El Hazzouri Salime, Al-Rifai Rawya, Surges Nicole, Rath Melanie, Singer Heike, Oldenburg Johannes, El-Maarri Osman
Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, 53127 Bonn, Germany.
Int J Mol Sci. 2025 Jul 1;26(13):6349. doi: 10.3390/ijms26136349.
Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP, CANX, and CALR cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the -Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies.
凝血因子VIII（FVIII）与内质网（ER）伴侣钙联蛋白（CANX）和钙网蛋白（CALR）相互作用，并与ER-高尔基体中间区室（ERGIC）转运蛋白、凝集素、甘露糖结合蛋白1（LMAN1）和多种凝血缺陷蛋白2（MCFD2）相互作用。我们之前报道过，γ-氨基丁酸受体相关蛋白（GABARAPs）也会影响FVIII的分泌。在此，我们使用上述伴侣蛋白以及在表达FVIII的HEK293细胞中的GABARAP蛋白的单基因和双基因CRISPR/Cas9敲除（KO）模型，进一步研究了FVIII的细胞内动力学。通过布雷菲德菌素A（BFA）、氯喹（CQ）、一种Rab7抑制剂来操纵细胞途径，并使其经历葡萄糖饥饿。在细胞处理之前和之后，通过两阶段显色测定法和免疫荧光（IF）显微镜评估每种基因敲除对FVIII分泌和细胞器分布的影响。使用这些方法，我们首先观察到每种研究蛋白对FVIII运输的不同影响。值得注意的是，细胞内定位模式显示，在基础条件和处理条件下，FVIII表型在GABARAP、CANX和CALR细胞中共同聚集，这一观察结果在它们各自的双基因敲除组合中也得到了体现。此外，内膜系统的其他成分明显参与其中，特别是在高尔基体空间，这通过FVIII与脑中类Ras蛋白（Rab8和Rab7）以及囊泡相关膜蛋白（VAMP8）的共定位得以体现，同时还观察到所选细胞处理对FVIII表型的影响。这些结果加深了我们对调节FVIII的分子机制的理解，并为新的研究视角铺平了道路，这些视角可能会进一步应用于FVIII替代、细胞和基因治疗。
