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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.

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Related in: MedlinePlus

Gal-3 residue C173 and 37LRP residue K166 are implicated in 37LRP–Gal-3 dimerization. (a) Ribbon diagram of the homodimer interaction of Gal-3 with a second Gal-3 showing the position of C173 (white arrows). (b) Ribbon diagram with superimposed molecular surface of the heterodimer interaction of Gal-3 with 37LRP with residue 166 substituted to alanine (white arrow). The mutation of K166 in 37LRP to alanine is predicted to disrupt the interaction between the two molecules (compare with figure 1a), whereas some (i.e. between R151 and K227 of 37LRP with D151 of Gal-3) are retained. (c) Superimposed structures of lactose-liganded (yellow) and non-lactose-liganded (blue) Gal-3 complexed with 37LRP highlighting the positions of K166A, R155 and Y139 (white arrows). Other features in the 37LRP/Gal-3 interface are not shown for clarity. (d) Following transfection of COS7 cells with the indicated 37LRP and Gal-3 -vYFP, -Yn or -Yc fusion proteins, cells were harvested and the number of fluorescent cells quantified by FACS and expressed as a percentage of the total number of cells counted. Expression of 37LRP-YFP, Gal-3-YFP and Gal-3C173A-YFP resulted in a significant increase in fluorescent cells compared with -Yn and -Yc control constructs (37LRP: 18.4 ± 1.1% compared with 0.28 ± 0.07% and 0.18 ± 0.05%; Gal-3: 28.4 ± 3.1% compared with 0.19 ± 0.04% and 0.21 ± 0.03%; Gal-3C173A: 25 ± 6.7% compared with 0.21 ± 0.07% and 0.26 ± 0.12%). Co-transfection of 37LRP-Yn and -Yc, which would form a 37LRP homodimer (37LRP HD), resulted in 10.89 ± 0.9% fluorescent cells, a similar number to cells co-transfected with either 37LRP-Yn and Gal-3-Yc (12.39 ± 1.6%) or Gal-3-Yn and Gal-3-Yc (12.65 ± 1.4%), which would form a heterodimer (37LRP/Gal-3 HTD) or self-associated Gal-3 (Gal-3 HD), respectively. Similar results were obtained when Gal-3-Yn and 37LRP-Yc constructs were investigated. Mutation of C173 on Gal-3 resulted in a significant inhibitory effect on 37LRP/Gal-3 HTD (3.29 ± 1.8%; approx. 73% inhibition) and Gal-3 HD formation (6.5 ± 0.9%; approx. 49% inhibition). Co-transfection of Gal-3C173A–Yn and Gal-3C173A–Yc resulted in negligible numbers of fluorescent cells (0.8 ± 0.6%) demonstrating complete disruption of protein–protein interactions under these conditions. Data are mean ± s.e.m. of ≥3 independent experiments. (e) Binding of Gal-3 to ELISA wells coated with recombinant 37LRP or its mutant derivatives. The mean value obtained from Gal-3 binding to BSA-coated wells was subtracted from all data points. (f) Flow cytometry analysis of COS7 cells transfected with indicated pairs of BiFC constructs and analysed 36 h post-transfection. Substitution of LAMR1 lysine 166 with alanine significantly reduced its heterodimerization with Gal-3. Fluorescence intensity was evaluated as a percentage of gated cells. Data analysed by one-way ANOVA and Tukey test. ****p < 0.0001; *p < 0.05. Error bars = mean of triplicate values on three occasions ± s.e.m.
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RSOB140053F5: Gal-3 residue C173 and 37LRP residue K166 are implicated in 37LRP–Gal-3 dimerization. (a) Ribbon diagram of the homodimer interaction of Gal-3 with a second Gal-3 showing the position of C173 (white arrows). (b) Ribbon diagram with superimposed molecular surface of the heterodimer interaction of Gal-3 with 37LRP with residue 166 substituted to alanine (white arrow). The mutation of K166 in 37LRP to alanine is predicted to disrupt the interaction between the two molecules (compare with figure 1a), whereas some (i.e. between R151 and K227 of 37LRP with D151 of Gal-3) are retained. (c) Superimposed structures of lactose-liganded (yellow) and non-lactose-liganded (blue) Gal-3 complexed with 37LRP highlighting the positions of K166A, R155 and Y139 (white arrows). Other features in the 37LRP/Gal-3 interface are not shown for clarity. (d) Following transfection of COS7 cells with the indicated 37LRP and Gal-3 -vYFP, -Yn or -Yc fusion proteins, cells were harvested and the number of fluorescent cells quantified by FACS and expressed as a percentage of the total number of cells counted. Expression of 37LRP-YFP, Gal-3-YFP and Gal-3C173A-YFP resulted in a significant increase in fluorescent cells compared with -Yn and -Yc control constructs (37LRP: 18.4 ± 1.1% compared with 0.28 ± 0.07% and 0.18 ± 0.05%; Gal-3: 28.4 ± 3.1% compared with 0.19 ± 0.04% and 0.21 ± 0.03%; Gal-3C173A: 25 ± 6.7% compared with 0.21 ± 0.07% and 0.26 ± 0.12%). Co-transfection of 37LRP-Yn and -Yc, which would form a 37LRP homodimer (37LRP HD), resulted in 10.89 ± 0.9% fluorescent cells, a similar number to cells co-transfected with either 37LRP-Yn and Gal-3-Yc (12.39 ± 1.6%) or Gal-3-Yn and Gal-3-Yc (12.65 ± 1.4%), which would form a heterodimer (37LRP/Gal-3 HTD) or self-associated Gal-3 (Gal-3 HD), respectively. Similar results were obtained when Gal-3-Yn and 37LRP-Yc constructs were investigated. Mutation of C173 on Gal-3 resulted in a significant inhibitory effect on 37LRP/Gal-3 HTD (3.29 ± 1.8%; approx. 73% inhibition) and Gal-3 HD formation (6.5 ± 0.9%; approx. 49% inhibition). Co-transfection of Gal-3C173A–Yn and Gal-3C173A–Yc resulted in negligible numbers of fluorescent cells (0.8 ± 0.6%) demonstrating complete disruption of protein–protein interactions under these conditions. Data are mean ± s.e.m. of ≥3 independent experiments. (e) Binding of Gal-3 to ELISA wells coated with recombinant 37LRP or its mutant derivatives. The mean value obtained from Gal-3 binding to BSA-coated wells was subtracted from all data points. (f) Flow cytometry analysis of COS7 cells transfected with indicated pairs of BiFC constructs and analysed 36 h post-transfection. Substitution of LAMR1 lysine 166 with alanine significantly reduced its heterodimerization with Gal-3. Fluorescence intensity was evaluated as a percentage of gated cells. Data analysed by one-way ANOVA and Tukey test. ****p < 0.0001; *p < 0.05. Error bars = mean of triplicate values on three occasions ± s.e.m.

Mentions: Cells were examined 24 h post-transfection by confocal microscopy after fixation without permeabilization. Transfection with full-length YFP-fused proteins (figure 3c,d, respectively; positive control) resulted in the appearance of punctate fluorescence; non-transfected cells (figure 3b) exhibited negligible fluorescence, as did cells transfected with Yn or Yc constructs alone (figure 5d shows FACS analysis of COS7 cells; see below). Co-transfection of cells with 37LRP–Gal-3, 37LRP-37LRP or Gal-3-Gal-3 (each pair carrying complementary Yn + Yc) yielded fluorescent cells, indicating both homo- and heterodimerization of both molecules (figure 3e–g). Similar results were observed in COS7 cells, whereas in hBMEC cells, punctate fluorescence could be observed in cells co-transfected with 37LRP–Gal-3 or 37LRP-37LRP, while cells co-transfected with Gal-3–Gal-3 did not fluoresce. The reason for this is not known. Quantification and statistical analysis of surface fluorescence of transfected cells, using confocal and FACS analysis, confirmed the statistical significance of homo- and heterodimerization of both 37LRP and Gal-3 (figure 5d shows data for COS7 cells).


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)

Gal-3 residue C173 and 37LRP residue K166 are implicated in 37LRP–Gal-3 dimerization. (a) Ribbon diagram of the homodimer interaction of Gal-3 with a second Gal-3 showing the position of C173 (white arrows). (b) Ribbon diagram with superimposed molecular surface of the heterodimer interaction of Gal-3 with 37LRP with residue 166 substituted to alanine (white arrow). The mutation of K166 in 37LRP to alanine is predicted to disrupt the interaction between the two molecules (compare with figure 1a), whereas some (i.e. between R151 and K227 of 37LRP with D151 of Gal-3) are retained. (c) Superimposed structures of lactose-liganded (yellow) and non-lactose-liganded (blue) Gal-3 complexed with 37LRP highlighting the positions of K166A, R155 and Y139 (white arrows). Other features in the 37LRP/Gal-3 interface are not shown for clarity. (d) Following transfection of COS7 cells with the indicated 37LRP and Gal-3 -vYFP, -Yn or -Yc fusion proteins, cells were harvested and the number of fluorescent cells quantified by FACS and expressed as a percentage of the total number of cells counted. Expression of 37LRP-YFP, Gal-3-YFP and Gal-3C173A-YFP resulted in a significant increase in fluorescent cells compared with -Yn and -Yc control constructs (37LRP: 18.4 ± 1.1% compared with 0.28 ± 0.07% and 0.18 ± 0.05%; Gal-3: 28.4 ± 3.1% compared with 0.19 ± 0.04% and 0.21 ± 0.03%; Gal-3C173A: 25 ± 6.7% compared with 0.21 ± 0.07% and 0.26 ± 0.12%). Co-transfection of 37LRP-Yn and -Yc, which would form a 37LRP homodimer (37LRP HD), resulted in 10.89 ± 0.9% fluorescent cells, a similar number to cells co-transfected with either 37LRP-Yn and Gal-3-Yc (12.39 ± 1.6%) or Gal-3-Yn and Gal-3-Yc (12.65 ± 1.4%), which would form a heterodimer (37LRP/Gal-3 HTD) or self-associated Gal-3 (Gal-3 HD), respectively. Similar results were obtained when Gal-3-Yn and 37LRP-Yc constructs were investigated. Mutation of C173 on Gal-3 resulted in a significant inhibitory effect on 37LRP/Gal-3 HTD (3.29 ± 1.8%; approx. 73% inhibition) and Gal-3 HD formation (6.5 ± 0.9%; approx. 49% inhibition). Co-transfection of Gal-3C173A–Yn and Gal-3C173A–Yc resulted in negligible numbers of fluorescent cells (0.8 ± 0.6%) demonstrating complete disruption of protein–protein interactions under these conditions. Data are mean ± s.e.m. of ≥3 independent experiments. (e) Binding of Gal-3 to ELISA wells coated with recombinant 37LRP or its mutant derivatives. The mean value obtained from Gal-3 binding to BSA-coated wells was subtracted from all data points. (f) Flow cytometry analysis of COS7 cells transfected with indicated pairs of BiFC constructs and analysed 36 h post-transfection. Substitution of LAMR1 lysine 166 with alanine significantly reduced its heterodimerization with Gal-3. Fluorescence intensity was evaluated as a percentage of gated cells. Data analysed by one-way ANOVA and Tukey test. ****p < 0.0001; *p < 0.05. Error bars = mean of triplicate values on three occasions ± s.e.m.
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RSOB140053F5: Gal-3 residue C173 and 37LRP residue K166 are implicated in 37LRP–Gal-3 dimerization. (a) Ribbon diagram of the homodimer interaction of Gal-3 with a second Gal-3 showing the position of C173 (white arrows). (b) Ribbon diagram with superimposed molecular surface of the heterodimer interaction of Gal-3 with 37LRP with residue 166 substituted to alanine (white arrow). The mutation of K166 in 37LRP to alanine is predicted to disrupt the interaction between the two molecules (compare with figure 1a), whereas some (i.e. between R151 and K227 of 37LRP with D151 of Gal-3) are retained. (c) Superimposed structures of lactose-liganded (yellow) and non-lactose-liganded (blue) Gal-3 complexed with 37LRP highlighting the positions of K166A, R155 and Y139 (white arrows). Other features in the 37LRP/Gal-3 interface are not shown for clarity. (d) Following transfection of COS7 cells with the indicated 37LRP and Gal-3 -vYFP, -Yn or -Yc fusion proteins, cells were harvested and the number of fluorescent cells quantified by FACS and expressed as a percentage of the total number of cells counted. Expression of 37LRP-YFP, Gal-3-YFP and Gal-3C173A-YFP resulted in a significant increase in fluorescent cells compared with -Yn and -Yc control constructs (37LRP: 18.4 ± 1.1% compared with 0.28 ± 0.07% and 0.18 ± 0.05%; Gal-3: 28.4 ± 3.1% compared with 0.19 ± 0.04% and 0.21 ± 0.03%; Gal-3C173A: 25 ± 6.7% compared with 0.21 ± 0.07% and 0.26 ± 0.12%). Co-transfection of 37LRP-Yn and -Yc, which would form a 37LRP homodimer (37LRP HD), resulted in 10.89 ± 0.9% fluorescent cells, a similar number to cells co-transfected with either 37LRP-Yn and Gal-3-Yc (12.39 ± 1.6%) or Gal-3-Yn and Gal-3-Yc (12.65 ± 1.4%), which would form a heterodimer (37LRP/Gal-3 HTD) or self-associated Gal-3 (Gal-3 HD), respectively. Similar results were obtained when Gal-3-Yn and 37LRP-Yc constructs were investigated. Mutation of C173 on Gal-3 resulted in a significant inhibitory effect on 37LRP/Gal-3 HTD (3.29 ± 1.8%; approx. 73% inhibition) and Gal-3 HD formation (6.5 ± 0.9%; approx. 49% inhibition). Co-transfection of Gal-3C173A–Yn and Gal-3C173A–Yc resulted in negligible numbers of fluorescent cells (0.8 ± 0.6%) demonstrating complete disruption of protein–protein interactions under these conditions. Data are mean ± s.e.m. of ≥3 independent experiments. (e) Binding of Gal-3 to ELISA wells coated with recombinant 37LRP or its mutant derivatives. The mean value obtained from Gal-3 binding to BSA-coated wells was subtracted from all data points. (f) Flow cytometry analysis of COS7 cells transfected with indicated pairs of BiFC constructs and analysed 36 h post-transfection. Substitution of LAMR1 lysine 166 with alanine significantly reduced its heterodimerization with Gal-3. Fluorescence intensity was evaluated as a percentage of gated cells. Data analysed by one-way ANOVA and Tukey test. ****p < 0.0001; *p < 0.05. Error bars = mean of triplicate values on three occasions ± s.e.m.
Mentions: Cells were examined 24 h post-transfection by confocal microscopy after fixation without permeabilization. Transfection with full-length YFP-fused proteins (figure 3c,d, respectively; positive control) resulted in the appearance of punctate fluorescence; non-transfected cells (figure 3b) exhibited negligible fluorescence, as did cells transfected with Yn or Yc constructs alone (figure 5d shows FACS analysis of COS7 cells; see below). Co-transfection of cells with 37LRP–Gal-3, 37LRP-37LRP or Gal-3-Gal-3 (each pair carrying complementary Yn + Yc) yielded fluorescent cells, indicating both homo- and heterodimerization of both molecules (figure 3e–g). Similar results were observed in COS7 cells, whereas in hBMEC cells, punctate fluorescence could be observed in cells co-transfected with 37LRP–Gal-3 or 37LRP-37LRP, while cells co-transfected with Gal-3–Gal-3 did not fluoresce. The reason for this is not known. Quantification and statistical analysis of surface fluorescence of transfected cells, using confocal and FACS analysis, confirmed the statistical significance of homo- and heterodimerization of both 37LRP and Gal-3 (figure 5d shows data for COS7 cells).

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.

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Related in: MedlinePlus