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.

Glycoproteins are proteins that are post-translationally modified with oligosaccharides. Due to the non-template-mediated biosynthesis of glycoproteins in the nature, glycoproteins always exist as heterogeneous mixtures with different glycan structures. In order to obtain the homogeneous glycoproteins with the well-defined glycan structures for an extensive investigation of the relationship between the structure and function of glycoproteins, synthetic strategies including chemical and chemoenzymatic synthesis have been employed and achieved great success over the past years. Among these approaches, our research group has developed a novel serine/threonine ligation (STL), which involved a chemoselective ligation between a peptide with a salicylaldehyde (SAL) ester at the C-terminus and an N-terminal serine or threonine of another peptide to generate the natural Xxx-Ser/Thr linkage (Xxx represents any amino acid) at the conjugation site. STL provides more possibilities for disconnection sites for convergent protein/glycoprotein synthesis. My research has been focused on the synthesis of MUC1 glycopeptides. MUC1 is a transmemberane glycoprotein expressed on the apical surface of most normal epithelial cells at low levels but highly overexpressed on the entire membrane of human epithelial tumor cells. In the extracellular part, MUC1 contains a variable number of tandem repeat (VNTR) units which consist of twenty amino acids with five potential O-glycosylation sites. As MUC1 has been shown asa promising target for the production of immunostimulating antigens, a variety of chemical assembly strategies have been applied for the development of MUC1 glycopeptide-based anticancer vaccines with high immunogenicity and tumor selectivity, including the construction of multivalent dendrimers presenting tumor-associated MUC1 glycopeptide antigens and the incorporation of various immunoadjuvants. In my studies, I have successfully synthesized the large MUC1 VNTR glycopeptides (40-mer and 80-mer sections) possessing tumor-associated Tn antigens via one and three consecutive STL reactions. On the other hand, the cyclic MUC1 glycopeptide-BSA conjugates has been successfully constructed. We are yet to test the immunological properties of synthetic MUC1 glycopeptide oligomers and MUC1-based glycoconjugates as anticancer vaccine candidates. In addition, inspired by STL, I have developed an aspartic acid ligation, in which a C-terminal peptide-SAL ester chemoselectively reacts with an N-terminal diol group of another peptide under the same conditions as STL to form a six-membered N,O-benzylidene acetal linked intermediate. Followed by treatment with acidsand selectiveoxidation, the natural Xxx-Asplinkage(Xxx represents any amino acid) is chemoselectively generated at the conjugation site. This STL-based aspartic acid ligation has been applied in the synthesis of a series of cyclic and linear peptides. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

We have chosen the murine immunoglobulin M (IgM) as system to study glycoprotein biosynthesis and carbohydrate processing. Secreted IgM heavy chain (m) has five glycosylation sites which location and structures have been determined. m chain variable region (VH) is involved in antigen binding, while the constant region (CH) is responsible for the effector functions in which the carbohydrate plays an important role. We have determined the carbohydrate structures present at each glycosylation site of IgM produced by a hybridoma cell line (PC 700) and its derived mutants and compared them to IgM from myeloma cell MOPC 104E. PC 700 mutants secrete altered IgM. The alterations include: deletion of one or more constant domains (mutants: 128, 313, and 562) and m chain hyperglycosylation (mutants 21 and 38). Gene analysis indicated that deletions can arise from two different mechanisms. One of these involve a major gene change (mutant 128), while others come from base point mutations (mutants 313 and 562). Cells 21 and 38 did not appear to have m gene insertions. Determination of purified single glycosylation site structures show that PC 700 m chain is processed only to biantennary. Heavy chain protein fragmentation and carbohydrate studies indicate that mutants 21 and 38 alterations are due to an increase in oligosaccharide processing and reduction of unprocessed structures. There is a trend of processing going from PC 700 < 21 < 38. In addition, our results show how growth cell conditions can affect the carbohydrate processing without altering the determinants of m chain oligosaccharide structures. Studies on the IgM molecule illustrate the need for precisely define structure-function relationships. This would allow the selection of the best antibodies for studies such as those involved in immunotherapy.

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.

Call number: LD2668 .T4 1985 B37 / Master of Science

No description available.

Ph.D. (Biochemistry) / Genetically modified (GM) crops were first introduced in the 1980s for the production of medicinal products. Since then, areas designated to GM crops have expanded drastically, with the GM crops grown to enhance agricultural productivity, improve agricultural practices, and as a tool to address potential pressures that will be faced by the agricultural sector and to address the issue of food security. Currently, cultivated GM crops include cotton, maize, rapeseed and soybean, carrying agronomic traits such as herbicide tolerance and insect resistance. Following the genetic modification of crops, three possible outcomes can be anticipated: these outcomes include the GM crop produced being equivalent to its untransformed counterpart, the GM crop differing from its untransformed counterpart with several well-defined characteristics, and the GM crop differing from its untransformed counterpart with a multitude of complex characteristics. In cases where the GM crop is equivalent to the untransformed counterpart, no further testing is needed. In instances where several well-defined and characterised differences are found between the GM crop and the untransformed counterpart, safety assessments are performed targeting these differences. The assessments will determine the impact of these unintended and unexpected alterations of the intended enhancement of the GM crops. However, methods currently used to assess GM crops have been found to be lacking, since they only focus on environmental and product-specific risks. Further evidence is essential, as part of GM crop safety assessment, on the molecular characterisation of these crops. This evidence is based on the potential impact of the transformation event, integration of the transgene into the host plant, as well as unintended alterations such as altered gene expression that may occur to the host plant. These events may assist in the further detection of potential dangers of the GM crop. As a result of these highlighted gaps, a project was formulated to study the unintended genomic alterations that may occur during and following the production of a transgenic plant...