OC 07.1 - Role of GABARAP protein in the intracellular distribution of FVIII

Saturday, July 9, 2022

1:00 PM – 1:15 PM

Location: ExCel Center, ICC Capital Suite Room 2-4

Presentation Format: In-Person

Presenting Author(s)

Salime El-Hazzouri

PhD candidate
Uniklinikum Bonn
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

HS

Heike Singer

Dr. rer. nat
University Clinic Bonn, Institue of experimental Haematology and Transfusion medicine
Bonn, Nordrhein-Westfalen, Germany
MI

Mira Ibrahim

PhD candidate
Uniklinikum Bonn
Bonn, Nordrhein-Westfalen, Germany
NN

Nicole Nüsgen

Medizinisch-technische Assistentin
Uniklinikum Bonn
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
OE

Osman EL-Maarri

Priv.-Doz. Dr.
Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
bonn, Nordrhein-Westfalen, Germany

Background: We previously showed intracellular interaction between GABARAP (an ATG8 protein) and FVIII; this finding suggested the involvement of the autophagy machinery in FVIII intracellular trafficking. However, the precise role of GABARAP on the intracellular distribution and transportation of FVIII remain to be elucidated.

Aims: To investigate the role of GABARAP and its effect on intracellular distribution of FVIII.

Methods: HEK293 cell lines harboring single CRISPR/Cas9 KOs in CANX, CALR, LMAN1, MCFD2 and GABARAP were subjected to immunofluorescent staining. Co-localization data and correlation analysis of FVIII with different cellular markers of different cell compartments (ER, ERGIC, Golgi, Autophagosome, Lysosome, Endosome and Proteasome) was clustered, evaluated and analyzed.

Results: We observed in the GABARAP-KO, relatively to wild type, a decrease in FVIII colocalization in ER (CALX, p< 0.0001, CALR, p=0.0086), Trans-Golgi (TGN46, p=0.0031), Autophagosomes (LC3, p< 0.0001), Lysosomes (LAMP1, p=0.0603) and proteasomes (26S p=0.0004); while a relative increase was observed at the ERGIC compartment (LMAN1, MCFD2), endosomal markers (RAB7, p< 0.0001, RAB11, p=0.24, VAMP8 p=0.0055), Furin (p=0.0003) and GABARAPL1 (p < 0.0001). The KO of Calnexin and Calreticulin showed relatively similar FVIII distribution to HEK28 while LMAN1 and MCFD2 KOs where largely characterized by a drop of FVIII co-localization with all markers except ER and GABARAPs markers.

Conclusion(s): Our detailed correlation analysis show a significant impact of GABARAP protein on the intracellular distribution of FVIII especially between autophagy/lysosomal vesicles and endosomal compartments