Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo.
We have investigated the consequences of introducing a bisecting GlcNAc moiety into biantennary N-glycans. Computational analysis of glycan conformation with prolonged simulation periods in vacuo and in a solvent box revealed two main effects: backfolding of the alpha1-6 arm and stacking of the bisecting GlcNAc and the neighboring Man/GlcNAc residues of both antennae. Chemoenzymatic synthesis produced the bisecting biantennary decasaccharide N-glycan and its alpha2-3(6)-sialylated variants. They were conjugated to BSA to probe the ligand properties of N-glycans with bisecting GlcNAc. To assess affinity alterations in glycan binding to receptors, testing was performed with purified lectins, cultured cells, tissue sections and animals. The panel of lectins, including an adhesion/growth-regulatory galectin, revealed up to a sixfold difference in affinity constants for these neoglycoproteins relative to data on the unsubstituted glycans reported previously [André, S., Unverzagt, C., Kojima, S., Dong, X., Fink, C., Kayser, K. & Gabius, H.-J. (1997) Bioconjugate Chem. 8, 845-855]. The enhanced affinity for galectin-1 is in accord with the increased percentage of cell positivity in cytofluorimetric and histochemical analysis of carbohydrate-dependent binding of labeled neoglycoproteins to cultured tumor cells and routinely processed lung cancer sections. Intravenous injection of iodinated neoglycoproteins carrying galactose-terminated N-glycans into mice revealed the highest uptake in liver and spleen for the bisecting compound compared with the unsubstituted or core-fucosylated N-glycans. Thus, this substitution modulates ligand properties in interactions with lectins, a key finding of this report. Synthetic glycan tailoring provides a versatile approach to the preparation of newly substituted glycans with favorable ligand properties for medical applications.
André
Sabine
S
Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität Munich, Germany. Sabine.Andre@lmu.de
Unverzagt
Carlo
C
Kojima
Shuji
S
Frank
Martin
M
Seifert
Joachim
J
Fink
Christian
C
Kayser
Klaus
K
von der Lieth
Claus-Wilhelm
CW
Gabius
Hans-Joachim
HJ
D000117:Acetylglucosamine; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry; D007202:Indicators and Reagents; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000138:chemical synthesis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006023:Glycoproteins; D007202:Indicators and Reagents; D011134:Polysaccharides; D000117:Acetylglucosamine