Jin L, Shieh J J, Grabbe E, Adimoolam S, Durbin D, Jonas A
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
Biochemistry. 1999 Nov 23;38(47):15659-65. doi: 10.1021/bi9916729.
Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.
卵磷脂胆固醇酰基转移酶（LCAT）与脂蛋白表面的结合是逆向胆固醇转运过程中的关键步骤，因为随后的胆固醇酯化反应促使胆固醇从组织中移除进入血浆。在本研究中，表面等离子体共振方法用于研究LCAT与脂蛋白的结合动力学和亲和力。含有载脂蛋白A-I或A-II（apoA-I或apoA-II）、低密度脂蛋白（LDL）以及带有生物素标签的小单层磷脂酰胆碱囊泡的重组高密度脂蛋白（rHDL）被固定在含有链霉亲和素的生物传感器芯片上，并检测纯重组LCAT的结合动力学随LCAT浓度的变化。此外，还检测了LCAT的三个突变体（T123I、N228K和(Delta53-71)）与LDL的相互作用。对于野生型LCAT，与所有脂质表面的结合具有相同的缔合速率常数k(a)，但解离速率常数k(d)不同，这取决于apoA-I的存在（k(d)降低）以及LDL中不同的脂质。此外，缓冲液离子强度的增加降低了LCAT与apoA-I rHDL结合的k(a)。对于LCAT突变体，Delta53-71（缺失环突变体）与LDL无结合，而LCAT缺陷突变体（T123I和N228K）与LDL的结合几乎正常。总之，LCAT与脂蛋白表面的缔合基本上与其组成无关，但有较小的静电作用，而由于apoA-I的存在，LCAT从脂蛋白上的解离减少，提示存在蛋白质-蛋白质相互作用。此外，LCAT中53至71位残基之间的区域对于界面结合至关重要。
