TY - JOUR
T1 - NMR-based insight into galectin-3 binding to endothelial cell adhesion molecule CD146
T2 - Evidence for noncanonical interactions with the lectin's CRD β-sandwich F-face
AU - Zhang, Zhongyu
AU - Miller, Michelle C.
AU - Xu, Xuejiao
AU - Song, Chengcheng
AU - Zhang, Fan
AU - Zheng, Yi
AU - Zhou, Yifa
AU - Tai, Guihua
AU - Mayo, Kevin H.
PY - 2019/7/19
Y1 - 2019/7/19
N2 - Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1-D5) and a shorter, D5-deleted version of CD146 (D1-D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1-D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding β-sheet S-face (β-strands 1, 10, 3, 4, 5, 6) of the Gal-3 β-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face β-sheet (β-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with β-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin's CRD F-face in the CD146 binding process.
AB - Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1-D5) and a shorter, D5-deleted version of CD146 (D1-D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1-D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding β-sheet S-face (β-strands 1, 10, 3, 4, 5, 6) of the Gal-3 β-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face β-sheet (β-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with β-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin's CRD F-face in the CD146 binding process.
KW - CD146
KW - NMR
KW - binding
KW - cell adhesion molecule
KW - galectin
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U2 - 10.1093/glycob/cwz036
DO - 10.1093/glycob/cwz036
M3 - Article
C2 - 31094416
SN - 0959-6658
VL - 29
SP - 608
EP - 618
JO - Glycobiology
JF - Glycobiology
IS - 8
ER -