Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany.
Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef, Leiden, The Netherlands.
Glycobiology. 2018 Oct 1;28(10):765-773. doi: 10.1093/glycob/cwy061.
Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6-8 and 19-20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19-20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH's intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH's sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.
补体因子 H（FH）是一种长链且高度糖基化的 20 个结构域蛋白，是补体替代途径（AP）的可溶性调节剂。它包含几个糖基结合位点，介导对 α2-3 连接的唾液酸（FH 结构域 20）和糖胺聚糖（结构域 6-8 和 19-20）的识别。FH 还结合补体 C3 激活产物 C3b，C3b 是一种强大的调理素和 AP 反馈环中 C3 转化酶形成的焦点。在游离循环的 FH 中，结构域 19-20 中的 C3b 结合位点被封闭，这种现象尚未完全理解，可能是由 FH 固有唾液酸化糖基化与其自身唾液酸结合位点之间的分子内相互作用介导的。为了评估这种可能性，我们对 FH 的糖基化进行了特征分析，包括糖苷键类型，并在 FH 及其 7 个结构域跨越的剪接变体和同源补体调节剂 FH 样-1（FHL-1）中寻找进一步的潜在、尚未表征的唾液酸结合位点。我们还探测了 FH 与非人类唾液酸 Neu5Gc 的结合，Neu5Gc 不在人类中表达，但在限制人类 AP 的异源红细胞和 FH 转基因小鼠中表达。我们发现 FH 主要含有 α2-6 连接的唾液酸，与 α2-3 唾液酸特异性结合位点形成分子内相互作用，相关的自身锁定机制不太可能，证实 FH 中只有一个唾液酸结合位点，FHL-1 中没有，并且证明 FH 与非人类唾液酸 Neu5Gc 的直接结合，支持使用 FH 转基因小鼠模型进行补体相关疾病的研究。
