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The CXCL12gamma chemokine displays unprecedented structural and functional properties that make it a paradigm of chemoattractant proteins.

Rueda P, Balabanian K, Lagane B, Staropoli I, Chow K, Levoye A, Laguri C, Sadir R, Delaunay T, Izquierdo E, Pablos JL, Lendinez E, Caruz A, Franco D, Baleux F, Lortat-Jacob H, Arenzana-Seisdedos F - PLoS ONE (2008)

Bottom Line: In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment.We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain.The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Experimental, Universidad de Jaén, Jaén, Spain.

ABSTRACT
The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

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Cell signalling through CXCR4 induced either by α-wt, γ-wt or derivative chemokines.(A) CXCL12-induced [35S]GTPγS binding to membranes from lymphoblastoid A3.01 T cells. Membranes were incubated in assay buffer containing 0.1 nM [35S]GTPγS and the indicated concentrations of the corresponding chemokine. Data represents the percentage (mean±SD) of the maximal [35S]GTPγS binding obtained (100%), and are representative of three independent experiments. (B) Dose-dependent CXCL12-induced chemotaxis of A3.01 cells (upper panel) or primary CD4+ T lymphocytes (lower panel). Results (mean±SD) are from two independent experiments and are expressed as percentage of input cells that migrated to the lower chamber.
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pone-0002543-g005: Cell signalling through CXCR4 induced either by α-wt, γ-wt or derivative chemokines.(A) CXCL12-induced [35S]GTPγS binding to membranes from lymphoblastoid A3.01 T cells. Membranes were incubated in assay buffer containing 0.1 nM [35S]GTPγS and the indicated concentrations of the corresponding chemokine. Data represents the percentage (mean±SD) of the maximal [35S]GTPγS binding obtained (100%), and are representative of three independent experiments. (B) Dose-dependent CXCL12-induced chemotaxis of A3.01 cells (upper panel) or primary CD4+ T lymphocytes (lower panel). Results (mean±SD) are from two independent experiments and are expressed as percentage of input cells that migrated to the lower chamber.

Mentions: The pharmacological properties of γ-wt regarding its interaction with CXCR4 were investigated on transformed A3.01 T cells and primary unstimulated CD4+ T lymphocytes (Figure 5). Both lymphoid cell types lack detectable levels of HS as assessed by immunostaining with the specific 10E4 anti-HS mAb (data not shown) and permits the strict analysis of CXCL12/CXCR4 interaction per se. The capacity of γ-wt to set in motion CXCR4-dependent activation cell pathways was first assessed by measuring the amount of the non-hydrolysable [S35]-GTPγ associated to activated Gα subunits, the earliest cell-signal event induced by GPCR agonists. We observed that γ-wt was less potent than α-wt to activate CXCR4 (Figure 5A), which is in full agreement with the reduced binding affinity (one order of magnitude) shown by γ-wt for CXCR4 as compared to α-wt [28]. When concentration of γ-wt was raised and the occupancy of CXCR4 was enhanced, G protein activation increased to levels comparable to those measured with α-wt. However, presumably due to the sustained reduced potency of γ-wt to induce GTPγS binding, we were unable ro reach saturation for γ-wt, thus precluding determination of Emax and EC50 values for this chemokine in ths assay. For α-wt isoform, we deduced an EC50 value equal to 19.65 nM. CXCL12α has been shown to bind to- and activates CXCR7 [3], [4], [33]. We thus analysed the ability of γ-wt to compete with a C-ter biotinylated CXCL12α chemokine for binding to CXCR7. As shown in Figure S2, α-wt and γ-wt isoforms similarly bound to CXCR7 (IC50 = 6.56 nM and 10.37 nM for α-wt and γ-wt, respectively).


The CXCL12gamma chemokine displays unprecedented structural and functional properties that make it a paradigm of chemoattractant proteins.

Rueda P, Balabanian K, Lagane B, Staropoli I, Chow K, Levoye A, Laguri C, Sadir R, Delaunay T, Izquierdo E, Pablos JL, Lendinez E, Caruz A, Franco D, Baleux F, Lortat-Jacob H, Arenzana-Seisdedos F - PLoS ONE (2008)

Cell signalling through CXCR4 induced either by α-wt, γ-wt or derivative chemokines.(A) CXCL12-induced [35S]GTPγS binding to membranes from lymphoblastoid A3.01 T cells. Membranes were incubated in assay buffer containing 0.1 nM [35S]GTPγS and the indicated concentrations of the corresponding chemokine. Data represents the percentage (mean±SD) of the maximal [35S]GTPγS binding obtained (100%), and are representative of three independent experiments. (B) Dose-dependent CXCL12-induced chemotaxis of A3.01 cells (upper panel) or primary CD4+ T lymphocytes (lower panel). Results (mean±SD) are from two independent experiments and are expressed as percentage of input cells that migrated to the lower chamber.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481281&req=5

pone-0002543-g005: Cell signalling through CXCR4 induced either by α-wt, γ-wt or derivative chemokines.(A) CXCL12-induced [35S]GTPγS binding to membranes from lymphoblastoid A3.01 T cells. Membranes were incubated in assay buffer containing 0.1 nM [35S]GTPγS and the indicated concentrations of the corresponding chemokine. Data represents the percentage (mean±SD) of the maximal [35S]GTPγS binding obtained (100%), and are representative of three independent experiments. (B) Dose-dependent CXCL12-induced chemotaxis of A3.01 cells (upper panel) or primary CD4+ T lymphocytes (lower panel). Results (mean±SD) are from two independent experiments and are expressed as percentage of input cells that migrated to the lower chamber.
Mentions: The pharmacological properties of γ-wt regarding its interaction with CXCR4 were investigated on transformed A3.01 T cells and primary unstimulated CD4+ T lymphocytes (Figure 5). Both lymphoid cell types lack detectable levels of HS as assessed by immunostaining with the specific 10E4 anti-HS mAb (data not shown) and permits the strict analysis of CXCL12/CXCR4 interaction per se. The capacity of γ-wt to set in motion CXCR4-dependent activation cell pathways was first assessed by measuring the amount of the non-hydrolysable [S35]-GTPγ associated to activated Gα subunits, the earliest cell-signal event induced by GPCR agonists. We observed that γ-wt was less potent than α-wt to activate CXCR4 (Figure 5A), which is in full agreement with the reduced binding affinity (one order of magnitude) shown by γ-wt for CXCR4 as compared to α-wt [28]. When concentration of γ-wt was raised and the occupancy of CXCR4 was enhanced, G protein activation increased to levels comparable to those measured with α-wt. However, presumably due to the sustained reduced potency of γ-wt to induce GTPγS binding, we were unable ro reach saturation for γ-wt, thus precluding determination of Emax and EC50 values for this chemokine in ths assay. For α-wt isoform, we deduced an EC50 value equal to 19.65 nM. CXCL12α has been shown to bind to- and activates CXCR7 [3], [4], [33]. We thus analysed the ability of γ-wt to compete with a C-ter biotinylated CXCL12α chemokine for binding to CXCR7. As shown in Figure S2, α-wt and γ-wt isoforms similarly bound to CXCR7 (IC50 = 6.56 nM and 10.37 nM for α-wt and γ-wt, respectively).

Bottom Line: In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment.We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain.The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Experimental, Universidad de Jaén, Jaén, Spain.

ABSTRACT
The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

Show MeSH
Related in: MedlinePlus