Pasman Y, Kaushik A K
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Scand J Immunol. 2016 Jul;84(1):28-38. doi: 10.1111/sji.12443.
We analysed contributions of variable heavy (FdVH ) and variable light (FdVL ) domains in comparison to scFv (FdVH +FdVL ) of naturally occurring polyspecific bovine IgM with an exceptionally long CDR3H and an induced monospecific bovine herpes virus-1 (BoHV-1) neutralizing IgG1 antibody in the context of to antigen-binding site and antibody function. Various recombinant FdVH , FdVL and scFv were constructed and expressed in Pichia pastoris from the bovine IgM and IgG1 antibody encoding cDNA. The scFv1H12 showed polyspecific antigen binding similar to parent IgM antibody, though subtle differences, for example, higher thyroglobulin recognition. Such differences reflect influence of the constant region on the antigen-binding site configuration. Unlike, variable light domain FdVL 1H12, the variable heavy domain FdVH 1H12 alone recognized multiple antigens that differed from the recognition pattern of scFv1H12 (FdVH +FdVL ) and the parent IgM antibody. Nonetheless, role of FdVL 1H12 in providing structural support to FdVH in antigen recognition is noted, apart from its intrinsic antigen recognition ability. Surface plasmon resonance analysis revealed low to moderate affinity of scFv1H12 to IgG antigen. By contrast, the individual FdVH 073 and FdVL 074, originating from induced BoHV-1 neutralizing IgG1 antibody, recognized target epitope on BoHV-1 weakly when compared to FdVH +FdVL (scFv3-18L). Interestingly, both the FdVH and FdVL domains of induced IgG antibody are required to achieve BoHV-1 neutralization. To conclude, there exist subtle functional differences in the contribution of FdVH and FdVL to antigen-binding site generation of polyspecific IgM and monospecific IgG antibodies relevant to antigen recognition and virus neutralization functions.
我们分析了可变重链（FdVH）和可变轻链（FdVL）结构域的作用，并将其与天然存在的多特异性牛IgM的单链抗体片段（scFv，FdVH + FdVL）进行比较。该多特异性牛IgM具有异常长的互补决定区3重链（CDR3H），并在抗原结合位点和抗体功能的背景下诱导产生了单特异性牛疱疹病毒1型（BoHV-1）中和IgG1抗体。构建了各种重组FdVH、FdVL和scFv，并在毕赤酵母中表达，其编码cDNA来自牛IgM和IgG1抗体。单链抗体片段1H12（scFv1H12）表现出与亲本IgM抗体相似的多特异性抗原结合能力，不过也存在细微差异，例如对甲状腺球蛋白的识别能力更强。这些差异反映了恒定区对抗原结合位点构象的影响。与可变轻链结构域FdVL 1H12不同，单独的可变重链结构域FdVH 1H12识别多种抗原，其识别模式不同于单链抗体片段1H12（scFv1H12，FdVH + FdVL）和亲本IgM抗体。尽管如此，除了其固有的抗原识别能力外，FdVL 1H12在抗原识别中为FdVH提供结构支持的作用也值得关注。表面等离子体共振分析显示，单链抗体片段1H12（scFv1H12）与IgG抗原的亲和力较低至中等。相比之下，源自诱导产生的BoHV-1中和IgG抗体的单个FdVH 073和FdVL 074，与FdVH + FdVL（单链抗体片段3-18L）相比，对BoHV-1上的靶表位的识别较弱。有趣的是，诱导产生的IgG抗体的FdVH和FdVL结构域都需要实现对BoHV-1的中和作用。总之，在多特异性IgM和单特异性IgG抗体的抗原结合位点形成过程中，FdVH和FdVL在与抗原识别和病毒中和功能相关方面的贡献存在细微的功能差异。
