Visualizing enveloping layer glycans during zebrafish early embryogenesis

Abstract

Developmental events can be monitored at the cellular and molecular levels by using noninvasive imaging techniques. Among the biomolecules that might be targeted for imaging analysis, glycans occupy a privileged position by virtue of their primary location on the cell surface. We previously described a chemical method to image glycans during zebrafish larval development; however, we were unable to detect glycans during the first 24 hours of embryogenesis, a very dynamic period in development. Here we report an approach to the imaging of glycans that enables their visualization in the enveloping layer during the early stages of zebrafish embryogenesis. We microinjected embryos with azidosugars at the one-cell stage, allowed the zebrafish to develop, and detected the metabolically labeled glycans with copper-free click chemistry. Mucin-type O-glycans could be imaged as early as 7 hours postfertilization, during the gastrula stage of development. Additionally, we used a nonmetabolic approach to label sialylated glycans with an independent chemistry, enabling the simultaneous imaging of these two distinct classes of glycans. Imaging analysis of glycan trafficking revealed dramatic reorganization of glycans on the second time scale, including rapid migration to the cleavage furrow of mitotic cells. These studies yield insight into the biosynthesis and dynamics of glycans in the enveloping layer during embryogenesis and provide a platform for imaging other biomolecular targets by microinjection of appropriately functionalized biosynthetic precursors.azide | cyclooctyne | bioorthogonal | oxime | cytokinesis

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

“…The impressive aglycon malleability of OleD (17)(18)(19), the demonstrated ability to expand the sugar scope of this catalyst, and the demonstrated superior ability of Loki to also catalyze "forward" coupled transglycosylation reactions suggest a range of exciting opportunities. Examples include extending applications toward (i) modified nucleoside-based NDPs (3) (reminiscent of the kinase "bump and hole" strategies) (38); (ii) specific sugardrug or sugar-natural product pairings (39)(40)(41); (iii) a broadened scope of deoxy, dideoxy, and/or uniquely functionalized sugars (e.g., sugars bearing amino-, N-akyl, O-alkyl, C-alkyl-, nitro, nitroso-, thio-modifications) (2,4,7,10,15,18,19); and (iv) other important biomolecules (e.g., proteins, polysaccharides) (42)(43)(44)(45).…”

Polysaccharides: The “Click” Chemistry Impact

“…Finally, the SPAAC was investigated on more complex living system: mice [82]. In this study, the "chemical reporters" were the unnatural azido-sugar Ac 4 ManNAz and several cyclooctynes ( Figure 48) have been used, conjugated to a small peptide (FLAG).…”

“…The most advanced work on SPAAC labeling was conducted by Bertozzi et al [82] as they managed to directly visualize glycans in membrane cells of zebrafish embryos during their development. Injection of Ac 4 GalNAz combined with a DIFO-fluorophore conjugates labeling led to very interesting observations about O-glycans distribution in zebrafish embryos.…”

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

“…This technique involves the incorporation of chemically modified sugars, such as azide-or alkyne-modified sugars, into cellular glycans via a normal biosynthetic pathway, and its subsequent detection with a probe, such as a FLAG-tag, biotin or fluorescent molecules [4][5][6] . Furthermore, application of this method for imaging of specific sugars in living cells and organisms has provided insights into the spatiotemporal expression and trafficking of glycans [7][8][9][10] . On the other hand, the currently available techniques face certain technical limitations, including the lack of selectivity, that is, there is no established method to detect a 'glycoform' (a protein with a specific glycan modification) of a given glycoprotein.…”