Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1

• Published in: ACS chemical biology Vol. 13; no. 9; pp. 2560 - 2567
• Main Authors: Moure, Maria J, Eletsky, Alexander, Gao, Qi, Morris, Laura C, Yang, Jeong-Yeh, Chapla, Digantkumar, Zhao, Yuejie, Zong, Chengli, Amster, I. Jonathan, Moremen, Kelley W, Boons, Geert-Jan, Prestegard, James H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.09.2018
• Subjects: Click Chemistry; Cyclic N-Oxides - analysis; Cyclic N-Oxides - metabolism; Electron Spin Resonance Spectroscopy; Glycoproteins - chemistry; Glycoproteins - metabolism; Glycosylation; HEK293 Cells; Heparitin Sulfate - chemistry; Heparitin Sulfate - metabolism; Humans; Ligands; Molecular Docking Simulation; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Receptors, Immunologic - chemistry; Receptors, Immunologic - metabolism; Roundabout Proteins
• ISSN: 1554-8929; 1554-8937; 1554-8937
• DOI: 10.1021/acschembio.8b00511

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper­(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with 15N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.