The endoplasmic reticulum protein SEC22B interacts with NBEAL2 and is required for megakaryocyte α-granule biogenesis

Abstract

Studies of inherited platelet disorders have provided many insights into platelet development and function. Loss of function of neurobeachin-like 2 (NBEAL2) causes Gray Platelet Syndrome (GPS), where the absence of platelet α-granules indicates NBEAL2 is required for their production by precursor megakaryocytes. The endoplasmic reticulum is a dynamic network that interacts with numerous intracellular vesicles and organelles and plays key roles in their development. The megakaryocyte endoplasmic reticulum is very extensive, and in this study we investigated its role in the biogenesis of α-granules by focusing on the membrane-resident trafficking protein SEC22B. Co-immunoprecipitation experiments using tagged proteins expressed in human HEK293 and megakaryocytic imMKCL cells established binding of NBEAL2 with SEC22B, and demonstrated that NBEAL2 can simultaneously bind SEC22B and P-selectin. NBEAL2-SEC22B binding was also observed for endogenous proteins in human megakaryocytes using co-immunoprecipitation, and immunofluorescence microscopy detected substantial overlap. SEC22B binding was localized to a region of NBEAL2 spanning amino acids 1798-1903, where two GPS-associated missense variants have been reported: E1833K and R1839C. NBEAL2 containing either variant did not bind SEC22B co-expressed in HEK292 cells. CRISPR/Cas9 mediated knockout of SEC22B in imMKCL cells resulted in decreased NBEAL2, but not vice versa. Loss of either SEC22B or NBEAL2 expression also resulted in failure of α-granule production and reduced granule proteins in imMKCL cells. We conclude that SEC22B is required for α-granule biogenesis in megakaryocytes, and that interactions with SEC22B and P-selectin facilitate the essential role of NBEAL2 in granule development and cargo stability.

Gray platelet syndrome: NBEAL2 mutations are associated with pathology beyond megakaryocyte and platelet function defects

“…In the absence of NBEAL2 α‐granule cargo is externalized via RAB11 mediated recycling endosomes 34 . Unexpectedly, two GPS missense variants not localized to a known functional domain of NBEAL2 were found to be required for interacting with SEC22B and thus α‐granule formation 39 …”

“…34 Unexpectedly, two GPS missense variants not localized to a known functional domain of NBEAL2 were found to be required for interacting with SEC22B and thus α-granule formation. 39 More recently, studies of NBEAL2-null mice noted reduced electron-dense granules in neutrophils, accompanied by a significant reduction in granule contents across all granule subsets, and decreased granule cargo release when stimulated. 43 Natural killer (NK) cells also showed reduced degranulation.…”

“…How these three NBEAL2‐interacting proteins facilitate MK α‐granule development remains to be determined. Our recent studies using mouse and human MKs, and human‐derived imMKCL cells, identified a tripartite interaction among NBEAL2, P‐selectin, 34 and the endoplasmic reticulum membrane protein SEC22B, in which NBEAL2 is the crucial link 39 . In the absence of NBEAL2 α‐granule cargo is externalized via RAB11 mediated recycling endosomes 34 .…”

“…Our recent studies using mouse and human MKs, and human-derived imMKCL cells, identified a tripartite interaction among NBEAL2, P-selectin, 34 and the endoplasmic reticulum membrane protein SEC22B, in which NBEAL2 is the crucial link. 39 In the absence of NBEAL2 α-granule cargo is externalized via RAB11 mediated recycling endosomes. 34 Unexpectedly, two GPS missense variants not localized to a known functional domain of NBEAL2 were found to be required for interacting with SEC22B and thus α-granule formation.…”

Neutrophil specific granule and NETosis defects in gray platelet syndrome

“…[4][5][6] In a recent study, NBEAL2 was found to simultaneously bind P-selectin and membrane-resident trafficking protein SEC22B, where these interactions facilitate the suggested essential role of NBEAL2 in granule development and cargo stability. 7 Furthermore, in a study using NBEAL2-Tag protein constructs in HEK cells or CHRFs (a megakaryoblastic cell line), NBEAL2 was found to be primarily localized in the cytoplasm. 8 In addition, proteins like Dock7, Sec16a, and Vac14, which are suggested to function in signaling, were found to be interactors of NBEAL2.…”