The goal of this project is to define the functional role of carbohydrate in the antibody molecule. We will address two fundamental questions: Firstly, we will determine how the structure of the carbohydrate attached to the variable and/or the constant region of the antibody molecule influences its functional properties. Secondly, we will attach carbohydrate to different positions in the variable region to define the mechanism by which the presence of V region carbohydrate influence antigen- antibody interactions; we will also determine if V region carbohydrate facilitates the purification of antibodies using lectins, and influences the biochemical properties of the antibody. To investigate the role of the variable region carbohydrate we will use anti-dextran antibodies in which it has been demonstrated that the presence of carbohydrate at residue 58 in CDR2 of the VH region leads to increased affinity for antigen and is available for lectin binding. We will place carbohydrate at different positions in the variable region using site directed mutagenesis coupled with transfection and expression into murine myeloma cell lines or Chinese hamster ovary cells. Carbohydrate will be positioned at exposed sites proximal and distal to residue 58. It will be ascertained whether the carbohydrate addition site is utilized, the effect of carbohydrate on antigen binding, and accessibility of the carbohydrate to binding by lectins. To assess the influence of carbohydrate structure on antibody function, we will use gene transfection techniques to produce antibodies in Chinese hamster ovary cells with different mutations in the N-linked glycosylation pathway. The antibodies produced will be evaluated with respect to their ability to bind their specific antigen, with respect to their effector functions such as complement activation and receptor binding and with respect to their biochemical and biologic properties such as serum half- life, ability to form immune complexes, and tendency to self-aggregate. These studies will define the influence of the carbohydrate structure on the functional properties of the antibody molecules. It will contribute to our understanding of how antibodies function and how abnormal function can contribute to disease. In addition, as more and more antibody molecules are produced using expression systems, it is important that we understand what carbohydrate structures are required for different functional properties so that the correct expression system can be utilized. Also, since some expression systems can change their glycosylation patterns, it is important that we understand the impact of alternated carbohydrate structures on the functional properties of the antibody molecule so that proper quality controls can be maintained during production.
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