Lipoprotein lipaseBethesda: a single amino acid substitution (Ala-176----Thr) leads to abnormal heparin binding and loss of enzymic activity.
Lipoprotein lipaseBethesda: a single amino acid substitution (Ala-176----Thr) leads to abnormal heparin binding and loss of enzymic activity.
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DOI：
10.1073/pnas.87.9.3474
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发表时间：
1990-05
影响因子：
11.1
通讯作者：
O. Beg;M. Meng;S. Skarlatos;L. Previato;J. Brunzell;H. Brewer;S. Fojo
中科院分区：
文献类型：
--
作者：
O. Beg;M. Meng;S. Skarlatos;L. Previato;J. Brunzell;H. Brewer;S. Fojo
在贝塞斯达先证者中，导致脂蛋白脂酶(LPL)活性缺陷的分子缺陷已被鉴定。先证者肝素前和肝素后血浆LPL质量与对照组相比升高；然而，没有检测到LPL活性，这表明存在缺陷酶(称为LPLBethesda)。用肝素-琼脂糖亲和层析法分析患者肝素后的血浆表明，突变的LPL对肝素的亲和力发生了变化。LPLBethesda基因的Southern印迹杂交显示没有重大的重排。对患者单核细胞来源的巨噬细胞LPLBethesda mRNA的Northern印迹分析显示，与对照巨噬细胞相比，大小正常的mRNAs(3.4和3.7kb)以及正常的细胞mRNA水平。聚合酶链式反应扩增的LPL基因的序列分析显示，正常LPL基因第781位发生G-A替换，导致第176位的丙氨酸替换为苏氨酸，并丢失了正常LPL基因中存在的SfaNI位点。用聚合酶链式反应扩增cDNA，然后用SfaNI消化，证实该患者是该突变的真正纯合子。LPL基因在COS-7细胞中的表达导致LPL酶的合成，证实Ala-Thr替换是导致LPL失活的突变。LPL基因突变的鉴定确定了LPL酶的一个区域，位于ALA-176，对于正常的肝素结合和催化活性是必不可少的。我们认为，LPLBethesda这一关键区域的氨基酸替换导致了一种非功能性酶的合成，从而导致了先证者表现出的乳糜体微粒血症综合征。
The molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in the proband from a Bethesda kindred has been identified. The pre- and post-heparin plasma LPL mass in the proband was elevated when compared to controls; however, there was no detectable LPL activity, indicating the presence of a defective enzyme (termed LPLBethesda). Analysis of the patient's post-heparin plasma by heparin-Sepharose affinity chromatography demonstrated that the mutant LPL had an altered affinity for heparin. Southern blot hybridization of the gene for LPLBethesda revealed no major rearrangements. Northern blot analysis of LPLBethesda mRNA from patient monocyte-derived macrophages revealed normal-sized mRNAs (3.4 and 3.7 kilobases) as well as normal cellular mRNA levels when compared to control macrophages. Sequence analysis of polymerase chain reaction-amplified LPL cDNA revealed a G----A substitution at position 781 of the normal LPL gene that resulted in the substitution of an alanine for a threonine at residue 176 and the loss of an SfaNI site present in the normal LPL gene. Amplification of cDNA by the PCR followed by digestion with SfaNI established that the patient was a true homozygote for the mutation. Expression of LPL cDNA in COS-7 cells resulted in the synthesis of a nonfunctional LPL enzyme establishing that the Ala----Thr substitution was the mutation responsible for the inactive LPL. The identification of this mutation in the LPL gene defines a region of the LPL enzyme, at Ala-176, that is essential for normal heparin-binding and catalytic activity. We propose that an amino acid substitution in this critical region of LPLBethesda results in the synthesis of a nonfunctional enzyme that leads to the chylomicronemia syndrome expressed in this proband.