The role of the GABARAP Protein in the Intracellular Trafficking of FVIII

Abstract

FVIII is a glycoprotein secreted by liver sinusoidal endothelial cells (LSECs) that plays an essential role in the intrinsic pathway of the coagulation cascade. Intracellularly, FVIII biosynthesis and processing are supported by known interactions with the Endoplasmic Reticulum chaperones, Calnexin (CANX), Calreticulin (CALR), and BiP, as well as ERGIC transport proteins, LMAN1 and MCFD2. However, compared to other heavily glycosylated proteins, like FV and vWF, FVIII transport through the ERGIC and its further trafficking appear limited. Despite its clinical importance, the overall intracellular lifecycle of FVIII remains poorly understood. Our team previously identified a novel FVIII secretion-modulating protein, "GABARAP" (Gamma-Aminobutyric Acid Type A Receptor-Associated Protein), using a yeast two-hybrid screen. Subsequent studies employing CRISPR/Cas9-knockouts and siRNA-based-knockdowns revealed the involvement of GABARAP in FVIII secretion. In this thesis, three key aspects were investigated: 1) the interaction between GABARAP and FVIII, 2) the intracellular localization of FVIII and GABARAP across subcellular compartments, and 3) the effects of CRISPR/Cas9-KOs of both FVIII-associated control proteins (Calnexin, Calreticulin, LMAN1, and MCFD2) and ATG8-family proteins (GABARAP, GABARAPL1, and GABARAPL2) on FVIII secretion and intracellular trafficking, under basal (untreated) and experimentally manipulated conditions. Here, it is demonstrated that the interaction between GABARAP and FVIII is weak, transient or potentially regulated via an intermediary protein. Despite this, GABARAP^KO re-distributed FVIII intracellularly in a manner similar to patterns observed in CANX^KO and CALR^KO clones (ER-chaperone-KOs), contrasting with patterns seen in GABARAPL1^KO and GABARAPL2^KO (GABARAP homologs). Consistently, pairwise double-KO combinations, including CANX, CALR and GABARAP showed as well comparable impacts on FVIII compartmental localization. Additionally, GABARAP localized to compartments involved in FVIII processing and appeared to also contribute to the maintenance of organized Golgi morphology in HEK cells. Finally, cell treatment with Brefeldin A, glucose starvation, chloroquine, and Rab7 inhibition—agents known to affect ER-to-Golgi transport and specific pathways of the endomembrane system, altered FVIII secretion and intracellular trafficking. These perturbations further aligned the effects of GABARAP^KO with those of CANX^KO and CALR^KO. Collectively, these findings provide deeper insights into FVIII intracellular processing in the endomembrane system and related vesicles in addition to the potential functional role of GABARAP in these mechanisms.