Missense mutation (Gly----Glu188) of human lipoprotein lipase imparting functional deficiency.
Missense mutation (Gly----Glu188) of human lipoprotein lipase imparting functional deficiency.
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DOI：
10.1016/s0021-9258(19)39449-9
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发表时间：
1990-04
期刊：
影响因子：
--
通讯作者：
Mitsuru Emi;Dana E. Wilson;P. Iverius;Lily L. Wu;Aaron N. Hata;Robert A. Hegele;Roger R. Williams;J. Lalouel
中科院分区：
文献类型：
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作者：
Mitsuru Emi;Dana E. Wilson;P. Iverius;Lily L. Wu;Aaron N. Hata;Robert A. Hegele;Roger R. Williams;J. Lalouel
从一个典型的脂蛋白脂酶缺乏症患者的脂肪组织中制备的脂蛋白脂酶（LPL）cDNA的克隆和测序显示，在所有测序的克隆中，在核苷酸818处的G到A的转变，导致成熟蛋白的残基188处的甘氨酸被谷氨酸取代。基因组DNA与等位基因特异性寡核苷酸的杂交证实了该患者是该突变的纯合子，并揭示了该患者亲属中该突变的携带者状态与高胆固醇血症显著相关。对患者血浆的免疫反应性酶和活性进行检测，证明存在循环的无活性酶蛋白，注射肝素后其浓度进一步增加。将突变体序列克隆到表达载体pSVL中，转染COS-1细胞。正常序列导致体外表达的酶结合肝素琼脂糖凝胶，并具有类似于正常肝素后血浆酶的特异性催化活性。相比之下，在体外表达的突变酶是催化失活的，并显示出较低的亲和力肝素比正常的酶。我们的结论是，这种单一氨基酸取代导致体内表达一种失活酶，这解释了患者中观察到的LPL缺乏症的表现。
Cloning and sequencing of lipoprotein lipase (LPL) cDNA prepared from the adipose tissue of a patient with classical LPL deficiency revealed a G to A transition at nucleotide 818 in all sequenced clones, leading to the substitution of glutamic acid for glycine at residue 188 of the mature protein. Hybridization of genomic DNA with allele-specific oligonucleotides confirmed that the patient was homozygous for this mutation and revealed that carrier status for this mutation among relatives of the patient was significantly associated with hypertriglyceridemia. Assay of the patient's plasma for immunoreactive enzyme and activity demonstrated the presence of a circulating inactive enzyme protein, the concentration of which was further increased by injection of heparin. The mutant sequence was produced by oligonucleotide-directed mutagenesis, and both normal and mutant sequences were cloned into the expression vector pSVL and transfected into COS-1 cells. The normal sequence led to the in vitro expression of an enzyme that bound to heparin-Sepharose and had a specific catalytic activity similar to that of normal postheparin plasma enzyme. By contrast, the mutant enzyme expressed in vitro was catalytically inactive and displayed a lower affinity for heparin than the normal enzyme. We conclude that this single amino acid substitution leads to the in vivo expression of an inactive enzyme accounting for the manifestations of LPL deficiency noted in the patient.