The study of cellular glycosylation presents many challenges due, in large part, to the non-template driven nature of glycan biosynthesis and their structural complexity. Chemoenzymatic glycan labeling (CEGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CEGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CEGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CEGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.
To monitor the kinetics of biological processes that take place within the minute time scale, simple and fast analytical methods are required. In this article, we present our discovery of an azide with an internal Cu(I)-chelating motif that enabled the development of the fastest protocol for Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) to date, and its application toward following the dynamic process of glycan biosynthesis. We discovered that an electron-donating picolyl azide boosted the efficiency of the ligand-accelerated CuAAC 20–38-fold in living systems with no apparent toxicity. With a combination of this azide and BTTPS, a tris(triazolylmethyl)amine-based ligand for Cu(I), we were 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 the dynamic glycan biosynthesis in mammalian cells and in early zebrafish embryogenesis. In live mammalian cells, we discovered that it takes approximately 30–45 min for a monosaccharide building block to be metabolized and incorporated into cell-surface glycoconjugates. In zebrafish embryos, the labeled glycans could be detected as early as the two-cell stage. To our knowledge, this was the first time that newly synthesized glycans were detected at the cleavage period (0.75–2 hpf) in an animal model using bioorthogonal chemistry.
Profiling specific glycans in histopathological samples is hampered by the lack of selective and sensitive tools for their detection. Here, we report on the development of chemoenzymatic histology of membrane polysaccharide (CHoMP)-based methods for the detection of O- and N-linked glycans on tissue sections via the use of sialyltransferases ST3Gal1 and ST6Gal1, respectively. Combining these two methods, we developed tandem labeling and double labeling strategies that permit the detection of unsialylated and sialylated glycans or the detection of O- and N-linked glycans on the same tissue section, respectively. We applied these methods to screen murine tissue specimens, human multiple-organ cancer arrays, and lymphoma and prostate cancer arrays. Using tandem labeling with ST6Gal1 to analyze N-glycans in a prostate cancer array, we found striking differences in expression patterns of both sialylated and unsialylated N-glycans between cancerous and healthy samples. Such differences were also observed between normal tissue from healthy donors and healthy tissue adjacent to tumors. Our double labeling technique identified significant differences in unsialylated O-glycans between B-cell and T-cell lymphomas and between B-cell lymphomas and normal adjacent lymph nodes. Remarkable differences were also detected between adjacent lymph nodes and spleen tissue samples. These new chemoenzymatic histology methods therefore provide valuable tools for the analysis of glycans in clinically relevant tissue samples.
During an acute infection, antigenic stimulation leads to activation, expansion, and differentiation of naïve CD8 + T cells, first into cytotoxic effector cells and eventually into long-lived memory cells. T cell antigen receptors (TCRs) detect antigens on antigen-presenting cells (APCs) in the form of antigenic peptides bound to major histocompatibility complex I (MHC-I)-encoded molecules and initiate TCR signal transduction network. This process is mediated by phosphorylation of many intracellular signaling proteins. Protein O-GlcNAc modification is another post-translational modification involved in this process, which often has either reciprocal or synergistic roles with phosphorylation. In this study, using a chemoenzymatic glycan labeling technique and proteomics analysis, we compared protein O-GlcNAcylation of murine effector and memory-like CD8+ T cells differentiated in vitro. By quantitative proteomics analysis, we identified 445 proteins that are significantly regulated in either effector- or memory-like T cell subsets. Furthermore, qualitative and quantitative analysis identified highly regulated protein clusters that suggest involvement of this post-translational modification in specific cellular processes. In effector-like T cells, protein O-GlcNAcylation is heavily involved in transcriptional and translational processes that drive fast effector T cells proliferation. During the formation of memory-like T cells, protein O-GlcNAcylation is involved in a more specific, perhaps more targeted regulation of transcription, mRNA processing, and translation. Significantly, O-GlcNAc plays a critical role as part of the “histone code” in both CD8+ T cells subgroups.
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