Giles Ian P, Haley Joanna, Nagl Sylvia, Latchman David S, Chen Pojen P, Chukwuocha Reginald U, Isenberg David A, Rahman Anisur
Department of Medicine, Centre for Rheumatology, University College London, Arthur Stanley House, 40-50 Tottenham Street, London W1T 4NJ, UK.
Mol Immunol. 2003 Sep;40(1):49-60. doi: 10.1016/s0161-5890(03)00100-7.
Previous studies have suggested the importance of somatic mutations and arginine, asparagine and lysine residues in the complementarity determining regions (CDRs) of antiphospholipid antibodies (aPL) implicated in the pathogenesis of the antiphospholipid antibody syndrome. The relative contributions of the heavy and light chains of aPL in binding to cardiolipin (CL) were assessed by pairing the heavy and light chains of two IgG, beta(2)GPI dependent aPL (IS4 and CL24) with different partner chains from other IgG, beta(2)GPI independent aPL (UK4) and anti-DNA antibodies (B3 and 33H11).
Four heavy (V(H)) and five light (V(L)) chain variable sequences from three aPL and two anti-DNA antibodies were cloned into expression vectors containing appropriate gamma(1), lambda or kappa constant region cDNA. Paired combinations of heavy and light chain expression plasmids were transfected into COS-7 cells allowing transient expression of whole IgG molecules, which were harvested and tested for the ability to bind CL and DNA by enzyme-linked immunosorbant assay (ELISA).
Whole IgG was produced from 19 heavy/light chain combinations. IS4V(H) was dominant in conferring the ability to bind CL with four of the five V(L) tested. The identity of the V(L) region paired with IS4V(H) was important in determining the strength of binding to CL. IS4V(H) contains multiple arginine residues in CDR3, which may have accumulated due to antigen driven selection. It is likely that these arginine residues may interact with CL. The combination B3V(H)/B3V(L) also bound CL, but none of the other 14 combinations showed any binding in this assay.
Whole IgG molecules capable of binding CL were produced by in vitro expression in COS-7 cells. Arginine residues play important roles in binding to CL and double-stranded DNA. However, different patterns of mutation to arginine are associated with binding to each of these antigens.
先前的研究表明，抗磷脂抗体（aPL）互补决定区（CDR）中的体细胞突变以及精氨酸、天冬酰胺和赖氨酸残基在抗磷脂抗体综合征的发病机制中具有重要意义。通过将两种IgG、β2糖蛋白（β2GPI）依赖性aPL（IS4和CL24）的重链和轻链与来自其他IgG、β2GPI非依赖性aPL（UK4）和抗DNA抗体（B3和33H11）的不同伙伴链配对，评估了aPL重链和轻链在与心磷脂（CL）结合中的相对贡献。
将来自三种aPL和两种抗DNA抗体的四个重链（V(H)）和五个轻链（V(L)）可变序列克隆到含有适当γ1、λ或κ恒定区cDNA的表达载体中。将重链和轻链表达质粒的配对组合转染到COS-7细胞中，使完整IgG分子瞬时表达，收获后通过酶联免疫吸附测定（ELISA）检测其结合CL和DNA的能力。
从19种重链/轻链组合中产生了完整的IgG。在测试的五种V(L)中的四种中，IS4V(H)在赋予结合CL的能力方面占主导地位。与IS4V(H)配对的V(L)区域的同一性对于确定与CL结合的强度很重要。IS4V(H)在CDR3中含有多个精氨酸残基，这可能是由于抗原驱动的选择而积累的。这些精氨酸残基可能与CL相互作用。组合B3V(H)/B3V(L)也结合CL，但其他14种组合在该测定中均未显示任何结合。
通过在COS-7细胞中体外表达产生了能够结合CL的完整IgG分子。精氨酸残基在与CL和双链DNA的结合中起重要作用。然而，精氨酸的不同突变模式与与每种这些抗原的结合相关。
