Kobayashi J, Applebaum-Bowden D, Dugi K A, Brown D R, Kashyap V S, Parrott C, Duarte C, Maeda N, Santamarina-Fojo S
Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland, 20892, USA.
J Biol Chem. 1996 Oct 18;271(42):26296-301.
Hepatic lipase (HL) and lipoprotein lipase (LPL) are key enzymes involved in the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Despite their similarities, the role that each of these two lipases play in the metabolism of triglyceride-rich lipoproteins and high density lipoproteins is distinct. In order to identify structural domains that may confer the different substrate specificities between HL and LPL, we have utilized a novel approach for performing structure-function analysis of a protein, in vivo, by using recombinant adenovirus vectors to express native and mutant enzymes in an animal model for a human genetic deficiency. HL-deficient mice (n = 19) characterized by increased plasma cholesterol and phospholipid concentrations were injected with adenovirus expressing luciferase (rLucif-AdV), native hepatic (rHL-AdV), and lipoprotein lipase (rLPL-AdV) or lipase mutants in which the lid covering the catalytic site of either enzyme was exchanged (rHL+LPL lid-AdV and rLPL+HL lid-AdV). Mice injected with rLucif-AdV had no changes in post-heparin HL and LPL activities (217 +/- 29 and 7 +/- 2 nmol/min/ml, respectively) as well as plasma lipids. Despite expression of similar levels of post-heparin plasma lipase activity on day 5 post-adenovirus infusion (9806 +/- 915 and 9677 +/- 2033 nmol/min/ml, respectively) mice injected with rHL-AdV or rHL+LPL lid-AdV demonstrated marked differences in the reduction of plasma phospholipids (70% and 32%, respectively, p < 0.005). Similarly, despite post-heparin plasma lipolytic activities of 4495 +/- 534 and 4844 +/- 1336 nmol/min/ml, injection of rLPL-AdV or rLPL+HL lid-AdV resulted in phospholipid reductions of 31% and 81% (p < 0.005). Exchange of the lipase lid did not significantly alter plasma triglyceride concentrations. Thus, preferential in vivo hydrolysis of phospholipids was demonstrated in animals expressing lipases containing the HL lid but not the LPL lid. These studies identify the lipase lid as a major structural motif responsible for conferring the different in vivo phospholipase activities between HL and LPL, a function which may modulate the distinct physiological roles of these two similar lipolytic enzymes in lipoprotein metabolism. The use of recombinant adenovirus to express mutant proteins in animal models for human genetic deficiencies represents a powerful, new approach for performing structure-function analysis of proteins in vivo.
肝脂肪酶（HL）和脂蛋白脂肪酶（LPL）是参与循环血浆脂蛋白中甘油三酯和磷脂水解的关键酶。尽管它们有相似之处，但这两种脂肪酶在富含甘油三酯的脂蛋白和高密度脂蛋白代谢中所起的作用是不同的。为了确定可能赋予HL和LPL不同底物特异性的结构域，我们采用了一种新方法，通过使用重组腺病毒载体在人类遗传缺陷动物模型中表达天然和突变酶，在体内对蛋白质进行结构功能分析。以血浆胆固醇和磷脂浓度升高为特征的HL缺陷小鼠（n = 19）被注射表达荧光素酶的腺病毒（rLucif-AdV）、天然肝脂肪酶（rHL-AdV）、脂蛋白脂肪酶（rLPL-AdV）或脂肪酶突变体，其中覆盖任一酶催化位点的盖子进行了交换（rHL+LPL盖子-AdV和rLPL+HL盖子-AdV）。注射rLucif-AdV的小鼠肝素后HL和LPL活性（分别为217±29和7±2 nmol/min/ml）以及血浆脂质没有变化。尽管在腺病毒输注后第5天肝素后血浆脂肪酶活性表达水平相似（分别为9806±915和9677±2033 nmol/min/ml），但注射rHL-AdV或rHL+LPL盖子-AdV的小鼠在血浆磷脂降低方面表现出显著差异（分别为70%和32%，p < 0.005）。同样，尽管肝素后血浆脂解活性分别为4495±534和4844±1336 nmol/min/ml，但注射rLPL-AdV或rLPL+HL盖子-AdV导致磷脂降低31%和81%（p < 0.005）。脂肪酶盖子的交换没有显著改变血浆甘油三酯浓度。因此，在表达含有HL盖子而非LPL盖子的脂肪酶的动物中证明了体内对磷脂的优先水解。这些研究确定脂肪酶盖子是赋予HL和LPL不同体内磷脂酶活性的主要结构基序，这一功能可能调节这两种相似脂解酶在脂蛋白代谢中的不同生理作用。利用重组腺病毒在人类遗传缺陷动物模型中表达突变蛋白代表了一种在体内对蛋白质进行结构功能分析的强大新方法。
