No description available.

No description available.

No description available.

No description available.

Glycoproteins play a key role in cellular growth and differentiation. In order to study glycoprotein biosynthesis and processing, we have chosen the murine Immunoglobulin M (IgM) system as a model. Our system utilizes hybridoma, lymphoma and plasmacytoma cell lines which synthesize intracellular, membrane-bound and secreted IgM. Each type of IgM is synthesized during a specific phase of B-cell differentiation. We have examined the kinetics of IgM synthesis and processing in cells at each developmental stage. The rate of synthesis of membrane-bound and soluble IgM are different. Characteristic rates for membrane versus soluble IgM may be dependent on the extent of oligosaccharide processing. The membrane-bound IgM contains more high-mannose oligosaccharide than does the secreted product. In addition, we have begun to determine how protein structural requirements can affect final glycosylation patterns on the glycoprotein. Two cell lines were studied which secreted smaller than normal IgM heavy chains in tissue culture. One cell line studied (208) contains one glycosylation site, while another (562) retains three sites on the molecule synthesized in tissue culture. Studies performed on these cell lines in tissue culture indicate greater processing of the oligosaccharides on these mutant IgM molecules when compared to the parental cell line (PC700). Studies on the 208 IgM molecules synthesized in the mouse and purified from ascites fluid confirm these results. Upon injection into the mouse, the 562 cell line reverts to produce protein and carbohydrate structures characteristic of the parental cell line. Studies on the 562 protein purified from ascites fluid illustrate the need for more precisely defined cell lines and genetic engineering for the study of altered protein structures.

In order to understand glycoprotein biosynthesis and processing, we have studied the glycosylation and intracellular assembly kinetics of murine IgM which are expressed functionally only at specific stages of B-cell differentiation by the corresponding tumor cell lines. We have shown that the majority of carbohydrate chains on intracellular IgM contain predominantly Man₈GlcNac₂ rate limiting step in the carbohydrate processing is the transport from the RER to the Golgi apparatus. We made comparisons of carbohydrate structures on secretory and membrane-bound u chains produced by different cell lines. Our results show that the carbohydrates on WEHI231 membrane-bound IgM are less processed, and the processing at individual glycosylation sites is different for IgMs produced by plasmacytoma (MOPC104E) and hybridoma (MPC11xW279.2) cell lines. In addition, we also show that the glycosylation and processing are dramatically altered by lowering the glucose concentration in the cell culture medium. These results are a beginning for our understanding of the influence of the polypeptide on the final glycosylation patterns of a glycoprotein, and the genetic and environmental control over the carbohydrate processing during intracellular transport. The kinetic studies on IgM synthesis and maturation in WEHI231 as well as WEHI279.1/12 cells have led to the conclusion that membrane bound IgM and soluble IgM are segregated and processed individually even in the same cell. These differences appear to lead to the changes in carbohydrate/processing for membrane-bound and soluble IgM.

No description available.

published_or_final_version / abstract / Chemistry / Master / Master of Philosophy

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy