合子交叉反应物质阴性因子XII缺陷患者，以鉴定基因组水平的突变。低离子强度单链构象多态性（SSCP）分析和序列分析表明，先证者的因子XII基因在第3外显子7832核苷酸位置发生A—>G置换，导致因子XII nh2末端II型结构域Tyr34向Cys置换。我们将这个突变命名为因子XII Tenri。诱变聚合酶链反应（PCR），随后的KpnI消化，显示先证基因纯合突变和杂合突变在他的父母和妹妹。对因子XII Tenri家族的血浆样本进行免疫沉淀和Western blot分析表明，先证者有微量的变异因子XII，其表观分子质量为115 kD，还原后转化为正常的80 kD形式，表明因子XII Tenri以二硫连接的异二聚体的形式分泌，其蛋白约为35 kD，我们通过免疫印迹鉴定为α - 1微球蛋白。用小仓鼠肾（BHK）细胞进行的脉冲追踪实验表明，Tenri-type factor XII在细胞内被广泛降解，但添加胱氨酸导致突变体的分泌增加。使用膜渗透性抑制剂，我们观察到降解发生在前高尔基体，非溶酶体室和蛋白酶体似乎在这一过程中起主要作用。基于这些体外实验结果，我们推测大部分XII Tenri因子通过内质网（ER）的质量控制机制在细胞内被降解，少量XII Tenri因子与α - 1微球蛋白形成二硫联异二聚体分泌到血流中。
A homozygous cross-reacting material negative factor XII-deficient patient with 3% antigen and activity levels of factor XII was screened for the identification of a mutation at the genomic level. Low-ionic strength single-stranded conformation polymorphism (SSCP) analysis and sequence analysis showed that the proband's gene for factor XII had an A-->G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and sister. Immunoprecipitation and Western blot analyses of plasma samples from the factor XII Tenri family indicated that the proband had a trace amount of variant factor XII with an apparent molecular mass of 115 kD, which was converted to the normal 80-kD form after reduction, suggesting that factor XII Tenri was secreted as a disulfide-linked heterodimer with a approximately 35-kD protein, which we identified as alpha1-microglobulin by immunoblotting. Pulse-chase experiments using baby hamster kidney (BHK) cells showed that Tenri-type factor XII was extensively degraded intracellularly, but the addition of cystine resulted in increased secretion of the mutant. Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in vitro results, we speculate that the majority of the factor XII Tenri is degraded intracellularly through a quality control mechanism in the endoplasmic reticulum (ER), and a small amount of factor XII Tenri that formed a disulfide-linked heterodimer with alpha1-microglobulin is secreted into the blood stream.