N-Azidoacetylmannosamine-mediated chemical tagging of gangliosides

Anton P Bussink,Paul F Van Swieten,Karen Ghauharali,Saskia Scheij,Marco Van Eijk,Tom Wennekes,Gijs A Van Der Marel,Rolf G Boot,Johannes M.F.G Aerts,Herman S Overkleeft

doi:10.1194/jlr.c700006-jlr200

Anton P Bussink, Paul F Van Swieten + Show 8 more

Open Access

https://doi.org/10.1194/jlr.c700006-jlr200

Copy DOI

Abstract

Peracetylated N-alpha-azidoacetylmannosamine (Ac(4)ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by approximately 25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by approximately 75%. In conclusion, exposure of cells to Ac(4)ManNAz allows in vivo chemical tagging of gangliosides.

Highlights

Peracetylated N-a-azidoacetylmannosamine (Ac4ManNAz) is metabolized by cells to CMP-azidosialic acid
We further show that cell surface labeling of azidosialic acid-containing glycoproteins and gangliosides can be suppressed independently by the proper selection of iminosugars: those that inhibit N-linked glycan processing mannosidases and those that inhibit glucosylceramide synthase, respectively
Our study reveals that exposure of Jurkat cells to Ac4ManNAz results in the formation of chemically tagged ganglioside, in particular the most abundant ganglioside, GM3

Summary

Introduction

Peracetylated N-a-azidoacetylmannosamine (Ac4ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. We first demonstrate that the same procedure results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by ?25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)pentyl]1-deoxynojirimycin reduces cell surface labeling by ?75%. Exposure of cells to Ac4ManNAz allows in vivo chemical tagging of gangliosides.—Bussink, A.

Methods

Results

Conclusion