Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling
Abstract
:1. Introduction
2. Results
2.1. Preparation of functionalized Poly-LacNAc glycans 1–8 and Neo-Glycoproteins 9–16
2.2. Binding Properties of Glycans 1–8 and Neo-Glycoproteins 9–16 to Gal-3 in ELISA Assay
2.3. Binding Kinetics of Neo-Glycoproteins 9–16 with Gal-3 Determined by Surface Plasmon Resonance
2.4. Molecular Dynamics of Disaccharide (1,2) and Tetrasaccharide (3,4) Glycans in Gal-3 CRD
3. Discussion
4. Materials and Methods
4.1. Synthesis of Glycans 1–8 and Neo-Glycoproteins 9–16
4.2. Protein Production
4.3. ELISA Assays with Gal-3
4.4. Surface Plasmon Resonance Measurements
4.5. Molecular Dynamics Simulations
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
ASF | Asialofetuin |
BSA | Bovine serum albumin |
CRD | Carbohydrate recognition domain |
ELISA | Enzyme-linked immunosorbent assay |
ESI-MS | Electrospray ionization mass spectrometry |
Gal-3 | Galectin-3 |
Gal-3-AVI | Galectin-3 containing AviTag peptide sequence |
HPLC | High-performance liquid chromatography |
IC50 | Half maximal inhibitory concentration |
LacNAc | N-Acetyllactosamine (β-d-Gal-(1→4)-d-GlcNAc) |
LacdiNAc | N,N′-Diacetyllactosamine (β-d-GalNAc-(1→4)-d-GlcNAc) |
NMR | Nuclear magnetic resonance |
SDS-PAGE | Sodium dodecyl sulfate polyacrylamide gel electrophoresis |
SPR | Surface plasmon resonance |
TMB | 3,3′,5,5′-tetramethylbenzidine |
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Neo-Glycoprotein (Respective Glycan) a | Mw (kDa) | m b | IC50 Glycan (µM) c | IC50 Neo-Glycoprotein (nM) | rp d | rp/me | Kd Neo-Glycoprotein (nM) |
---|---|---|---|---|---|---|---|
9 (glycan 1) | 76.4 | 17 | 42 ± 2 | 2026 ± 147 | 20.7 | 1.2 | 6290 ± 530 |
10 (glycan 2) | 78.2 | 18 | 36 ± 1 | 344 ± 38 | 104.7 | 5.8 | 4780 ± 1240 |
11 (glycan 3) | 87.4 | 21 | 7 ± 1 f | 11 ± 2 | 642.9 | 30.6 | 30 ± 4 f |
12 (glycan 4) | 84.7 | 19 | 13 ± 3 f | 31 ± 1 | 419.4 | 22.1 | 300 ± 60 f |
13 (glycan 5) | 86.6 | 16 | 6.20 ± 0.02 g | 37 ± 7 g | 169.9 | 10.6 | 76 ± 19 g |
14 (glycan 6) | 86.6 | 17 | 20.1 ± 0.1 g | 76 ± 8 g | 263.4 | 15.5 | 350 ± 110 g |
15 (glycan 7) | 85.9 | 17 | 12.5 ± 0.1 g | 212 ± 22 g | 58.9 | 3.5 | 700 ± 100 g |
16 (glycan 8) | 89.6 | 18 | 8.4 ± 0.1 g | 65 ± 4 g | 128.2 | 7.1 | 290 ± 90 g |
Compound | Attached Glycan a | ka (M−1·s−1) | kd (s−1) | KD (M) |
---|---|---|---|---|
9 | LacdiNAc | N.D. | N.D. | N.D. |
10 | LacNAc | N.D. | N.D. | N.D. |
11 | LacdiNAc-LacNAc | (5.9 ± 1.3) × 106 | (8.5 ± 2.5) × 10−5 | (1.4 ± 0.4) × 10−11 |
12 | LacNAc-LacNAc | (6.2 ± 2.1) × 106 | (4.2 ± 3.1) × 10−4 | (6.8 ± 3.9) × 10−11 |
13 | LacdiNAc-LacNAc-LacNAc | (5.8 ± 2.2) × 106 | (1.5 ± 0.4) × 10−4 | (2.6 ± 0.9) × 10−11 |
14 | LacNAc-LacNAc-LacNAc | (7.8 ± 2.9) × 106 | (5.1 ± 2.8) × 10−4 | (6.5 ± 2.6) × 10−11 |
15 | LacNAc type 1-LacNAc-LacNAc | (4.9 ± 1.8) × 106 | (1.3 ± 0.3) × 10−3 | (2.7 ± 1.0) × 10−10 |
16 | Galili-LacNAc-LacNAc | (9.3 ± 3.1) × 106 | (6.3 ± 2.0) × 10−4 | (6.8 ± 3.5) × 10−11 |
ASF | Positive control standard | (4.8 ± 1.9) × 104 | (4.0 ± 1.7) × 10−4 | (8.3 ± 3.1) × 10−9 |
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Bumba, L.; Laaf, D.; Spiwok, V.; Elling, L.; Křen, V.; Bojarová, P. Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling. Int. J. Mol. Sci. 2018, 19, 372. https://doi.org/10.3390/ijms19020372
Bumba L, Laaf D, Spiwok V, Elling L, Křen V, Bojarová P. Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling. International Journal of Molecular Sciences. 2018; 19(2):372. https://doi.org/10.3390/ijms19020372
Chicago/Turabian StyleBumba, Ladislav, Dominic Laaf, Vojtěch Spiwok, Lothar Elling, Vladimír Křen, and Pavla Bojarová. 2018. "Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling" International Journal of Molecular Sciences 19, no. 2: 372. https://doi.org/10.3390/ijms19020372