Meng Q H, Calabresi L, Fruchart J C, Marcel Y L
Lipoproteins and Atherosclerosis Group, University of Ottawa Heart Institute, Ontario, Canada.
J Biol Chem. 1993 Aug 15;268(23):16966-73.
The reaction of highly purified lecithin:cholesterol acyltransferase (LCAT) with defined reconstituted discoidal apoA-I-containing lipoproteins (LpA-I) with 2, 3, or 4 apoA-I molecules/particle (Lp2, 3, or 4A-I) has been studied in the presence of a number of specific anti apoA-I antibodies. Among nine anti-apoA-I monoclonal antibodies (mAbs) reacting with epitopes distributed over 80% of the sequence, three significantly inhibit the LCAT reaction with all particles. The position of their epitopes located in the middle to COOH-terminal region between residues 96-121 (3G10), 135-148 (A03), and 149-186 (A44) is compatible with an inhibition by steric hindrance over a central domain. Antibody 4H1 binding to the NH2 terminus (residues 2-8) profoundly increases (5-fold) the LCAT reaction with Lp2A-I (7.8 nm), but not with other particles. Other mAbs, A11 and 5F6, binding to epitopes (residues 99-139 and 118-141) enhance LCAT reactivity with the small Lp2A-I (7.8 nm) and Lp3A-I (10.8 nm) but not with their larger counterparts. Most mAbs have similar effects on LCAT reaction with native high density lipoprotein3 as with LpA-I. The inhibitory or enhancing effects of these mAbs are also observed with Fab fragments and not related to their binding affinity for apoA-I containing reconstituted lipoprotein particles. The intercalation of epitopes for mAbs that inhibit or enhance LCAT reaction with small LpA-I is compatible not with steric hindrance but with conformational modifications of apoA-I and indirectly of the lipids in small particles. We propose that enhancing mAbs act by stabilization of an apoA-I conformation which is not favored in small LpA-I, i.e. by increasing binding of amphipathic helices to lipids or by interfering with the mobility of a hinged domain. The epitopes for the inhibitory mAbs can be shown to overlap on several LpA-I models, indicating that steric hindrance over a single site is a possible mechanism of inhibition.
在多种特异性抗载脂蛋白A-I（apoA-I）抗体存在的情况下，研究了高纯度卵磷脂胆固醇酰基转移酶（LCAT）与含有2、3或4个apoA-I分子/颗粒（Lp2、3或4A-I）的特定重构盘状含apoA-I脂蛋白（LpA-I）的反应。在与分布于80%序列上的表位发生反应的9种抗apoA-I单克隆抗体（mAb）中，有3种显著抑制LCAT与所有颗粒的反应。它们的表位位于96 - 121位（3G10）、135 - 148位（A03）和149 - 186位（A44）之间的中间至COOH末端区域，这与通过中心结构域的空间位阻抑制作用相一致。与NH2末端（2 - 8位）结合的抗体4H1使LCAT与Lp2A-I（7.8纳米）的反应显著增加（5倍），但与其他颗粒的反应无此作用。其他与表位（99 - 139位和118 - 141位）结合的mAb，即A11和5F6，增强了LCAT与小的Lp2A-I（7.8纳米）和Lp3A-I（10.8纳米）的反应性，但与较大的对应物无此反应。大多数mAb对LCAT与天然高密度脂蛋白3的反应和对LpA-I的反应具有相似的作用。这些mAb的抑制或增强作用在Fab片段中也能观察到，且与它们对含apoA-I重构脂蛋白颗粒的结合亲和力无关。抑制或增强LCAT与小LpA-I反应的mAb表位的插入与空间位阻不相符，而是与apoA-I的构象改变以及小颗粒中脂质的间接构象改变有关。我们提出，增强性mAb通过稳定小LpA-I中不占优势的apoA-I构象起作用，即通过增加两亲性螺旋与脂质的结合或通过干扰铰链结构域的流动性。抑制性mAb的表位在几种LpA-I模型上显示有重叠，表明单个位点的空间位阻是一种可能的抑制机制。
