The Mechanism Research Of FUT8's Impact On Early B Cell Differentiation And Development

Glycosylation is one of the most common type of post-translational modification of proteins.Half of all known proteins are found glycosylated.In the process of protein glycosylation,glycosyltransferases are enzymes that transfer mono-or oligosaccharides from donor molecules to growing oligosaccharide chains or proteins.Core fucosyltransferase(FUT8)is a key glycotransferase,which catalyzes the transfer of a fucose residue from GDP-fucose to the innermost N-acetylglucosamine(Glc NAc)residue of hybrid and complex N-glycans via alpha1,6-linkage(core fucosylation)in the Golgi apparatus in mammals.The core fucosylation on N-glycans of glycoprotein is only catalyzed by FUT8,which is able to regulate the protein-protein interaction and cell-cell interaction with the change of thier core fucosylation.Previous studies demonstrate that FUT8 knock out(FUT8-/-)mice have higher mortality than the wild type,show emphysema-like phenotype and growth retardation.Most key proteins involved in the innate and addaptive immune responses are glycoproteins.In previous studies,it is found that the N46 glycosylation in the CH1 domain of pre-BCR is essential for pre-B cell development.Also,lack of fucose on IgG1 N-Linked oligosaccharide improves its binding to Fc?RIII and antibody-dependent cellular toxicity(ADCC)by 50-100 fold.In this study,we aims at exploring FUT8's role in humoral immunity.Flow cytometry analysis revealed that the population of CD45R-CD19-cells was markedly reduced in FUT8-/-BM.Also,the CD19-CD43-(pre-B enriched)and CD19-Ig M-(immature B enriched)populations were significantly decreased in FUT8-/-BM,whereas the CD19-CD43-(pro-B enriched)population was sustained.To investigate the mechanism involved in FUT8 regulation of B cell generation,we inserted a FUT8 si RNA into a retrovirus expression vector and established FUT8-knockdown 70Z/3 cells(70Z/3-KD).Reintroduction of the FUT8 gene into70Z/3-KD cells resulted in recovery of FUT8 m RNA expression in 70Z/3-KD-re cells.We found that loss of core fucosylation of ?HC impaired the interaction between ?HC and ?5.The formation of pre-BCR on the cell surface was down-regulated in 70Z/3-KD,and was restored in 70Z/3-KD-re cells.It is also noteworthy that Ig? phosphorylation was attenuated in 70Z/3-KD cells and the reintroduction of the FUT8 gene to 70Z/3-KD cells potently rescued pre-BCR-mediated signaling.In 70Z/3-KD cells,the frequency of clonable pre-B cell progenitors was reduced,compared with mock cells and 70Z/3-KD-re cells.In 12-week-old FUT8-/-mice sera,the levels of IgG1,Ig G2 a,Ig G2 b,Ig A,Ig G3,and Ig M were significantly reduced.Also the combined down-regulation of those genes: Ig?,Ig?,Ebf1,and Tcfe2 a,which promote the activation of B cells,were down-regulated in FUT8-/-B progenitors.This present study is the first to clearly demonstrate that core fucosylation of ?HC influences the pre-BCR assembly so as to regulate pre-BCR signaling and pre-B cell proliferation using the models of FUT8-/-and FUT8+/+ mice and 70Z/3,70Z/3-KD and 70Z/3-KD-re cells.