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

肝脂酶(HL)和脂蛋白脂酶(LPL)是脂解酶 它们在正常的甘油三酯代谢中起着重要作用。作为 脂肪酶家族，这两种酶具有高度的同源性；然而， LPL和HL具有不同的辅因子要求和底物 具体细节。虽然LPL和HL都是三酰甘油水解酶， HL也是一种磷脂酶。 我们研究了不同衬底的结构基础。 LPL和HL在体外和体内的特异性 利用定点突变和重组技术获得突变脂肪酶 腺病毒载体。此前，我们已经证明了LPL和HL 调节底物对催化部位的访问的盖子发挥作用 在调节脂质-底物相互作用中起着至关重要的作用。中断 存在于脂肪酶盖中的两个两亲性螺旋导致 使用三油酸脂或磷脂(PL)底物的活性 对短链水溶性底物的酯酶活性 三丁酸甘油酯被保存下来。含LPL主链的嵌合脂肪酶 对于HL盖，具有LPL盖和氨基末端的HL主干 具有HL羧基末端的LPL具有明显不同的能力 对磷光底物进行水解。HL盖子的存在增加了PL 而LPL LID则促进了甘油三酯的水解。 这些体外研究现在已经扩展到体内使用重组 表达天然HL或带有LPL盖子的HL的腺病毒。这些 将重组体注射到血浆升高的HL缺陷小鼠中 导致肝素后血浆活性的TC和PL水平(第4天 8,953和9,133毫摩尔/分钟/毫升。 自然感染的HL病毒的血浆PL降低了30%，并有明显的变化 在脂蛋白谱中；然而，注射了LPL盖子的HL组有 血浆PL水平无明显变化。这些研究表明， 带有LPL盖子的HL在体内改变脂肪酶的能力 水解磷脂酶，从而确立了脂肪酶盖在 调节脂肪酶底物专一性。

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.