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Metabolic labeling of fucosylated glycans in developing zebrafish.

Dehnert KW, Beahm BJ, Huynh TT, Baskin JM, Laughlin ST, Wang W, Wu P, Amacher SL, Bertozzi CR - ACS Chem. Biol. (2011)

Bottom Line: This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis.We used this technique to image fucosylated glycans in the enveloping layer of zebrafish embryos during the first 5 days of development.This work provides a method to study the biosynthesis of fucosylated glycans in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, and Howard Hughes Medical Institute, University of California-Berkeley, CA 94720, United States.

ABSTRACT
Many developmental processes depend on proper fucosylation, but this post-translational modification is difficult to monitor in vivo. Here we applied a chemical reporter strategy to visualize fucosylated glycans in developing zebrafish. Using azide-derivatized analogues of fucose, we metabolically labeled cell-surface glycans and then detected the incorporated azides via copper-free click chemistry with a difluorinated cyclooctyne probe. We found that the fucose salvage pathway enzymes are expressed during zebrafish embryogenesis but that they process the azide-modified substrates inefficiently. We were able to bypass the salvage pathway by using an azide-functionalized analogue of GDP-fucose. This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis. We used this technique to image fucosylated glycans in the enveloping layer of zebrafish embryos during the first 5 days of development. This work provides a method to study the biosynthesis of fucosylated glycans in vivo.

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Microinjection of GDP-FucAz followed by copper-free click chemistry enables imaging of fucosylated glycans during the first 5 days of development. Zebrafish embryos were microinjected with vehicle alone (top) or 75 pmol of GDP-FucAz (bottom), allowed to develop, then treated with DIFO-488, and imaged at the time indicated. Shown are z-projection DIFO-488 fluorescence images of the EVL (rows 1 and 3) and corresponding brightfield images (rows 2 and 4). Scale bar: 200 μm.
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fig4: Microinjection of GDP-FucAz followed by copper-free click chemistry enables imaging of fucosylated glycans during the first 5 days of development. Zebrafish embryos were microinjected with vehicle alone (top) or 75 pmol of GDP-FucAz (bottom), allowed to develop, then treated with DIFO-488, and imaged at the time indicated. Shown are z-projection DIFO-488 fluorescence images of the EVL (rows 1 and 3) and corresponding brightfield images (rows 2 and 4). Scale bar: 200 μm.

Mentions: We used GDP-FucAz as a metabolic substrate to determine the onset and extent of fucose labeling during early development. Embryos were microinjected with GDP-FucAz or vehicle alone, then reacted with DIFO-488, and imaged at several stages of development. Cell-surface labeling of the enveloping layer was observed as early as 65% epiboly, or 7 hpf (Supplementary Figure 4). The labeling intensity increased from midgastrulation to early segmentation periods (Figure 4). These dynamics are similar to those observed for expression of mucin-type O-glycans (16) and N-glycans,(36) both of which can be modified by fucosylation. Fluorescence derived from FucAz-labeled glycans in the enveloping layer decreased from 24 hpf onward (Figure 4). This decrease in signal could be due to depletion or degradation of the GDP-FucAz substrate, or it could reflect decreasing levels of endogenous fucosylation. Indeed, labeling of fucosylated glycans in the enveloping layer by the fucose-binding Aleuria aurantia lectin (AAL) also decreased from 12 to 24 hpf, as determined by flow cytometry analysis (Supplementary Figure 5). Nevertheless, signal from FucAz-labeled glycans remained strong enough to observe FucAz-dependent signal over the first 5 days of development.


Metabolic labeling of fucosylated glycans in developing zebrafish.

Dehnert KW, Beahm BJ, Huynh TT, Baskin JM, Laughlin ST, Wang W, Wu P, Amacher SL, Bertozzi CR - ACS Chem. Biol. (2011)

Microinjection of GDP-FucAz followed by copper-free click chemistry enables imaging of fucosylated glycans during the first 5 days of development. Zebrafish embryos were microinjected with vehicle alone (top) or 75 pmol of GDP-FucAz (bottom), allowed to develop, then treated with DIFO-488, and imaged at the time indicated. Shown are z-projection DIFO-488 fluorescence images of the EVL (rows 1 and 3) and corresponding brightfield images (rows 2 and 4). Scale bar: 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117394&req=5

fig4: Microinjection of GDP-FucAz followed by copper-free click chemistry enables imaging of fucosylated glycans during the first 5 days of development. Zebrafish embryos were microinjected with vehicle alone (top) or 75 pmol of GDP-FucAz (bottom), allowed to develop, then treated with DIFO-488, and imaged at the time indicated. Shown are z-projection DIFO-488 fluorescence images of the EVL (rows 1 and 3) and corresponding brightfield images (rows 2 and 4). Scale bar: 200 μm.
Mentions: We used GDP-FucAz as a metabolic substrate to determine the onset and extent of fucose labeling during early development. Embryos were microinjected with GDP-FucAz or vehicle alone, then reacted with DIFO-488, and imaged at several stages of development. Cell-surface labeling of the enveloping layer was observed as early as 65% epiboly, or 7 hpf (Supplementary Figure 4). The labeling intensity increased from midgastrulation to early segmentation periods (Figure 4). These dynamics are similar to those observed for expression of mucin-type O-glycans (16) and N-glycans,(36) both of which can be modified by fucosylation. Fluorescence derived from FucAz-labeled glycans in the enveloping layer decreased from 24 hpf onward (Figure 4). This decrease in signal could be due to depletion or degradation of the GDP-FucAz substrate, or it could reflect decreasing levels of endogenous fucosylation. Indeed, labeling of fucosylated glycans in the enveloping layer by the fucose-binding Aleuria aurantia lectin (AAL) also decreased from 12 to 24 hpf, as determined by flow cytometry analysis (Supplementary Figure 5). Nevertheless, signal from FucAz-labeled glycans remained strong enough to observe FucAz-dependent signal over the first 5 days of development.

Bottom Line: This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis.We used this technique to image fucosylated glycans in the enveloping layer of zebrafish embryos during the first 5 days of development.This work provides a method to study the biosynthesis of fucosylated glycans in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, and Howard Hughes Medical Institute, University of California-Berkeley, CA 94720, United States.

ABSTRACT
Many developmental processes depend on proper fucosylation, but this post-translational modification is difficult to monitor in vivo. Here we applied a chemical reporter strategy to visualize fucosylated glycans in developing zebrafish. Using azide-derivatized analogues of fucose, we metabolically labeled cell-surface glycans and then detected the incorporated azides via copper-free click chemistry with a difluorinated cyclooctyne probe. We found that the fucose salvage pathway enzymes are expressed during zebrafish embryogenesis but that they process the azide-modified substrates inefficiently. We were able to bypass the salvage pathway by using an azide-functionalized analogue of GDP-fucose. This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis. We used this technique to image fucosylated glycans in the enveloping layer of zebrafish embryos during the first 5 days of development. This work provides a method to study the biosynthesis of fucosylated glycans in vivo.

Show MeSH