The Mechanism Of Vps33b Involved In Platelet ?-granule's Generation

ARC syndrome is an autosomal recessive inherited disorder,which was characterized with arthrogryposis,renal dysfunction and cholestasis syndrome and a common feature of lack of ?-granules in the patients' platelets.Vacuolar protein sorting protein 33b(Vps33b)mutations were investigated as one of the molecular mechanism of this disease.So we speculate that Vps33 b maybe be involved in the process of alpha-granule formation.In this study,we use HEK293 T and Meg01 cell line to find the combination partner with Vps33 b in vitro.HA-tagged Vps33 b and different truncate Vps33 b fusion proteins were obtained in HEK293 T.Vps33b association with Vps16 b,?-tubulin and Sec22 b was identified by co-immunoprecipitation,mass spectra and immunoblotting in Human embryonic kidney HEK293 T cells and confirmed in Meg01 cells by immunoprecipitation using endogenous Vps33 b antibody.Also,pull-down experiments revealed that Vps16 b bound to intact Vps33b;in contrast,?-tubulin and Sec22 b interacted with the sec1-like domains of Vps33 b separately.The immunofluorescence staining revealed that Vps33 b complex distributed as filaments along the proplatelet elongation in differentiated megakaryoblast Meg01 cells.Furthermore,von Willebrand factor(vWF)positive vesicles obviously spread along the Vps33 b complex in the differentiated MEG01 cells.Conditional Vps33 b knockout mice were developed to investigate the function(s)of Vps33 b in platelet ?-granule formation.We found that early embryonic deletion of Vps33 b was lethal.PF4-Cre driven megakaryocytic Vps33 b gene deletion significantly eliminated Vps33 b expression in platelets,but had no effect on platelet ?-granule formation and protein content.Tamoxifen induced hematopoietic stem cell(HSC)specific Vps33 b deletion completely depleted Vps33 b in platelets and caused the absence of ?-granules and increased numbers of vacuoles in platelets.Transportation of the vWF positive vesicles to proplatelets was arrested in the differentiated primary Vps33 b deficient megakaryocyte,suggesting that Vps33 b complex regulates ?-granule formation by the incorporation of thevWF-positive vesicles into the proplatelets.This study provides new insight into the biogenesis of ?-granules.