Department of Metabolism, Endocrinology and Molecular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan.
Department of Nephrology, Osaka City University Graduate School of Medicine, Osaka, Japan.
Sci Rep. 2021 Apr 5;11(1):7486. doi: 10.1038/s41598-021-86881-0.
Fetuin-A is an inhibitor of ectopic calcification that is expressed mainly in hepatocytes and is secreted into the circulation after posttranslational processing, including glycosylation and phosphorylation. The molecular weight (MW) of fully modified fetuin-A (FM-fetuin-A) is approximately 60 kDa in an immunoblot, which is much higher than the estimated MW by amino acid sequence. Under conditions of calcification stress such as advanced stage chronic kidney disease, fetuin-A prevents calcification by forming colloidal complexes, which are referred to as calciprotein particles (CPP). Since the significance of CPP in this process is unclear, we investigated the effect of synthetic secondary CPP on the level of FM-fetuin-A in HepG2 cells. Secondary CPP increased the level of FM-fetuin-A in dose- and time-dependent manners, but did not affect expression of mRNA for fetuin-A. Treatment with O- and/or N-glycosidase caused a shift of the 60 kDa band of FM-fetuin-A to a lower MW. Preincubation with brefeldin A, an inhibitor of transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus, completely blocked the secondary CPP-induced increase in FM-fetuin-A. Treatment with BAPTA-AM, an intracellular calcium chelating agent, also inhibited the CPP-induced increase in the FM-fetuin-A level. Secondary CPP accelerate posttranslational processing of fetuin-A in HepG2 cells.
胎球蛋白 A 是异位钙化的抑制剂，主要在肝细胞中表达，经翻译后加工（包括糖基化和磷酸化）后分泌到循环中。经免疫印迹检测，完全修饰的胎球蛋白 A（FM-fetuin-A）的分子量（MW）约为 60 kDa，远高于氨基酸序列估计的 MW。在钙化应激条件下（如晚期慢性肾脏病），胎球蛋白 A 通过形成胶体复合物来防止钙化，这些复合物被称为钙磷蛋白颗粒（CPP）。由于 CPP 在这个过程中的意义尚不清楚，我们研究了合成的次级 CPP 对 HepG2 细胞中 FM-fetuin-A 水平的影响。次级 CPP 以剂量和时间依赖的方式增加 FM-fetuin-A 的水平，但不影响胎球蛋白 A mRNA 的表达。用 O-和/或 N-糖苷酶处理会导致 FM-fetuin-A 的 60 kDa 带向低 MW 迁移。用布雷非德菌素 A（一种从内质网到高尔基体运输新合成蛋白质的抑制剂）预先孵育完全阻断了次级 CPP 诱导的 FM-fetuin-A 增加。用 BAPTA-AM（一种细胞内钙螯合剂）处理也抑制了 CPP 诱导的 FM-fetuin-A 水平增加。次级 CPP 加速了 HepG2 细胞中胎球蛋白 A 的翻译后加工。
