One-step biotinylation procedure for carbohydrates to study carbohydrate-protein interactions.
Protein-carbohydrate interactions play crucial roles in numerous biological processes. To study these interactions, we developed a simple and fast procedure for the biotinylation of carbohydrates based on reductive amination. The method allows complete and stable biotinylation of small quantities of oligosaccharides and includes a rapid and simple procedure to remove excess labeling reagent. After biotinylation, the structural and biological integrity of the glycans was intact as determined by HPLC, mass spectrometry, and a plant lectin assay. By using the human C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin), we demonstrate that the biotinylated glycans can be used in a glycan array to determine binding specificities of lectins. Moreover, we show that fluorescent beads coated with selected biotinylated glycans bind to DC-SIGN-expressing dendritic cells in vitro. Finally, by using biotinylated high-mannose N-glycans, we could visualize DC-SIGN-expressing cells in lymph node tissue. The availability of easy biotinylation methods for oligosaccharides such as those described here greatly facilitates the functional analysis of lectins. In addition, the biotinylated glycans will be great tools for investigating functional lectin receptors in situ.
Grün
Christian H
CH
Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, 1081 BT Amsterdam, The Netherlands. christian.grun@unilever.com
van Vliet
Sandra J
SJ
Schiphorst
Wietske E C M
WE
Bank
Christine M C
CM
Meyer
Sandra
S
van Die
Irma
I
van Kooyk
Yvette
Y
D000345:Affinity Labels; D019921:Biotinylation / Q000379:methods*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis / Q000737:chemistry*; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry; D006801:Humans; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism; D008198:Lymph Nodes / Q000166:cytology / Q000378:metabolism; D013058:Mass Spectrometry; D037121:Plant Lectins / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D000345:Affinity Labels; D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D037181:Lectins, C-Type; D037121:Plant Lectins; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin