Rouis M, Lohse P, Dugi K A, Lohse P, Beg O U, Ronan R, Talley G D, Brunzell J D, Santamarina-Fojo S
Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Lipid Res. 1996 Mar;37(3):651-61.
Familial lipoprotein lipase (LPL) deficiency is an inherited disorder of lipoprotein metabolism characterized by hypertriglyceridemia and recurrent episodes of abdominal pain and pancreatitis. We have studied the genetic basis of LPL deficiency in a 62-year-old black male with undetectable pre- and post-heparin plasma LPL mass and activity, DNA sequence analysis of the patient's LPL cDNA and gene as well as digestion with Bcl I and Asu I revealed that the proband is a homozygote for two separate gene defects. One mutation changed a G to an A, resulting in the conversion of amino acid 9 of the mature protein, aspartic acid (GAC), to asparagine (AAC). The second substitution, a C for a T, replaced tyrosine (TAC) at residue 262 with histidine (CAC). Northern blot analysis of monocyte-derived macrophage RNA demonstrated the presence of LPL mRNA of approximately normal size and quantity when compared to control. Expression of both mutations separately (pCMV-9 and pCMV-262) or in combination (pCMV-9+262) in human embryonal kidney-293 cells demonstrated that LPL-9 had approximately 80% the specific activity of wild type LPL, but LPL-262 and LPL-9+262 had no enzymic activity, thus establishing the functional significance of the LPL-262 defect. Despite an absolute deficiency of LPL mass and activity demonstrated by analysis of patient post-heparin plasma, in vitro expression of both LPL mutants was normal, suggesting that the absence of LPL in patient post-heparin plasma was a result of altered in vivo processing. Analysis of the heparin binding properties of the mutant enzymes by heparin-Sepharose affinity chromatography indicated that most of the LPL-262 mass was present in an inactive peak, which like the normal LPL monomer, eluted at 0.8 M NaCl. Thus, the Tyr262 --> His mutation may alter the stability of the LPL dimer, leading to the formation of inactive LPL-262 monomer which exhibits reduced heparin affinity. Based on these results, we propose that, in vivo, enhanced formation of LPL-9+262 monomer leads to abnormal binding of the mutant lipase to endothelial glycosaminoglycans ultimately resulting in enhanced catabolism of the mutant enzyme and lower enzyme mass in post-heparin plasma.
家族性脂蛋白脂肪酶（LPL）缺乏症是一种遗传性脂蛋白代谢紊乱疾病，其特征为高甘油三酯血症以及反复发作的腹痛和胰腺炎。我们对一名62岁黑人男性LPL缺乏症的遗传基础进行了研究，该患者肝素化前后血浆中的LPL质量和活性均检测不到。对患者的LPL cDNA和基因进行DNA序列分析以及用Bcl I和Asu I酶切后发现，先证者是两个不同基因缺陷的纯合子。一个突变使G变为A，导致成熟蛋白第9位氨基酸天冬氨酸（GAC）转变为天冬酰胺（AAC）。第二个替换是C替换T，使第262位残基的酪氨酸（TAC）被组氨酸（CAC）取代。对单核细胞衍生的巨噬细胞RNA进行Northern印迹分析表明，与对照相比，存在大小和数量大致正常的LPL mRNA。在人胚肾-293细胞中分别单独表达这两个突变（pCMV-9和pCMV-262）或联合表达（pCMV-9+262）表明，LPL-9具有野生型LPL约80%的比活性，但LPL-262和LPL-9+262没有酶活性，从而确定了LPL-262缺陷的功能意义。尽管对患者肝素化后血浆分析显示LPL质量和活性绝对缺乏，但两种LPL突变体的体外表达正常，这表明患者肝素化后血浆中LPL的缺失是体内加工改变的结果。通过肝素-琼脂糖亲和色谱分析突变酶的肝素结合特性表明，大多数LPL-262质量存在于一个无活性峰中，该峰与正常LPL单体一样，在0.8M NaCl处洗脱。因此，Tyr262→His突变可能改变LPL二聚体的稳定性，导致形成无活性的LPL-262单体，其肝素亲和力降低。基于这些结果，我们提出，在体内，LPL-9+262单体形成增加导致突变脂肪酶与内皮糖胺聚糖异常结合，最终导致突变酶的分解代谢增强以及肝素化后血浆中酶质量降低。
