RESUMEN
Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.
Asunto(s)
Virus Hendra , Polisacáridos/química , Proteínas del Envoltorio Viral/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cristalografía por Rayos X , Ensayo de Cambio de Movilidad Electroforética , Glicosilación , Células HEK293 , Células HeLa , Humanos , Lectinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Estructura Cuaternaria de Proteína , Receptor EphB2/química , Proteínas Recombinantes/química , Análisis de Secuencia de ProteínaRESUMEN
Retinoids are potent inducers of cell cycle arrest and differentiation of numerous cell types, notably granulocytes. However the mechanisms by which retinoids mediate cell cycle arrest during differentiation remain unclear. We have used myeloid differentiation to characterize the molecular pathways that couple cell cycle withdrawal to terminal differentiation. Using primary cells from mice deficient for either the cyclin-dependent kinase inhibitor (CDKi) p27(Kip1), the Myc antagonist Mad1, or both Mad1 and p27(Kip1), we observed that signals mediated through retinoic acid receptor alpha (RAR alpha), but not RAR beta or gamma, required both Mad1 and p27(Kip1) to induce cell cycle arrest and to accelerate terminal differentiation of granulocytes. Although RAR alpha did not directly regulate Mad1 or p27(Kip1), the RAR alpha target gene C/EBP epsilon directly regulated transcription of Mad1. Induction of C/EBP epsilon activity in granulocytic cells led to rapid induction of Mad1 protein and transcript, with direct binding of C/EBP epsilon to the Mad1 promoter demonstrated through chromatin immunoprecipitation assay. These data demonstrate that cell cycle arrest in response to RAR alpha specifically requires Mad1 and p27(Kip1) and that Mad1 is transcriptionally activated by CCAAT/enhancer-binding protein epsilon (C/EBP epsilon). Moreover, these data demonstrate selectivity among the RARs for cell cycle arrest pathways and provide a direct mechanism to link differentiation induction and regulation of the Myc antagonist Mad1.