IN VITRO AND IN VIVO STRUCTURE-FUNCTION ANALYSIS OF LPL AND HL

肝脂酶（HL）和脂蛋白脂酶（LPL）是脂肪分解酶 其在正常甘油三酯代谢中起主要作用。的成员 脂肪酶家族中，两种酶具有高度的同源性;然而， LPL和HL具有不同的辅因子需求和底物 特殊性虽然LPL和HL都是三酰甘油水解酶， HL还作为磷脂酶发挥作用。 我们研究了不同基质的结构基础 LPL和HL在体外和体内的特异性， 使用定点诱变的突变脂肪酶以及重组脂肪酶 腺病毒载体以前，我们已经证明，LPL和HL 调节底物进入催化位点的盖子， 介导脂质-底物相互作用的关键作用。破坏 存在于脂肪酶盖中的2个两亲性螺旋导致 使用三油酸甘油酯或磷脂（PL）底物的活性， 针对短链水溶性底物的酯酶活性 三丁酸甘油酯被保存。含有LPL骨架的嵌合脂肪酶 具有HL盖，具有LPL盖和氨基末端的HL骨架 LPL的羧基端与HL的羧基端具有显著不同的能力， 以水解PL底物。HL盖的存在增强了PL 然而，LPL盖增强了甘油三酯水解。 这些体外研究现在已经在体内使用重组 表达天然HL或具有LPL盖的HL的腺病毒。这些 将构建体注射到具有升高的血浆浓度的HL缺陷小鼠中， 导致肝素后血浆活性的TC和PL水平（第4天 输注后）的8，953和9，133 nmol/min/ml。动物注射 天然HL病毒的血浆PL降低了30%， 然而，那些注射HL和LPL盖的人， 血浆PL水平无变化。这些研究表明， 具有LPL盖的HL在体内改变脂肪酶的能力， 水解PL，从而确立了脂肪酶盖在 介导脂肪酶底物特异性。

Hepatic lipase (HL) and lipoprotein lipase (LPL) are lipolytic enzymes which play a major role in normal triglyceride metabolism. As members of the lipase family, both enzymes share a high degree of homology; however, LPL and HL have different cofactor requirements and substrate specificities. Although both LPL and HL are triacylglycerol hydrolases, HL also functions as a phospholipase. We have investigated the structural basis for the different substrate specificities of LPL and HL both in vitro and in vivo by generating mutant lipases using site-directed mutagenesis as well as recombinant adenoviral vectors. Previously, we have demonstrated that the LPL and HL lids, which modulate access of the substrate to the catalytic site, play a crucial role in mediating lipid-substrate interaction. Disruption of the 2 amphipathic helices present in the lipase lids results in loss of activity using either triolein or phospholipid (PL) substrates while esterase activity against the short-chained watersoluble substrate tributyrin is preserved. Chimeric lipases containing the LPL backbone with the HL lid, the HL backbone with the LPL lid and the amino terminus of LPL with the carboxyl-terminus of HL had a markedly different ability to hydrolyze PL substrates. The presence of the HL lid augmented PL hydrolysis whereas the LPL lid enhanced triglyceride hydrolysis. These in vitro studies have now been extended in vivo using recombinant adenovirus expressing either native HL or HL with the LPL lid. These constructs were injected in HL-deficient mice who have elevated plasma levels of TC and PL resulting in post-heparin plasma activities (day 4 post-infusion) of 8,953 and 9,133 nmoles/min/ml. Animals injected with the native HL virus had a 30% reduction in plasma PL and a marked change in lipoprotein profile; however, those injected with HL with LPL lid had no change in plasma PL levels. These studies indicate that exchange of the HL with the LPL lid modifies the ability of the lipase, in vivo, to hydrolyze PL and thus, establish an important role of the lipase lid in mediating lipase substrate specificity.