University of Pittsburgh, School of Medicine, Center for Vaccine Research, 9047 Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA.
Immunol Lett. 2010 Mar 10;129(1):32-8. doi: 10.1016/j.imlet.2009.12.022. Epub 2010 Jan 12.
The final degradation product of the complement protein C3, C3d, has been used as a molecular adjuvant to various antigens. Chimera proteins of the antigen and multiple copies of C3d were developed to test the adjuvant effect of this molecule. The main mechanism by which C3d enhances the immune response is interaction with CR2. In vitro studies showed that the avidity of C3d for CR2 is affected by residues located at the interacting surface (e.g. 170N) as well as by residues located in other areas. The role of the latter residues has been proposed to depend on the electrostatic nature of the C3d-CR2 interaction, where the charges of the whole molecules are responsible for their binding. C3d is primarily a negatively charged molecule, while CR2 is a positive one. Previous experiments demonstrated that elimination of a positive charge (K162A) in C3d enhanced its avidity for CR2, while elimination of negative charges or addition positives ones (D163A, N170R, respectively), impaired the avidity for CR2. Despite the extensive in vitro research, the role of these residues in the adjuvant effect of C3d is unclear. To study the role of residues at the interacting and non-interacting surface of C3d on the adjuvanticity, single as well as a double residue substitutions were engineered in the murine C3d (R162A, D163A, N170R and D163A-N170R) gene. Two copies of these mutant molecules were fused to HIV-1 Env(gp120) and the proteins were tested for their avidity to bind CR2 (sCR2). Later, these DNA constructs were tested in mice to determine their adjuvant capability. Mutation at residue 162 (R162A) neither enhanced nor impaired the avidity of Env(gp120)-C3d(2) for sCR2 in vitro. Mutations at residues D163A and N170R, on the other hand, reduced the binding affinity of Env(gp120)-C3d(2) for sCR2. Furthermore, these mutations synergized and abolished the interaction of C3d for CR2. The data correlated with the adjuvant capability of these molecules in the mouse model. In summary, residues that alter the electronegative status of C3d (D163A and N170R) impair the binding of chimera proteins to CR2, reducing the adjuvant activity of this molecule.
补体蛋白 C3 的最终降解产物 C3d 已被用作各种抗原的分子佐剂。抗原和多个 C3d 拷贝的嵌合体蛋白被开发出来，以测试该分子的佐剂效应。C3d 增强免疫反应的主要机制是与 CR2 相互作用。体外研究表明，C3d 与 CR2 的亲和力受位于相互作用表面的残基（例如 170N）以及位于其他区域的残基的影响。后一种残基的作用被认为取决于 C3d-CR2 相互作用的静电性质，其中整个分子的电荷负责它们的结合。C3d 主要是带负电荷的分子，而 CR2 是带正电荷的分子。先前的实验表明，消除 C3d 中的一个正电荷（K162A）可增强其与 CR2 的亲和力，而消除负电荷或添加正电荷（分别为 D163A、N170R）则会损害其与 CR2 的亲和力。尽管有广泛的体外研究，但这些残基在 C3d 的佐剂效应中的作用尚不清楚。为了研究 C3d 相互作用和非相互作用表面上的残基在佐剂中的作用，在小鼠 C3d（R162A、D163A、N170R 和 D163A-N170R）基因中设计了单个和双残基取代。这些突变分子的两个拷贝融合到 HIV-1Env（gp120）中，并测试其与 sCR2（sCR2）结合的亲和力。后来，这些 DNA 构建体在小鼠中进行了测试，以确定它们的佐剂能力。残基 162（R162A）的突变既没有增强也没有削弱 Env（gp120）-C3d（2）与 sCR2 的体外亲和力。另一方面，D163A 和 N170R 突变降低了 Env（gp120）-C3d（2）与 sCR2 的结合亲和力。此外，这些突变协同作用并消除了 C3d 与 CR2 的相互作用。数据与这些分子在小鼠模型中的佐剂能力相关。总之，改变 C3d 电负性状态的残基（D163A 和 N170R）会损害嵌合体蛋白与 CR2 的结合，从而降低该分子的佐剂活性。
