Biosynthetic Glycan Labeling

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. Moreo-ver, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generaliza-ble 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 stud-ies 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.

Mining the human gut microbiome identifies mycobacterial d-arabinan degrading enzymes

“…We next assessed whether DUF4185 endo- d -arabinofuranases can cleave AG in the context of an intact cell wall. We utilized metabolic arabinogalactan labelling with the azide-modified lipid-linked Ara f donor 5-AzFPA and then fluorescently labelled intact bacteria with DBCO-conjugated AF647 using click chemistry ( Figure S16 ) 36–38 . Both Mab GH4185 and Rv3707c released fluorescently labelled material from cell wall, with greater activity and a greater range of products formed by the former enzyme, as observed by HPAEC-PAD.…”

“…Fluorescently labeled mycolyl-arabinogalactan-peptidoglycan complex (mAGP) was isolated from Corynebacterium glutamicum ATCC13032 following previously reported methods 36–38 . In brief, cells were grown in the presence of 5-AzFPA to saturation, reacted with DBCO-conjugated AF647 and then the mAGP was isolated.…”

Azido Inositol Probes Enable Metabolic Labeling of Inositol-Containing Glycans and Reveal an Inositol Importer in Mycobacteria

“…In the present work, our screen of three regioisomeric InoAz analogues revealed that the azido group position had a critical impact on metabolic incorporation, which is consistent with previous findings on azide-modified substrates for labeling of trehalose mycolates 14 and arabinogalactan. 29 Our current study mainly focused on the 5-InoAz isomer, which incorporated into early PIMs, LM, and LAM in Msmeg strains that were rationally engineered to favor probe incorporation. Furthermore, we took an unbiased genetic approach to improve the inositol analogue uptake by growing an inositol auxotroph on 5-InoAz over an extended period, which selected for strains with mutations that enhanced utilization of 5-InoAz.…”

“…12 Synthetic unnatural biosynthetic precursors have been developed to metabolically label an array of mycobacterial cell envelope components, including trehalose-and arabinogalactan-linked mycolates, 13−25 peptidoglycan, 26−28 and arabinogalactan. 29 No such probes have been developed for PIMs or the PIM-anchored derivatives LM and LAM in mycobacteria. The ability to metabolically incorporate inositolbased probes, such as azido inositol (InoAz) analogues, would yield a single substitution in the glycan core of PIMs (or additionally in terminal phospho-inositol capping residues of LAM in relevant organisms) and, in conjunction with bioorthogonal chemistry, would enable researchers to address many of the questions about PIM, LM, and LAM biosynthesis, transport, and dynamics raised above.…”

Profile of Carolyn R. Bertozzi, Morten Meldal, and K. Barry Sharpless: 2022 Nobel laureates in Chemistry

“…Examples include monitoring dynamic processes in cells, such as the general synthesis of nucleic acids ( 29 ), proteins ( 30 ), and glycans ( 25 ). By tailoring the substrate, these reactions can be used even to assess the reactivity and accessibility of protein side chains ( 31 ), the location of small molecules or specific lipids ( 32 ), and the biosynthesis of very specific glycans ( 33 , 34 ).…”