.6 A8 Gene Function Studies: Protein Glycosylation And Lymphocyte Death

Majority of proteins in organisms are existed in a modulation of glycoprotein, also named glycoconjugates. The glycan in the glycoproteins is not only related to the biological function of glycoproteins, but also has important biological significance itself. As an information molecule, the glycans plays an important role in cell-cell, cell-matrix, cell-molecule and molecule-molecule interaction, and in cell activation, proliferation, differentiation and death. The modulation of glycosylation and the structure of glycans are quit complicated, which makes the studies on the rules of glycosylation, and its function and structure in difficulty. Glycosylation is catalyzed by a series of glycosyltransferases and glycosidases. α-mannosidase is an important type of glycosidase, which plays an important part in the processing and maturation of N-linked glycosylation by trimming the mannose from the glycan in glycosylation process. Several techniques have been developed for the study of the biological significance of N-linked glycosylation. The antisense technique is one of them.In our lab, a 1358 bp cDNA was isolated from a human tonsil cell λgt11 cDNA library by using monoclonal antibody 6A8 as a probe. 6A8 cDNA is highly homologous to a rat ER a-mannosidase. The isolation of 6A8 cDNA gives us an opportunity of studying the relationship between aberrant glycosylation and cell functioning. Our previous study showed that the metastasis and invasiveness of human nasopharyngeal carcinoma cell line CNE-2L2 were decreased upon transfection with an antisense 6A8 DNA when the cells were subcutaneously inoculated in nude mice.In my Ph D thesis project, I first investigated the nature of the protein encoded by 6A8 cDNA, and then constructed an adeno-associated virus vector and utilized the vector to mediate the sense or the antisense 6A8 DNA into the human lymphocytes, especially EBV-transformed B cells for studying the possible relationship between 6A8 a-mannosidase expression and cell death.Based on the fact that the receptor of Con A is the mannoses and that the function of a-mannosidase is trimming of mannoses in the oligosaccharide chains of glycoproteins, Con A binding assay was carried out on COS-7 cells and CNE-2L2 cells, with a transfection of pRc/CMV-sense 6A8 DNA or pRc/CMV-antisense 6A8 DNA. In addition, mAb 6A8 staining of the cells was also detected. The data demonstrated that with transfection of pRc/CMV-sense 6A8 DNA, the binding of Con A to the cells was decreased , but the staining of the cells with mAb 6A8 was enhanced , and vise versa for the cells with transfection of pRc/CMV-antisense 6A8 cDNA, suggesting that protein encoded by 6A8 cDNA might be a type of a-mannosidase recognized by mAb 6A8.Then I detected the 6A8 mRNA expression in various human immune cell lines...