Smelyansky Stephanie R, Ma Chi-Wang, Marando Victoria M, Babunovic Gregory H, Lee So Young, Bryson Bryan D, Kiessling Laura L
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.
Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 2025 May 13;122(19):e2422185122. doi: 10.1073/pnas.2422185122. Epub 2025 May 5.
() is a leading cause of death worldwide. cell envelope glycans are potent virulence factors that play key roles in mediating infection of host tissues and modulating the host immune response. However, there are few ways to site-selectively modify and label these or any glycans to study their functions in disease. This gap arises because glycans generally lack functional groups amenable to bioconjugation strategies. Methylthioxylofuranose (MTX), a rare monosaccharide in select pathogenic mycobacteria, is an exception. MTX is appended to mannose-capped lipoarabinomannan (ManLAM), an antigenic glycolipid in the cell envelope implicated in downregulating the host immune system during infection. MTX is unique not only in its prevalence but also in its functionality-it contains a thioether not present in other glycans. We envisioned exploiting the MTX thioether to selectively label ManLAM with an oxaziridine probe. Here, we show that MTX-containing glycans can be labeled selectively in the test tube and live cells, highlighting the reactivity and accessibility of this motif. Our approach labels ManLAM efficiently despite the presence of protein methionine residues and can distinguish between different mycobacterial species. Using an oxaziridine equipped with a reporter, we could visualize ManLAM localization in live cells and a macrophage infection model, highlighting the stability of the label and the cell envelope in this environment. These studies will enable investigations of dynamic changes in a critical cell envelope component during infection. Moreover, the selective reactivity of thioethers can be leveraged to expand the repertoire of glycan bioconjugation strategies.
（）是全球主要的死亡原因。细胞包膜聚糖是强大的毒力因子，在介导宿主组织感染和调节宿主免疫反应中起关键作用。然而，几乎没有方法可以位点选择性地修饰和标记这些聚糖或任何聚糖来研究它们在疾病中的功能。出现这种差距是因为聚糖通常缺乏适合生物共轭策略的官能团。甲硫氧基呋喃糖（MTX）是某些致病性分枝杆菌中一种罕见的单糖，是个例外。MTX附着于甘露糖封端的脂阿拉伯甘露聚糖（ManLAM），这是细胞包膜中的一种抗原性糖脂，在感染期间参与下调宿主免疫系统。MTX不仅在其普遍性上而且在其功能上都是独特的——它含有其他聚糖中不存在的硫醚。我们设想利用MTX硫醚用氮杂环丙烷探针选择性标记ManLAM。在这里，我们表明含MTX的聚糖可以在试管和活细胞中被选择性标记，突出了这个基序的反应性和可及性。尽管存在蛋白质甲硫氨酸残基，我们的方法仍能有效地标记ManLAM，并且可以区分不同的分枝杆菌物种。使用配备有报告基团的氮杂环丙烷，我们可以在活细胞和巨噬细胞感染模型中可视化ManLAM的定位，突出了这种环境中标记物和细胞包膜的稳定性。这些研究将有助于研究感染期间关键细胞包膜成分的动态变化。此外，硫醚的选择性反应性可用于扩展聚糖生物共轭策略的范围。
