Limits...
Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

Alqahtani F, Mahdavi J, Wheldon LM, Vassey M, Pirinccioglu N, Royer PJ, Qarani SM, Morroll S, Stoof J, Holliday ND, Teo SY, Oldfield NJ, Wooldridge KG, Ala'Aldeen DA - Open Biol (2014)

Bottom Line: Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization.Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE.This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Show MeSH

Related in: MedlinePlus

37LRP/Gal-3 hetero- and 37LRP/37LRP homodimerization is confirmed by BiFC analysis. (a) A schematic showing the different constructs used for transfection and subsequent BiFC analysis. LamR (magenta) or galectin-3 (red) cDNA was cloned in frame with Yn, YFP or Yc. Cells were then transfected as indicated (b–g) and allowed to express for 24 h prior to fixation and confocal analysis. Merged images also show Hoechst 33258 DNA staining. Scale bar, 10 mm. Insets are shown at 4× original magnification. Images are representative of ≥3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4221890&req=5

RSOB140053F3: 37LRP/Gal-3 hetero- and 37LRP/37LRP homodimerization is confirmed by BiFC analysis. (a) A schematic showing the different constructs used for transfection and subsequent BiFC analysis. LamR (magenta) or galectin-3 (red) cDNA was cloned in frame with Yn, YFP or Yc. Cells were then transfected as indicated (b–g) and allowed to express for 24 h prior to fixation and confocal analysis. Merged images also show Hoechst 33258 DNA staining. Scale bar, 10 mm. Insets are shown at 4× original magnification. Images are representative of ≥3 independent experiments.

Mentions: To test the hypothesis that 37LRP is capable of forming homodimers and also heterodimers with Gal-3, as suggested by our model, we employed a bimolecular fluorescence complementation (BiFC) technique. A fluorescing union is formed when two separate non-fluorescent YFP subfragments are brought together by the close association of two intimately interacting proteins [39]. In addition to the full-length YFP, its N- or C-terminal domains (Yn and Yc, respectively) were fused to the C-terminal end of Gal-3 and 37LRP (figure 3a), and transfected into N2a, hBMECs and COS7 cells (figure 3b–g shows N2a cells). Immunoblot analysis of host cells (electronic supplementary material, figure S3) confirmed transfection and protein expression. Although 37LRP was readily observed in COS7 cells by fluorescence microscopy (electronic supplementary material, figure S2) and immunoblotting (electronic supplementary material, figure S3), no 67LR was observed in these cells, suggesting that under the experimental conditions tested, 67LR did not form. As the tagged proteins do not form a higher molecular mass protein equivalent to 67LR, it is possible that additional factors may be required subsequent to homodimerization (as detected by our BiFC experiments) for the maturation into the SDS-stable 67LR form of the protein, which are not present in these cells. This would be consistent with the observation that endogenous 37LRP was apparent in these cells (electronic supplementary material, figures S2 and S3), whereas 67LR was not detected. It should also be noted that in cells expressing endogenous 37LRP and 67LR the latter species runs in SDS–PAGE gels with an apparent molecular mass of around 60 kDa, which is lower than might be expected of a homodimer of 37LRP, which has an apparent molecular mass of around 42 kDa (electronic supplementary material, figure S1). The most likely explanation for this is that 67LR is resistant to denaturation in SDS, and thus maintains a more compact structure than during SDS–PAGE and runs with a higher electrophoretic mobility.Figure 3.


Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

Alqahtani F, Mahdavi J, Wheldon LM, Vassey M, Pirinccioglu N, Royer PJ, Qarani SM, Morroll S, Stoof J, Holliday ND, Teo SY, Oldfield NJ, Wooldridge KG, Ala'Aldeen DA - Open Biol (2014)

37LRP/Gal-3 hetero- and 37LRP/37LRP homodimerization is confirmed by BiFC analysis. (a) A schematic showing the different constructs used for transfection and subsequent BiFC analysis. LamR (magenta) or galectin-3 (red) cDNA was cloned in frame with Yn, YFP or Yc. Cells were then transfected as indicated (b–g) and allowed to express for 24 h prior to fixation and confocal analysis. Merged images also show Hoechst 33258 DNA staining. Scale bar, 10 mm. Insets are shown at 4× original magnification. Images are representative of ≥3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221890&req=5

RSOB140053F3: 37LRP/Gal-3 hetero- and 37LRP/37LRP homodimerization is confirmed by BiFC analysis. (a) A schematic showing the different constructs used for transfection and subsequent BiFC analysis. LamR (magenta) or galectin-3 (red) cDNA was cloned in frame with Yn, YFP or Yc. Cells were then transfected as indicated (b–g) and allowed to express for 24 h prior to fixation and confocal analysis. Merged images also show Hoechst 33258 DNA staining. Scale bar, 10 mm. Insets are shown at 4× original magnification. Images are representative of ≥3 independent experiments.
Mentions: To test the hypothesis that 37LRP is capable of forming homodimers and also heterodimers with Gal-3, as suggested by our model, we employed a bimolecular fluorescence complementation (BiFC) technique. A fluorescing union is formed when two separate non-fluorescent YFP subfragments are brought together by the close association of two intimately interacting proteins [39]. In addition to the full-length YFP, its N- or C-terminal domains (Yn and Yc, respectively) were fused to the C-terminal end of Gal-3 and 37LRP (figure 3a), and transfected into N2a, hBMECs and COS7 cells (figure 3b–g shows N2a cells). Immunoblot analysis of host cells (electronic supplementary material, figure S3) confirmed transfection and protein expression. Although 37LRP was readily observed in COS7 cells by fluorescence microscopy (electronic supplementary material, figure S2) and immunoblotting (electronic supplementary material, figure S3), no 67LR was observed in these cells, suggesting that under the experimental conditions tested, 67LR did not form. As the tagged proteins do not form a higher molecular mass protein equivalent to 67LR, it is possible that additional factors may be required subsequent to homodimerization (as detected by our BiFC experiments) for the maturation into the SDS-stable 67LR form of the protein, which are not present in these cells. This would be consistent with the observation that endogenous 37LRP was apparent in these cells (electronic supplementary material, figures S2 and S3), whereas 67LR was not detected. It should also be noted that in cells expressing endogenous 37LRP and 67LR the latter species runs in SDS–PAGE gels with an apparent molecular mass of around 60 kDa, which is lower than might be expected of a homodimer of 37LRP, which has an apparent molecular mass of around 42 kDa (electronic supplementary material, figure S1). The most likely explanation for this is that 67LR is resistant to denaturation in SDS, and thus maintains a more compact structure than during SDS–PAGE and runs with a higher electrophoretic mobility.Figure 3.

Bottom Line: Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization.Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE.This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Show MeSH
Related in: MedlinePlus