Developing chemoenzymatic tools for studying cell-surface glycans

Abstract

Glycosylation is a posttranslational modification, not under direct genetic control. The combinatorial action of many glycosyltransferases in competing biosynthetic pathways renders cellular glycans difficult to predict from genetic information alone. It is still challenging to monitor the biosynthesis and the spatiotemporal distribution of glycans in living systems, and many questions remain unanswered. For example, 1) How long does it take for a monosaccharide to be imported and incorporated into cell-surface glycoconjugates? 2) How can we specifically detect higher-order glycans (e.g., disaccharides or oligosaccharides) on the surface of living cells? 3) How can we profile cellular proteins that are modified with a specific glycan structure? In this thesis, I developed new chemical tools, which are complementary to the traditional genetic and molecular biological methods, to answer these questions.;In the first part of thesis, I discovered that an electron-donating picolyl azide boosted the efficiency of the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), a prototypical example of bioorthogonal click chemistry, 20-38 fold in living systems with no apparent toxicity. By combining this azide and BTTPS, a tris(triazolylmethyl)amine-based ligand for Cu(I), I was able to detect newly synthesized cell-surface glycans by flow cytometry using as low as 1 nM of a metabolic precursor. This supersensitive chemistry enabled us to monitor dynamic glycan biosynthesis in mammalian cells and in early zebrafish embryogenesis. In live mammalian cells, I discovered that it takes approximately 30-45 minutes for a monosaccharide building block to be metabolized and incorporated into cell-surface glycoconjugates. In zebrafish embryos, the labeled glycans could be detected at as early as the two-cell stage.;In the second part of my thesis, I developed a chemoenzymatic approach for labeling cell-surface polysaccharides bearing N-acetyllactosamine (LacNAc; Ga1beta1,4G1cNAc). Many mammalian glycans associated with signaling receptors contain terminal or penultimate LacNAc, which functions in the regulation of growth factor signaling. My labeling method mimics the natural glycan biosynthetic pathway, utilizing a recombinant H. pylori a(1,3)fucosyltransferase to transfer a C-6 azide- or alkyne-tagged fucose residue to the 3-OH of GlcNAc of the LacNAc disaccharide. The tag may then be selectively derivatized with imaging probes via bioorthogonal click chemistry. I used this strategy to detect LacNAc-bearing glycans on the surface of live mammalian cells, and characterized the specificity of labeling using Chinese hamster ovary (CHO) cell glycosylation mutants. Ex vivo I discovered that spleen lymphocytes with an activated/memory phenotype exhibited higher LacNAc levels compared to their naive counterparts. This method also allowed, for the first time, noninvasive imaging of LacNAc in glycans of zebrafish embryos at late gastrula and tissue segmentation stages. Therefore, this chemoenzymatic method serves as a general approach for the detection of LacNAc-containing glycans in living systems, and may be used to reveal changes in LacNAc status that occur during cellular differentiation.;Bioorthogonal chemical tags (e.g., alkyne) introduced into target glycans either metabolically or enzymatically enable covalent ligation with "clickable" biotin probes for protein enrichment. However, elution of these glycoprotein targets from solid supports remains challenging due to the high affinity of biotin for streptavidin. In the third part of this thesis, I developed a new class of cleavable linker based on automatically synthesized, single-stranded DNAs. I incorporated a DNA oligo into an azide-functionalized biotin (biotin-DNA-N 3) and used the probe to enrich for alkyne-tagged glycoproteins from mammalian cell lysates. Highly efficient and selective release of the captured proteins from streptavidin agarose resins was achieved using DNase treatment under very mild conditions. A total of 36 sialylated glycoproteins were identified from the lysates of HL60 cells, an acute human promyeloid leukemia cell line. These sialylated glycoproteins were involved in many different biological processes ranging from glycan biosynthesis to cell adhesion events. (Abstract shortened by UMI.).