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A Model for Cell‐Surface‐Exposed Carbohydrate Moieties Suitable for Structural Studies by NMR Spectroscopy
Author(s) -
Mares Jiri,
Müller Jan U.,
Skirgailiene Audrone,
Neumoin Alexey,
Bewley Carole A.,
Schmidt Richard R.,
Zerbe Oliver
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600219
Subject(s) - glycolipid , micelle , chemistry , mannose , linker , nuclear magnetic resonance spectroscopy , stereochemistry , ligand (biochemistry) , biochemistry , organic chemistry , receptor , aqueous solution , computer science , operating system
In the present study a synthetic glycolipid system is presented that can be readily incorporated into phospholipid micelles and that allows the study of cell‐surface‐exposed carbohydrate units by high‐resolution NMR techniques. Here, we present an efficient route for the synthesis of glycolipid compounds that contain mannose, mannobiose, or mannotriose coupled either directly to an alkyl chain or through a poly(ethylene glycol) linker. Furthermore, we have validated our model system by measuring the binding of cyanovirin N (CV‐N), a cyanobacterial protein that binds with nanomolar affinity to the terminal arms of high‐mannose structures of the HIV surface‐envelope glycoprotein gp120, to glycolipids the carbohydrate portions of which comprise the corresponding high‐mannose moieties. From the results of chemical‐shift mapping with uniformly 15 N‐labelled CV‐N, we conclude that binding to the protein occurs at sites similar to those involved in binding the nonconjugated carbohydrates. We characterized the insertion of the glycolipids into dodecylphosphocholine (DPC) micelles by measuring translational diffusion, and we observed that the diffusion constants of the glycolipids were very similar to those of the DPC micelles themselves, but significantly deviated from those of the free glycolipids. We also present experimental proof that the glycolipids remain inserted in the micelles while binding to CV‐N. Finally, by addition of a ligand that had a higher affinity to CV‐N but which was not attached did not couple to a lipid anchor, CV‐N could be released from the glycolipid and, hence, from the micelle‐associated state.