Giles Ian, Lambrianides Nancy, Pattni Nisha, Faulkes David, Latchman David, Chen Pojen, Pierangeli Silvia, Isenberg David, Rahman Anisur
Centre for Rheumatology, Department of Medicine, University College, London, UK.
J Immunol. 2006 Aug 1;177(3):1729-36. doi: 10.4049/jimmunol.177.3.1729.
In the antiphospholipid syndrome (APS), antiphospholipid Abs (aPL) bind to anionic phospholipids (PL) and various associated proteins, especially beta(2)-glycoprotein I (beta2GPI) and prothrombin. In the present study, we show that altering specific Arg residues in the H chain of a human pathogenic beta2GPI-dependent aPL, IS4, has major effects on its ability to bind these clinically important Ags. We expressed whole human IgG in vitro by stable transfection of Chinese hamster ovary cells with expression plasmids containing different V(H) and V(L) sequences. V(H) sequences were derived from IS4 by altering the number of Arg residues in CDR3. V(L) sequences were those of IS4, B3 (anti-nucleosome Ab), and UK4 (beta2GPI-independent aPL). Binding of the expressed H/L chain combinations to a range of anionic, neutral, and zwitterionic PL, as well as prothrombin, beta2GPI, dsDNA, and chicken OVA, was determined by ELISA. Of four Arg residues in IS4VH CDR3 substituted to Ser, two at positions 100 and 100g, reduced binding to all Ags, while two at positions 96 and 97 reduced binding to beta2GPI but increased or decreased binding to different PL. Eleven of 14 H/L chain combinations displayed weak binding to OVA with Arg to Ser replacements of all four Arg residues enhancing binding to this Ag. Only one H/L chain combination bound neutral PL and none bound dsDNA; hence, these effects are particularly relevant to Ags important in antiphospholipid syndrome. We hypothesize that these four Arg residues have developed as a result of somatic mutations driven by an Ag containing both PL and beta2GPI.
在抗磷脂综合征（APS）中，抗磷脂抗体（aPL）与阴离子磷脂（PL）及各种相关蛋白结合，尤其是β2糖蛋白I（β2GPI）和凝血酶原。在本研究中，我们发现改变人类致病性β2GPI依赖性aPL（IS4）重链中的特定精氨酸残基，对其结合这些临床重要抗原的能力有重大影响。我们通过用含有不同重链可变区（V(H)）和轻链可变区（V(L)）序列的表达质粒稳定转染中国仓鼠卵巢细胞，在体外表达完整的人IgG。V(H)序列源自IS4，通过改变互补决定区3（CDR3）中的精氨酸残基数量获得。V(L)序列来自IS4、B3（抗核小体抗体）和UK4（β2GPI非依赖性aPL）。通过酶联免疫吸附测定（ELISA）确定表达的重链/轻链组合与一系列阴离子、中性和两性离子PL以及凝血酶原、β2GPI、双链DNA（dsDNA）和鸡卵清蛋白（OVA）的结合情况。在IS4VH CDR3中被替换为丝氨酸的四个精氨酸残基中，位于100和100g位置的两个残基减少了与所有抗原的结合，而位于96和97位置的两个残基减少了与β2GPI的结合，但增加或减少了与不同PL的结合。14种重链/轻链组合中有11种对OVA显示出弱结合，将所有四个精氨酸残基替换为丝氨酸增强了与该抗原的结合。只有一种重链/轻链组合结合中性PL，没有一种结合dsDNA；因此，这些效应与抗磷脂综合征中重要的抗原特别相关。我们推测这四个精氨酸残基是由同时含有PL和β2GPI的抗原驱动的体细胞突变产生的。
