Chahinian Henri, Bezzine Sofiane, Ferrato Francine, Ivanova Margarita G, Perez Barbara, Lowe Mark E, Carrière Frédéric
Laboratoire de Lipolyse Enzymatique du CNRS, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
Biochemistry. 2002 Nov 19;41(46):13725-35. doi: 10.1021/bi0257944.
The structural similarities between the C-terminal domain of human pancreatic lipase (C-HPL) and C2 domains suggested a similar function, the interaction with lipids. The catalytic N-terminal domain (N-HPL) and C-HPL were produced as individual proteins, and their partitioning between the water phase and the triglyceride-water interface was assessed using trioctanoin emulsions (TC8). N-HPL did not bind efficiently to TC8 and was inactive. C-HPL did bind to TC8 and to a phospholipid monolayer with a critical surface pressure of penetration similar to that of HPL (15 mN m(-1)). These experiments, performed in the absence of colipase and bile salts, support an absolute requirement of C-HPL for interfacial binding of HPL. To refine our analysis, we determined the contribution to lipid interactions of a hydrophobic loop (beta 5') in C-HPL by investigating a HPL mutant in which beta 5' loop hydrophobicity was increased by introducing the homologous lipoprotein lipase (LPL) beta 5' loop. This mutant (HPL-beta 5'LPL) penetrated into phospholipid monolayers at higher surface pressures than HPL, and its level of binding to TC8 was higher than that of HPL in the presence of serum albumin (BSA), an inhibitory protein that competes with HPL for interfacial adsorption. The beta 5' loop of LPL is therefore tailored for an optimal interaction with the surface of triglyceride-rich lipoproteins (VLDL and chylomicrons) containing phospholipids and apoproteins. These observations support a major contribution of the beta 5' loop in the interaction of LPL and HPL with their respective substrates.
人胰脂肪酶（C-HPL）的C末端结构域与C2结构域之间的结构相似性表明其功能相似，即与脂质相互作用。催化性N末端结构域（N-HPL）和C-HPL作为单独的蛋白质产生，并使用三辛酸甘油酯乳液（TC8）评估它们在水相和甘油三酯-水界面之间的分配。N-HPL不能有效地结合到TC8上且无活性。C-HPL确实能结合到TC8和具有与HPL相似的临界穿透表面压力（15 mN m(-1)）的磷脂单层上。这些在无辅脂酶和胆汁盐存在的情况下进行的实验，支持了C-HPL对于HPL在界面结合方面的绝对需求。为了完善我们的分析，我们通过研究一种HPL突变体来确定C-HPL中一个疏水环（β5'）对脂质相互作用的贡献，在该突变体中，通过引入同源的脂蛋白脂肪酶（LPL）的β5'环来增加β5'环的疏水性。在存在血清白蛋白（BSA）（一种与HPL竞争界面吸附的抑制性蛋白质）的情况下，该突变体（HPL-β5'LPL）比HPL在更高的表面压力下渗透到磷脂单层中，并且其与TC8的结合水平高于HPL。因此，LPL的β5'环经过调整以与富含甘油三酯的脂蛋白（极低密度脂蛋白和乳糜微粒）表面含有磷脂和载脂蛋白的部分实现最佳相互作用。这些观察结果支持了β5'环在LPL和HPL与其各自底物相互作用中的主要作用。
