Author

Xuan FuFollow

Date of Award

8-9-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jun Yin

Second Advisor

Lei Li

Third Advisor

Binghe Wang

Abstract

Protein post-translational modifications (PTMs) increase the functional diversity of the proteome and over 400 different types of PTMs have been identified so far. In this thesis, we focus on glycosylation and ubiquitination, and developed new methods and tools to study these modifications.

Glycosylation is one of the most common PTMs. Accessing large numbers of structural-defined glycoconjugates, and their derivatives is essential for their structural and functional studies to understand glycan-involved biological processes. In Chapter 2, we showed general tolerance of galactosyltransferases toward uncommon donor uridine-diphosphate-galactosamine (UDP-GalN). Such a property of galactosyltransferase was harnessed to develop a two-step chemoenzymatic strategy for facile synthesis of novel N-acetylgalactosamine (GalNAc)-glycosides and derivatives. The discovery and the application of the new catalytic property of glycosyltransferases expand their capabilities in generating novel carbohydrate linkages, thus facilitating the synthesis of diverse glycans and glycoconjugates for biological studies.

Sialylation is an important type of glycosylation and involved in many biological processes. The terminal sialic acids of glycoproteins are essential communicating molecules and are recognized by many viruses as receptors for cell entry. In Chapter 3, we expressed A domains of various coronavirus, and worked with our collaborator to prepare a microarray coating with various sialic acids to screen sialoside targets of each A domain. In another direction, we developed a phage display strategy to screen A domain variants with engineered binding specificity to sialic acid. The identified A domain variants may be able to trace the evolution of coronavirus and be used as diagnostic tools to identify glycan structures on cell surface.

In Chapter 4 we developed an efficient method for linkage-specific synthesis of diUB probes enabled by UAA incorporation. We incorporated the Nε-L-thiaprolyl-L-Lys (L-ThzK) into ubiquitin for ligation with another ubiquitin at a defined Lys position. The linkage-specific diUB formed has a Dha group and it can be readily used for activity-based protein profiling to uncover E3 ubiquitin ligase and deubiquitinases of the UB code. This method provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by ubiquitination.

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