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Glycan dependence of Galectin-3 self-association properties.

Halimi H, Rigato A, Byrne D, Ferracci G, Sebban-Kreuzer C, ElAntak L, Guerlesquin F - PLoS ONE (2014)

Bottom Line: This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD).Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association.NMR mapping clearly established that the N-terminal domain interacts with the CRD.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Ingénierie des Systèmes Moléculaires, UMR 7255, CNRS, Aix-Marseille Université, Marseille, France.

ABSTRACT
Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.

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Complex formation involving Galectin-3.A] 15N-HSQC spectra carried out on a 600 MHz NMR spectrometer equipped with a TCI cryoprobe at 300 K. Top left, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 1.5 mM in red; top right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of lactose at 1.5 mM in red; bottom left, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 80 µM in red; bottom right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of LNnT at 80 µM in red. B] Zoom in of HSQC: in left box, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 0.1 mM in red, 0.8 mM in green and 1.5 mM in blue; in the right box, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 28 µM in red and 90 µM in blue. C] Chemical shift variations observed on Galectin-3 CRD NH, top, induced by the N-terminal domain within FL (Table S2); center, induced by lactose (Table S3); bottom, induced by LNnT (Table S4). Values shown were calculated using the equation Δδobs =  [(ΔδHN2 + ΔδN2/25)]1/2. In green boxes are highlighted residues affected by lactose. In red boxes are highlighted residues affected by the N-terminal domain within the full length protein.
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pone-0111836-g004: Complex formation involving Galectin-3.A] 15N-HSQC spectra carried out on a 600 MHz NMR spectrometer equipped with a TCI cryoprobe at 300 K. Top left, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 1.5 mM in red; top right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of lactose at 1.5 mM in red; bottom left, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 80 µM in red; bottom right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of LNnT at 80 µM in red. B] Zoom in of HSQC: in left box, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 0.1 mM in red, 0.8 mM in green and 1.5 mM in blue; in the right box, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 28 µM in red and 90 µM in blue. C] Chemical shift variations observed on Galectin-3 CRD NH, top, induced by the N-terminal domain within FL (Table S2); center, induced by lactose (Table S3); bottom, induced by LNnT (Table S4). Values shown were calculated using the equation Δδobs =  [(ΔδHN2 + ΔδN2/25)]1/2. In green boxes are highlighted residues affected by lactose. In red boxes are highlighted residues affected by the N-terminal domain within the full length protein.

Mentions: To access the interactions between the CRD and N-terminal domains we compared 1H-15N HSQC spectra of the free CRD with that of CRD within the FL (Fig. 4C and Fig. 5A). Chemical shift variation analysis indicates that the markedly shifted resonances of CRD belong to some residues close to the sugar binding site and residues located at the backside of the lectin (residues Ile132, Leu135, Val138, Lys139, Phe192, Glu193, Phe198, Ile200, Gln201, Val202, Leu203, Glu205, Lys210, Ala212, Asp215, Ala216, Asp241, Thr243 and Ser244) (Fig. 5A). This result is in perfect agreement with the peptide analysis already reported by NMR spectroscopy indicating that the N-terminal domain of Galectin-3 interacts with residues of CRD located at the back of the molecule [17]. Moreover, residues located at the N- and C-terminal extremities of the CRD (residues Ile115 and Val116, and Tyr247, Thr248 and Met249) were also perturbed by the presence of the N-terminal domain as previously predicted by modeling of Galectin-3 involving the β-strands S1 and S12 [18]. The binding of the N-terminal domain of Galectin-3 on the N- and C-terminal segments of the CRD may explain the monomeric status of this galectin in solution.


Glycan dependence of Galectin-3 self-association properties.

Halimi H, Rigato A, Byrne D, Ferracci G, Sebban-Kreuzer C, ElAntak L, Guerlesquin F - PLoS ONE (2014)

Complex formation involving Galectin-3.A] 15N-HSQC spectra carried out on a 600 MHz NMR spectrometer equipped with a TCI cryoprobe at 300 K. Top left, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 1.5 mM in red; top right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of lactose at 1.5 mM in red; bottom left, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 80 µM in red; bottom right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of LNnT at 80 µM in red. B] Zoom in of HSQC: in left box, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 0.1 mM in red, 0.8 mM in green and 1.5 mM in blue; in the right box, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 28 µM in red and 90 µM in blue. C] Chemical shift variations observed on Galectin-3 CRD NH, top, induced by the N-terminal domain within FL (Table S2); center, induced by lactose (Table S3); bottom, induced by LNnT (Table S4). Values shown were calculated using the equation Δδobs =  [(ΔδHN2 + ΔδN2/25)]1/2. In green boxes are highlighted residues affected by lactose. In red boxes are highlighted residues affected by the N-terminal domain within the full length protein.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219786&req=5

pone-0111836-g004: Complex formation involving Galectin-3.A] 15N-HSQC spectra carried out on a 600 MHz NMR spectrometer equipped with a TCI cryoprobe at 300 K. Top left, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 1.5 mM in red; top right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of lactose at 1.5 mM in red; bottom left, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 80 µM in red; bottom right, free full length Galectin-3 (FL) at 30 µM concentration in black and FL at 30 µM concentration in the presence of LNnT at 80 µM in red. B] Zoom in of HSQC: in left box, free CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of lactose at 0.1 mM in red, 0.8 mM in green and 1.5 mM in blue; in the right box, CRD at 40 µM concentration in black and CRD at 40 µM concentration in the presence of LNnT at 28 µM in red and 90 µM in blue. C] Chemical shift variations observed on Galectin-3 CRD NH, top, induced by the N-terminal domain within FL (Table S2); center, induced by lactose (Table S3); bottom, induced by LNnT (Table S4). Values shown were calculated using the equation Δδobs =  [(ΔδHN2 + ΔδN2/25)]1/2. In green boxes are highlighted residues affected by lactose. In red boxes are highlighted residues affected by the N-terminal domain within the full length protein.
Mentions: To access the interactions between the CRD and N-terminal domains we compared 1H-15N HSQC spectra of the free CRD with that of CRD within the FL (Fig. 4C and Fig. 5A). Chemical shift variation analysis indicates that the markedly shifted resonances of CRD belong to some residues close to the sugar binding site and residues located at the backside of the lectin (residues Ile132, Leu135, Val138, Lys139, Phe192, Glu193, Phe198, Ile200, Gln201, Val202, Leu203, Glu205, Lys210, Ala212, Asp215, Ala216, Asp241, Thr243 and Ser244) (Fig. 5A). This result is in perfect agreement with the peptide analysis already reported by NMR spectroscopy indicating that the N-terminal domain of Galectin-3 interacts with residues of CRD located at the back of the molecule [17]. Moreover, residues located at the N- and C-terminal extremities of the CRD (residues Ile115 and Val116, and Tyr247, Thr248 and Met249) were also perturbed by the presence of the N-terminal domain as previously predicted by modeling of Galectin-3 involving the β-strands S1 and S12 [18]. The binding of the N-terminal domain of Galectin-3 on the N- and C-terminal segments of the CRD may explain the monomeric status of this galectin in solution.

Bottom Line: This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD).Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association.NMR mapping clearly established that the N-terminal domain interacts with the CRD.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Ingénierie des Systèmes Moléculaires, UMR 7255, CNRS, Aix-Marseille Université, Marseille, France.

ABSTRACT
Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.

Show MeSH