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

Abstract

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 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.

In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy

“…Azidosugars have been used as tracers to report the sialic acid biosynthesis in living animals (12,13), but the BBB impedes their access to the brain. The LABOR strategy developed herein overcomes this obstacle by exploiting liposomes to shuttle 9AzSia into the brain.…”

“…3A). Copper-free click chemistry has been performed in living mice and rats and exhibited no apparent toxicity (13,22,23). Using mice treated with empty liposomes (LP), we determined that DBCO-Cy5 had been cleared from the bloodstream 3 h after the injection (SI Appendix, Fig.…”

“…injected into living animals and metabolically converted to the corresponding azido sialic acid, which was incorporated into the sialoglycans in a panel of organs, including the heart, kidney, and liver (12). Reacting the azides with an alkynecontaining fluorescent probe via bioorthogonal chemistry (e.g., copper-free click chemistry) enabled imaging of cardiac sialoglycans in intact rat hearts and revealed the up-regulation of sialylation during cardiac hypertrophy (13). However, sialoglycans in the brain cannot be labeled or visualized by using this strategy, presumably due to the inability of azidosugars to cross the blood−brain barrier (BBB).…”

In situ click chemistry generation of cyclooxygenase-2 inhibitors

“…Click chemistry 32,33 , including 1,3-dipolar cycloaddition between alkyne and azide (Huisgen cycloaddition), have attracted much attention because of their remarkable efficiency, simplicity, and its ability to be employed for the synthesis of a wide range of compounds such as molecular imaging agents 34,35 and drugs 36 , protein modification 37,38 , DNA and RNA targeting 39,40 , and glycan imaging 41,42 . Considering its versatility, click chemistry has found numerous synthesis applications not only performed in traditional reaction vessels, but also in living systems.…”

Chemical Glycoproteomics

“…207 After administration of Ac 4 ManNAz to saline- or isoprenaline-treated rats, heart tissue was isolated, lysed, and treated with an alkyne-biotin probe. Following enrichment of biotinylated glycoproteins with streptavidin beads and in-gel digestion, the authors used tandem MS to identify newly synthesized sialylated glycoproteins in hypertrophic hearts.…”