A model for cell-surface-exposed carbohydrate moieties suitable for structural studies by NMR spectroscopy.
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.
Mares
Jiri
J
Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Müller
Jan U
JU
Skirgailiene
Audrone
A
Neumoin
Alexey
A
Bewley
Carole A
CA
Schmidt
Richard R
RR
Zerbe
Oliver
O
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry*; D004058:Diffusion; D008055:Lipids / Q000138:chemical synthesis / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins; D008055:Lipids; D008823:Micelles; D010743:Phospholipids; C107733:cyanovirin N