NBEAL2 (Neurobeachin-Like 2) Is Required for Retention of Cargo Proteins by α-Granules During Their Production by Megakaryocytes

Abstract

Objective- Human and mouse megakaryocytes lacking NBEAL2 (neurobeachin-like 2) produce platelets where α-granules lack protein cargo. This cargo is mostly megakaryocyte-synthesized, but some proteins, including FGN (fibrinogen), are endocytosed. In this study, we examined the trafficking of both types of cargo within primary megakaryocytes cultured from normal and NBEAL2-null mice, to determine the role of NBEAL2 in α-granule maturation. We also examined the interaction of NBEAL2 with the granule-associated protein P-selectin in human megakaryocytes and platelets. Approach and Results- Fluorescence microscopy was used to compare uptake of labeled FGN by normal and NBEAL2-null mouse megakaryocytes, which was similar in both. NBEAL2-null cells, however, showed decreased FGN retention, and studies with biotinylated protein showed rapid loss rather than increased degradation. Intracellular tracking via fluorescence microscopy revealed that in normal megakaryocytes, endocytosed FGN sequentially associated with compartments expressing RAB5 (Ras-related protein in brain 5), RAB7 (Ras-related protein in brain 7), and P-selectin, where it was retained. A similar initial pattern was observed in NBEAL2-null megakaryocytes, but then FGN passed from the P-selectin compartment to RAB11 (Ras-related protein in brain 11)-associated endosomes before release. Megakaryocyte-synthesized VWF (Von Willebrand factor) was observed to follow the same route out of NBEAL2-null cells. Immunofluorescence microscopy revealed intracellular colocalization of NBEAL2 with P-selectin in human megakaryocytes, proplatelets, and platelets. Native NBEAL2 and P-selectin were coimmunoprecipitated from platelets and megakaryocytes. Conclusions- NBEAL2 is not required for FGN uptake by megakaryocytes. NBEAL2 is required for the retention of both endocytosed and megakaryocyte-synthesized proteins by maturing α-granules, and possibly by platelet-borne granules. This function may involve interaction of NBEAL2 with P-selectin.

Keywords: P-selectin; Von Willebrand factor; endosomes; fibrinogen; megakaryocytes.