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. 2021 Oct 13;143(40):16337-16342.
doi: 10.1021/jacs.1c07430. Epub 2021 Oct 4.

Biosynthetic Glycan Labeling

Victoria M Marando  1 , Daria E Kim  1 , Phillip J Calabretta  1   2 , Matthew B Kraft  2 , Bryan D Bryson  3   4 , Laura L Kiessling  1   2
Affiliations

Biosynthetic Glycan Labeling

Victoria M Marando et al. J Am Chem Soc. .

Abstract

Glycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of d-arabinofuranose (d-Araf), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including Mycobacterium tuberculosis. The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of d-Araf residues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(A) Monosaccharides within glycans derive their identity from polyol stereo- and constitutional isomerism. (B) The core cell wall structure of mycobacteria and corynebacteria is comprised of six unique monomers. This structure, termed the mycolyl-arabinogalactan-peptidoglycan complex (mAGP) is a dense glycolipid matrix that protects cells from environmental stresses, including antibiotics. Biosynthetic incorporation directly leverages the activity of cellular glycosyltransferases for specific monosaccharides to introduce modifications into cell surface glycans.
Figure 2.
Figure 2.
To harness the catalytic activity of arabinofuranosyltransferase GT-Cs for bioconjugation, an azide-modified substrate surrogate was designed based on structural homology to the endogenous d-Araf donor (DPA). Three azide regioisomers were produced (1–3). Exogenous delivery of AzFPA was designed to result in substrate incorporation, which could subsequently be detected and quantified using SPAAC-mediated fluorophore conjugation.
Figure 3.
Figure 3.
(A) Flow cytometry analysis of AzFPA (250 μM) labeled C. glutamicum and M. smegmatis treated with DBCO-AF647. Mean fluorescence intensity (MFI) was calculated using the geometric mean and plotted relative to a dye-only control. Error bars denote the standard error of the mean of three replicate experiments. (B) Fluorescence emission (633 nm) from isolated mAGP from AzFPA (250 μM) labeled C. glutamicum and M. smegmatis reacted with DBCO-AF647. Error bars denote the standard error of the mean of two replicate experiments.
Figure 4.
Figure 4.
(A) Localization analysis of M. smegmatis grown with 2-AzFPA (250 μM) and labeled with AF647 (500 μM). Each line denotes an individual cell (n=10). (B) Confocal fluorescence microscopy images of C. glutamicum grown with 5-AzFPA (250 μM) for 2 or 5 hours. (C) Confocal fluorescence microscopy images of C. glutamicum grown with 5-AzFPA (250 μM) and HADA (500 μM) for 2 hours. (Scale bars: 3 μm).
Figure 5.
Figure 5.
(A) Schematic of the macrophage uptake workflow. First M. smegmatis cells are exposed to AzFPA and then AF647. The resulting labeled cells were mixed with THP1-derived macrophages. (B) Confocal fluorescence microscopy images of labeled M. smegmatis (cyan) that had been taken up by THP1-derived macrophages (MOI: 10:1). Fluorophore-conjugated (405 nm) Wheat germ agglutinin was used to stain the plasma membrane (white) (Scale bars: 3 μm).
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References

    1. Boutureira O; Bernardes GJ, Advances in chemical protein modification. Chem Rev 2015, 115, 2174–95. - PubMed
    1. Gunnoo SB; Madder A, Bioconjugation - using selective chemistry to enhance the properties of proteins and peptides as therapeutics and carriers. Org Biomol Chem 2016, 14, 8002–8013. - PubMed
    1. Roy S; Cha JN; Goodwin AP, Nongenetic Bioconjugation Strategies for Modifying Cell Membranes and Membrane Proteins: A Review. Bioconjug Chem 2020, 31, 2465–2475. - PMC - PubMed
    1. Gilormini PA; Batt AR; Pratt MR; Biot C, Asking more from metabolic oligosaccharide engineering. Chem Sci 2018, 9, 7585–7595. - PMC - PubMed
    1. Cioce A; Bineva-Todd G; Agbay AJ; Choi J; Wood TM; Debets MF; Browne WM; Douglas HL; Roustan C; Tastan O; Kjaer S; Bush JT; Bertozzi CR; Schumann B, Metabolic Engineering Optimizes Bioorthogonal Glycan Labeling in Living Cells. 2021. ChemRxiv. DOI:10.26434/chemrxiv.13514365.v1. (accessed 2021-09-07) - DOI

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