Bruin T, Tuzgöl S, van Diermen D E, Hoogerbrugge-van der Linden N, Brunzell J D, Hayden M R, Kastelein J J
Centre for Hemostasis, Thrombosis, Atherosclerosis and Inflammation Research, Academic Medical Centre, University of Amsterdam, The Netherlands.
J Lipid Res. 1993 Dec;34(12):2109-19.
We report the molecular basis of familial chylomicronemia and recurrent pancreatitis in five members of a large Dutch family. All patients had normal plasma hepatic lipase and apoC-II levels, but absent lipoprotein lipase (LPL) catalytic activity and low LPL mass in postheparin plasma. The mutation in the LPL gene was characterized as a G715-->A substitution in the last nucleotide of exon 4, resulting in a substitution of Ser for Gly154. PCR amplification of exons 4 + 5 from the patients' mRNA, followed by direct sequencing, revealed normal splicing of intron 4. The mutation creates a BfaI restriction site that allows rapid screening of family members for the mutation. Reproduction of this mutation in LPL-cDNA by site-directed mutagenesis, followed by transient expression in COS-B cells, revealed production of a catalytically inactive enzyme. The Gly154-->Ser substitution appears in a conserved beta-sheet region, in close proximity to Asp156, which is part of the catalytic triad. These studies show that changes to residues close to Asp156 can have profound effects on catalytic activity of LPL.
我们报告了一个荷兰大家庭中五名成员发生家族性乳糜微粒血症和复发性胰腺炎的分子基础。所有患者的血浆肝脂酶和载脂蛋白C-II水平正常，但肝素后血浆中的脂蛋白脂肪酶（LPL）催化活性缺失且LPL质量较低。LPL基因的突变特征为外显子4最后一个核苷酸发生G715→A替换，导致第154位甘氨酸被丝氨酸取代。从患者mRNA中对4 + 5外显子进行PCR扩增，随后直接测序，结果显示内含子4剪接正常。该突变产生了一个BfaI限制性酶切位点，可用于快速筛查家族成员中的该突变。通过定点诱变在LPL-cDNA中重现此突变，随后在COS-B细胞中瞬时表达，结果显示产生了一种无催化活性的酶。第154位甘氨酸→丝氨酸的替换出现在一个保守的β折叠区域，紧邻作为催化三联体一部分的天冬氨酸156。这些研究表明，靠近天冬氨酸156的残基变化可对LPL的催化活性产生深远影响。
