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

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Many protein-linked oligosaccharides are believed to impart biological specificities to the molecules. The knowledge of detailed structural characteristics of oligosaccharides is essential for understanding their functions. In order to develop methodology for characterization of oligosaccharides of glycoproteins, and to compare glycosylation patterns of different immunoglobulins, oligosaccharides of IgM from two cell lines, MOPC 104E and PC 700, were analyzed. Homogeneous preparations of glycopeptides carrying individual glycosylation sites of the heavy chain were obtained from the two IgM's. The oligosaccharides of these glycopeptides were prepared by hydrazinolysis, and fractionated by HPLC under conditions that resolve oligosaccharides by charge and size, and by affinity chromatography on Concavalin A-Sepharose. Structures of some of these oligosaccharides were determined by 400 MHz NMR spectroscopy. HPLC fractionation by charge resolved oligosaccharides with zero, one, two, and three sialic acids. As indicated by HPLC analyses, oligosaccharides at all the glycosylation sites of both the IgM's were highly heterogeneous. A comparative study on oligosaccharides prepared by peptide-N-glycosidase F digestion of glycopeptides showed a similar degree of heterogeneity. Therefore, it was concluded that the observed heterogeneity of oligosaccharides was not an artefact caused by hydrazinolysis. Major differences between the glycosylation patterns of the two IgM's were evident from analyses of the oligosaccharides by both chromatographic techniques and NMR spectroscopy. MOPC IgM contained a high proportion of sialylated oligosaccharides when compared to PC IgM. Furthermore, the major oligosaccharide structures of MOPC IgM were of triantennary type whereas PC IgM contained biantennary oligosaccharides as its major species. In both the IgM's, a decreased trend of oligosaccharide processing was observed from the N-terminus to the C-terminus.

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.

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