Castro-Núñez Lydia, Koornneef Johanna M, Rondaij Mariska G, Bloem Esther, van der Zwaan Carmen, Mertens Koen, Meijer Alexander B, Meems Henriet
From the Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands.
From the Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands; Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Int J Biochem Cell Biol. 2017 Aug;89:34-41. doi: 10.1016/j.biocel.2017.05.027. Epub 2017 May 27.
Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding.
低密度脂蛋白受体相关蛋白1（LRP1）参与多种配体的分解代谢，包括凝血因子VIII（FVIII）和α2巨球蛋白（α2M）。目前认为，FVIII向LRP1的转移之前会先在细胞表面进行预浓缩，通过与一种尚未确定的成分相互作用。在本研究中，我们使用共聚焦显微镜和流式细胞术，以U87MG细胞比较FVIII和α2M的内吞作用。结果表明，α2M迅速被内化，并且不竞争LRP1介导的FVIII内化。在存在受体相关蛋白（RAP）的情况下，FVIII的内吞作用未发生，但FVIII在细胞边界处仍可见为条纹状荧光模式。在存在血管性血友病因子（VWF）的情况下，在细胞上或细胞内未观察到FVIII，这表明VWF会阻断与细胞表面和LRP1的相互作用。此前针对FVIII C1结构域定向单克隆抗体KM33也观察到了相同的双重抑制作用。然而，通过将FVIII C1结构域的2091 - 2095位和2155 - 2160位残基替换为因子V（FV）的同源残基来消除KM33表位，并不损害FVIII的内吞作用。仅这些膜尖峰不足以实现细胞摄取，因为FV既未被U87MG细胞内化，也无法有效竞争FVIII的内吞作用。这些结果表明，FVIII的内吞作用是由与LRP1的相互作用驱动的，但同时涉及C1结构域中与脂质结合有关的尖峰。
