Date of Award
Doctor of Philosophy (PhD)
Dr. Peng George Wang
Dr. Binghe Wang
Dr. Jenny Yang
Dr. Jun Yin
Carbohydrates are crucial biomacromolecules found in organisms across all domains of life and play pivotal roles in biological processes. To better understand the biological function of glycans, structurally-defined glycopeptides/glycoproteins and glycans are highly demanded. In this dissertation, the study has been focusing on acquiring homogenous glycopeptides/glycoproteins and glycans which are good candidates for decoding their function.
Chapter 1 provides a brief introduction of function of glycosylation for proteins, significance of glycosylation for therapeutic protein and artificial glycosylation approaches; glycan synthetic approaches and glycan microarray and an overview of synthesis of oligosaccharides in large scale in vitro and in vivo.
Chapter 2 describes two techniques for synthesis of glycopeptide/glycoprotein. (1) A facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. (2) Peptides can be GlcNAcylated at one or two natural N-glycosylation sites via two-step enzymatic reactions catalyzed by an evolved N-glycosyltransferase (ApNGT Q469A) and a glucosamine N-acetyltransferase (GlmA), respectively. Then, the resulting GlcNAc-peptides were further modified by an endo-β-N-acetylglucosaminidase M mutant (EndoM N175Q) to generate glycopeptides carrying complex-type N-glycans.
In Chapter 3, two focused glycan libraries were synthesized which were N-glycans containing tandem epitopes and a new class of asymmetric N-glycans. (1) A focused library of 36 biantennary asparagine (Asn)-linked glycans with some presenting tandem glycan epitopes was synthesized and glycan microarray study showed that the internal epitopes and/or modifications of terminal epitopes influence the binding of glycans. (2) A class of asymmetric N-glycans was synthesized by coupling glycan-oxazoline and N-glycans. Branch-biased binding and spacial inhibition caused by bulky group on the other branch were observed in glycan protein interactions involving lectins and these glycans.
In Chapter 4, a microbial glycosyltrasferases Hp1,3FTα66 were readily expressed in Escherichia coli (E. coli) was biochemically characterized and applied as a diverse catalyst in the facile synthesis of fucosylated human milk oligosaccharides. In addition, multi-gram scale of fucosylated human milk oligosaccharides, lacto-N-fucopentaose (LNFP) III and lacto-N-neofucopentaose (LNnFP) V were synthesized by three major combined engineered E. coli strains.
Wu, Zhigang, "Understanding Function of Carbohydrates by Synthesis of Structurally-defined Glycopeptides/Glycoproteins and Glycans." Dissertation, Georgia State University, 2017.
Available for download on Sunday, November 17, 2019