Cell-Selective Metabolic Glycan Labeling Based on Ligand-Targeted Liposomes

A cell-specific metabolic glycan labeling strategy has been developed using azidosugars encapsulated in ligand-targeted liposomes. The ligands are designed to bind specific cell-surface receptors that are only expressed or up-regulated in target cells, which mediates the intracellular delivery of azidosugars. The delivered azidosugars are metabolically incorporated into cell-surface glycans, which are then imaged via a bioorthogonal reaction

Sonic hedgehog-heat shock protein 90β axis promotes the development of nonalcoholic steatohepatitis in mice

Abstract Sonic hedgehog (SHH) and heat shock protein 90β (HSP90β) have been implicated in nonalcoholic steatohepatitis (NASH) but their molecular mechanisms of action remain elusive. We find that HSP90β is a key SHH downstream molecule for promoting NASH process. In hepatocytes, SHH reduces HSP90β ubiquitylation through deubiquitylase USP31, thus preventing HSP90β degradation and promoting hepatic lipid synthesis. HSP90β significantly increases in NASH mouse model, leading to secretion of exosomes enriched with miR-28-5p. miR-28-5p directly targetes and decreases Rap1b levels, which in turn promotes NF-κB transcriptional activity in macrophages and stimulates the expression of inflammatory factors. Genetic deletion, pharmacological inhibition of the SHH-HSP90β axis, or delivery of miR-28-5p to macrophages in the male mice liver, impairs NASH symptomatic development. Importantly, there is a markedly higher abundance of miR-28-5p in NASH patient sera. Taken together, the SHH-HSP90β-miR-28-5p axis offers promising therapeutic targets against NASH, and serum miR-28-5p may serve as a NASH diagnostic biomarker

Glycan Imaging in Intact Rat Hearts and Glycoproteomic Analysis Reveal the Upregulation of Sialylation during Cardiac Hypertrophy

In the heart, glycosylation is involved in a variety of physiological and pathological processes. Cardiac glycosylation is dynamically regulated, which remains challenging to monitor in vivo. Here we describe a chemical approach for analyzing the dynamic cardiac glycome by metabolically labeling the cardiac glycans with azidosugars in living rats. The azides, serving as a chemical reporter, are chemoselectively conjugated with fluorophores using copper-free click chemistry for glycan imaging; derivatizing azides with affinity tags allows enrichment and proteomic identification of glycosylated cardiac proteins. We demonstrated this methodology by visualization of the cardiac sialylated glycans in intact hearts and identification of more than 200 cardiac proteins modified with sialic acids. We further applied this methodology to investigate the sialylation in hypertrophic hearts. The imaging results revealed an increase of sialic acid biosynthesis upon the induction of cardiac hypertrophy. Quantitative proteomic analysis identified multiple sialylated proteins including neural cell adhesion molecule 1, T-kininogens, and alpha(2)-macroglobulin that were upregulated during hypertrophy. The methodology may be further extended to other types of glycosylation, as exemplified by the mucin-type O-linked glycosylation. Our results highlight the applications of metabolic glycan labeling coupled with bioorthogonal chemistry in probing the biosynthesis and function of cardiac glycome during pathophysiological responses

A Bioorthogonal Raman Reporter Strategy for SERS Detection of Glycans on Live Cells

National Instrumentation Program [2011YQ030124]; National Basic Research Program of China (973 Program) [2012CB917303]; National Natural Science Foundation of China [21172013

Bifunctional Unnatural Sialic Acids for Dual Metabolic Labeling of Cell-Surface Sialylated Glycans

Sialic acid analogues containing a unique chemical functionality or chemical reporter have been metabolically incorporated into sialylated glycans. This process, termed metabolic glycan labeling, has emerged as a powerful tool for studying sialylation as well as other types of glycosylation. Currently, this technique can install only a single functionality. Here we describe a strategy for dual labeling of sialylated glycans using a new class of bifunctional sialic acid analogues containing two distinct chemical reporters at the <i>N</i>-acyl and C9 positions. These bifunctional unnatural sialic acids were metabolically incorporated into cellular glycans, where the two chemical reporters exerted their distinct functions. This approach expands the capability of metabolic glycan labeling to probe sialylation and glycan–protein interactions

Glycan Imaging in Intact Rat Hearts and Glycoproteomic Analysis Reveal the Upregulation of Sialylation during Cardiac Hypertrophy

In the heart, glycosylation is involved in a variety of physiological and pathological processes. Cardiac glycosylation is dynamically regulated, which remains challenging to monitor <i>in vivo</i>. Here we describe a chemical approach for analyzing the dynamic cardiac glycome by metabolically labeling the cardiac glycans with azidosugars in living rats. The azides, serving as a chemical reporter, are chemoselectively conjugated with fluorophores using copper-free click chemistry for glycan imaging; derivatizing azides with affinity tags allows enrichment and proteomic identification of glycosylated cardiac proteins. We demonstrated this methodology by visualization of the cardiac sialylated glycans in intact hearts and identification of more than 200 cardiac proteins modified with sialic acids. We further applied this methodology to investigate the sialylation in hypertrophic hearts. The imaging results revealed an increase of sialic acid biosynthesis upon the induction of cardiac hypertrophy. Quantitative proteomic analysis identified multiple sialylated proteins including neural cell adhesion molecule 1, T-kininogens, and α₂-macroglobulin that were upregulated during hypertrophy. The methodology may be further extended to other types of glycosylation, as exemplified by the mucin-type O-linked glycosylation. Our results highlight the applications of metabolic glycan labeling coupled with bioorthogonal chemistry in probing the biosynthesis and function of cardiac glycome during pathophysiological responses

Glycan Imaging in Intact Rat Hearts and Glycoproteomic Analysis Reveal the Upregulation of Sialylation during Cardiac Hypertrophy

In the heart, glycosylation is involved in a variety of physiological and pathological processes. Cardiac glycosylation is dynamically regulated, which remains challenging to monitor <i>in vivo</i>. Here we describe a chemical approach for analyzing the dynamic cardiac glycome by metabolically labeling the cardiac glycans with azidosugars in living rats. The azides, serving as a chemical reporter, are chemoselectively conjugated with fluorophores using copper-free click chemistry for glycan imaging; derivatizing azides with affinity tags allows enrichment and proteomic identification of glycosylated cardiac proteins. We demonstrated this methodology by visualization of the cardiac sialylated glycans in intact hearts and identification of more than 200 cardiac proteins modified with sialic acids. We further applied this methodology to investigate the sialylation in hypertrophic hearts. The imaging results revealed an increase of sialic acid biosynthesis upon the induction of cardiac hypertrophy. Quantitative proteomic analysis identified multiple sialylated proteins including neural cell adhesion molecule 1, T-kininogens, and α₂-macroglobulin that were upregulated during hypertrophy. The methodology may be further extended to other types of glycosylation, as exemplified by the mucin-type O-linked glycosylation. Our results highlight the applications of metabolic glycan labeling coupled with bioorthogonal chemistry in probing the biosynthesis and function of cardiac glycome during pathophysiological responses