Department of Pediatrics, Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta, Canada.
Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
J Lipid Res. 2019 Mar;60(3):516-527. doi: 10.1194/jlr.M089193. Epub 2019 Jan 7.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDL receptor (LDLR) degradation, increasing plasma levels of LDL cholesterol and the risk of cardiovascular disease. We have previously shown that, in addition to the epidermal growth factor precursor homology repeat-A of LDLR, at least three ligand-binding repeats (LRs) of LDLR are required for PCSK9-promoted LDLR degradation. However, how exactly the LRs contribute to PCSK9's action on the receptor is not completely understood. Here, we found that substitution of Asp at position 172 in the linker between the LR4 and LR5 of full-length LDLR with Asn (D172N) reduced PCSK9 binding at pH 7.4 (mimic cell surface), but not at pH 6.0 (mimic endosomal environment). On the other hand, mutation of Asp at position 203 in the LR5 of full-length LDLR to Asn (D203N) significantly reduced PCSK9 binding at both pH 7.4 and pH 6.0. D203N also significantly reduced the ability of LDLR to mediate cellular LDL uptake, whereas D172N had no detectable effect. These findings indicate that amino acid residues in the LRs of LDLR play an important role in PCSK9 binding to the receptor.
前蛋白转化酶枯草溶菌素 9（PCSK9）可促进 LDL 受体（LDLR）降解，增加 LDL 胆固醇的血浆水平，并增加心血管疾病的风险。我们之前已经表明，除了 LDLR 的表皮生长因子前体同源重复 A 外，LDLR 的至少三个配体结合重复（LR）对于 PCSK9 促进 LDLR 降解是必需的。然而，LR 究竟如何促进 PCSK9 对受体的作用尚不完全清楚。在这里，我们发现全长 LDLR 的 LR4 和 LR5 之间的连接子中位置 172 的天冬氨酸替换为天冬酰胺（D172N）可降低 pH7.4（模拟细胞表面）时 PCSK9 的结合，但不能降低 pH6.0（模拟内体环境）时的结合。另一方面，全长 LDLR 的 LR5 中位置 203 的天冬氨酸突变为天冬酰胺（D203N）显著降低了在 pH7.4 和 pH6.0 时 PCSK9 的结合。D203N 还显著降低了 LDLR 介导细胞 LDL 摄取的能力，而 D172N 则没有可检测到的影响。这些发现表明 LDLR 的 LR 中的氨基酸残基在 PCSK9 与受体结合中发挥重要作用。
