Abstract：
Phospholipase A2\'s (PLA2\'s) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA2\'s, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA2\'s toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P2). The in vitro assay revealed that Group IVA cPLA2 (GIVA cPLA2) showed relatively high activity toward PI and PI(4)P among the tested PLA2\'s; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA2 and GVIA iPLA2 showed detectable activity toward PI(4,5)P2, but it appeared to be a poorer substrate for all of the PLA2\'s tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA2 may best account for the PI and PIP hydrolysis in living cells.
摘要：
磷脂酶A2(PLA2)构成水解甘油磷脂sn-2脂肪酰基链的酶超家族。我们以前报道过，每种PLA2类型对其水解的分子物种显示出独特的底物特异性，特别是从sn-2位置裂解的酰基链和一定程度的极性基团。然而,磷脂酰肌醇（PI）和磷酸PI（PIP）没有像其他磷脂那样作为底物进行很好的研究，因为PIP需要调整标准分析方法。但它们在体内很重要。我们确定了三种主要类型的人PLA2的体外活性，即胞质(C)，不依赖钙的(I)，并向PI分泌PLA2，PI-4-磷酸（PI（4）P），和PI-4,5-双磷酸酯(PI(4,5)P2)。体外测定显示，在测试的PLA2中，IVA组cPLA2(GIVAcPLA2)对PI和PI(4)P显示出相对较高的活性；然而，高度亲水的头基破坏了脂质表面和酶之间的相互作用。GIVAcPLA2和GVIAiPLA2对PI（4,5）P2显示出可检测的活性，但对于所有测试的PLA2，它似乎是较差的底物。此外，分子动力学（MD）模拟表明，GIVAcPLA2的Thr416和Glu418显着有助于容纳PI和PI（4）P的亲水头基，这可以解释PI和PI(4)P的某些选择性。这些结果表明，GIVAcPLA2可以在其活性位点容纳PI和PI（4）P并水解它们，这表明GIVAcPLA2可能最好地解释了活细胞中的PI和PIP水解。
