The Glycosylation Shaped Body Of Fc¦Ár Functional Effects

This thesis contains two parts. PartⅠstudied effects of FcαR glycosylation on its binding to IgA. PartⅡstudied expression and localization of FcαR splicing isoforms.PartⅠFcαR (CD89) is the Fc receptor for IgA and plays important roles in IgA mediated immune responses. It is a heavily glycosylated protein with six potential N-linked glycosylation sites. Previous reports showed that abnormal glycosylation of FcαR was involved in some diseases, including HIV infection, alcoholic liver cirrhosis and IgA nephropathy. In this study, we examined the effects of N-glycosylation on interaction between FcαR and IgA. We found that depletion of N-glycosylation of FcαR transfected in CHO cells by tunicamycin resulted in increased IgA binding. To identify which glycosylation site is responsible for increased IgA binding, we performed site-directed mutagenesis at each N-linked glycosylation site by changing asparagine to glutamine. Flow cytometry analysis of IgA binding to CHO cells transfected with mutated FcαR showed that deglycosylation of FcαR at individual N44, N120, N156, N165 or N177 site did not affect IgA binding but deglycosylation at N58 resulted in marked increase of IgA binding. Similar result was shown for N58Q-FcαR transfected RBL2H3, a rat basophilic leukemia cell line. Furthermore, increased IgA binding was also observed on desialylated FcαR after neuraminidase treatment and desialylation of N58 contributed most to the increased IgA binding. These data demonstrated that glycosylation at N58 site influenced FcαR binding to IgA.PartⅡFcαR (named as A1), the Fc receptor for IgA, is essential for IgA mediated immune responses. Previous studies identified two fcαr splice transcripts,△66EC2 (named as A2) and△EC2 (named as A3). The A2 transcript lacks 66 nucleotides proximal of the membrane and the A3 transcript lacks the entire immunoglobulin-like extracellular domain 2 (EC2). The corresponding mRNA transcripts have been detected in human myeloid cells by others. In this study, we investigated the expression and localization of FcaR protein isoforms in transfected CHO, COS7 and RBL2H3 cells. Compared to Al, A2 and A3 were absent from cell surface and primarily localized in the cytoplasm. To identify which amino acid (s) was responsible for membrane surface expression, we performed various deletion and substitution mutations by site-directed mutagenesis. Our results demonstrated that deletion mutations of FcaR except for deletion at Thr216 were either unable to be expressed on cell surface or expressed at a severely reduced level at cell surface. In comparison, substitution mutations of FcaR could be expressed on cell surface at a normal level. In addition, we failed to detect any A2 and A3 protein in CHO cells by Western Blot, which might be due to misfolded proteins in ER. Therefore, integrity of extracellular region of FcaR may be important in stable expression of the receptor.