| Antibody,a kind of glycoprotein secreted by plasma cells and capable of recognizing antigens,is an important component of the immune system.The study of antibodies has opened up broad prospects for disease diagnosis and treatment of various diseases such as cancer and autoimmune diseases.Antibody research has played an important role in the prevention and control of major diseases.Duing to the development of antibody vaccines,various high-risk diseases such as rabies have been effectively controlled.Current studies have shown that there is a conserved N-glycosylation-modified Asn-X-Ser/Thr(X?Pro)motif in position 297 of the IgG antibody heavy chain Fc fragment,and there is an N-glycosylation modification group on asparagine.The N-glycosylation modification linked to the conserved Asn motif plays an important role in the binding of the Fc fragment to the receptor,physiological and biochemical reactions such as recognition and regulation between cells and antibodies.Aberrant glycosylation is related to certain autoimmune diseases,and this abnormal glycosylation modification is used as an early diagnostic marker.These findings suggest that glycosylation plays a key role in the biological effects of antibody Fc fragments.Our previous study found a murine-derived monoclonal antibody,15A11,directed against cartilage oligomeric matrix protein(COMP),which showed a potent activity in inducing arthritis models in experimental animals.However,the glycosylation of the Fc fragment of the 15A11 antibody and the effect of glycosylation on the stimulating effect of immune cells are not yet clear.In this study,we used different N-glycans separation and analysis methods to detect the characteristics of N-glycosylation of 15 A11,and then detected the binding of 15A11 antibody to its target protein COMP,and to detect the immune complex formed by antibody 15A11.The substance changes macrophage activation-differentiation,phagocytosis,and secretion of cytokines(TNF-?,IL-1).The results of the study are as follows:(1)The heavy chain constant region sequence and glycosylation site of 15A11 antibody were detected by liquid chromatography coupled with high resolution mass spectrometry.The 15A11 glycosylation was analyzed and the results showed that 14 glycoforms were detected in the 15A11 samples.Three different detection methods,including LC-ESI-MS/MS for glycopeptides,MALDI-TOF-MS for sugar chains,and LC-ESI-MS/MS,showed that the main type of sugar chain is fucosylated sugar chains also have some sialylation and galactosylation modifications,and devoid of high mannose type glycan.The results showed that the presence of glycosylation at 15A11 reflects species specificity of the murine antibody.The results of the analysis are corroborated with some of the results in the literature.(2)Immunohistochemistry analysis showed that the specific binding capacity of 15A11 to articular cartilage was not significantly different from two control mAb,16B5 and M2139.Macrophages stimulated by 15A11 produced obvious morphological changes,and the joint tissues produced a clear loosening tendency.The macrophages stimulated by the two control mAb,16B5 and M2139 did not produce significant changes.Results showed that 15A11 immune complex can stimulate macrophages to activate it and cause damage to the joint tissue.Results indicating that the 15A11-antigen complex has a strong role in activating macrophages via Fc fragment.(3)The results of macrophage phagocytosis showed that the 15A11 immune complex stimulated the exudation of macrophages in the peritoneal cavity.The morphology of the macrophages was changed.And 15A11 immune complexes can induce macrophages to produce significantly enhanced phagocytosis,macrophage phagocytosis index and phagocytosis rate have significantly improved.However,there was no significant increase in phagocytosis after deglycosylation of immune complexes.Elevated glycosylation was associated with this stimulatory effect.(4)Flow cytometric analysis can detect macrophage surface activation markers.Two active markers for macrophage differentiation to Ml were CD86,F4/80 molecule.15A11,16B5 immune complexes stimulated macrophage cells to express CD86+F4/80+,and the proportion of cells was 22.3%,18.2%,respectively,whereas the proportion of deglycosylated 15A11,16B5 immune complex was 11.4%,12.5%.The results showed that the ratio of double positive cells was significantly increased after stimulation with 15A11 immune complex,but the deglycosylated immune complex didn't produce significant stimulation.These data indicates that the 15A11 immune complex stimulates macrophages to induce macrophage differentiation to M1,and that glycosylation is associated with this stimulatory effect.(5)Enzyme-linked immunosorbent assay(ELISA)showed that the 15A11 immune complex induced the stronger phagocytosis and higher secretion of cytokines of macrophages.After removal of the glycosylation,we didn't find that the 15A11 immune complex produced a significant stimulating effect on macrophages.Results suggest that the stimulation of the 15A11 immune complex is related to its N-glycans.The results showed that 15A11 stimulated macrophages significantly stronger than 16B5 and M2139 immune complexes.The strong induction of experimental animal arthritis model activity of 15A11 is not related to its ability to bind to target protein,but comes from the combination of its Fc segment and effector cell FcR.The glycosylation of the 15A11 mAb Fe fragment has the specificity of fucose and galactose modification.Further studies revealed that the antibody's stimulatory effect on macrophages after the loss of the sugar chain was significantly reduced.The results showed that the glycosylation of the Fc fragment of the 15A11 monoclonal antibody has certain specificity,and the specific glycosylation affects the activity of the antibody. |
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