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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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Angiogenic effect of α-wt or γ-wt and derivatives chemokines.(A) Haematoxylin-eosin staining of Matrigel plugs containing 10 nM of the indicated chemokine and analyzed at day 10 from implantation in BALB/c mice. Data are representative of ten independent experiments. In frame, vessel-like structures forming arround a central lumen. In the table, quantification of the number of migrated cells (±SD) into the Matrigel after 6 (p = 0.0459) or ten days (p = 0.0009) from implantation. (B) Immunofluorescent detection of PECAM-1/CD31+ endothelial cells in Matrigel neovessels with DAPI nuclear counter-labelling. Original magnifications ×100 (A) and ×600 (B).
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pone-0002543-g007: Angiogenic effect of α-wt or γ-wt and derivatives chemokines.(A) Haematoxylin-eosin staining of Matrigel plugs containing 10 nM of the indicated chemokine and analyzed at day 10 from implantation in BALB/c mice. Data are representative of ten independent experiments. In frame, vessel-like structures forming arround a central lumen. In the table, quantification of the number of migrated cells (±SD) into the Matrigel after 6 (p = 0.0459) or ten days (p = 0.0009) from implantation. (B) Immunofluorescent detection of PECAM-1/CD31+ endothelial cells in Matrigel neovessels with DAPI nuclear counter-labelling. Original magnifications ×100 (A) and ×600 (B).

Mentions: CXCL12α has the capacity to promote de novo formation of vessels, a property related to the ability of this chemokine to regulate both the traffic and survival of stem and progenitor cells [19], [35]. Thus, we compared the ability of γ-wt and α-wt to attract endothelial progenitors and initiate the angiogenic process. To this purpose, Matrigel plugs loaded with an endotoxin-free, 10 nM solution of either γ-wt or α-wt were implanted subcutaneously in BALB/c mice. Whereas virtually no infiltrating cells were detectable in control PBS Matrigel plugs (data not shown), γ-wt induced a more robust response (3-fold increase, p = 0.0009 Figure 7A) than α-wt regarding the total number of cells attracted at day 10 post-implantation. Vessel-like cellular tubes within Matrigel implants were particularly abundant in γ-wt-loaded implants. These vessel-like structures were mainly composed of endothelial cells expressing CD31/Platelet endothelial cell adhesion molecule (PECAM-1) (Figure 7B), a molecule that defines endothelial cells. Similar results were observed 6 days post-implantation, the minimal time-point required to observe angiogenesis using this technique [36] (p = 0.0459, Figure 7A table). Of note, both α-m and γ-m2 display a reduced capacity to promote cell infiltration and angiogenesis in Matrigel implants, demonstrating the importance of GAG binding for this process.


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)

Angiogenic effect of α-wt or γ-wt and derivatives chemokines.(A) Haematoxylin-eosin staining of Matrigel plugs containing 10 nM of the indicated chemokine and analyzed at day 10 from implantation in BALB/c mice. Data are representative of ten independent experiments. In frame, vessel-like structures forming arround a central lumen. In the table, quantification of the number of migrated cells (±SD) into the Matrigel after 6 (p = 0.0459) or ten days (p = 0.0009) from implantation. (B) Immunofluorescent detection of PECAM-1/CD31+ endothelial cells in Matrigel neovessels with DAPI nuclear counter-labelling. Original magnifications ×100 (A) and ×600 (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481281&req=5

pone-0002543-g007: Angiogenic effect of α-wt or γ-wt and derivatives chemokines.(A) Haematoxylin-eosin staining of Matrigel plugs containing 10 nM of the indicated chemokine and analyzed at day 10 from implantation in BALB/c mice. Data are representative of ten independent experiments. In frame, vessel-like structures forming arround a central lumen. In the table, quantification of the number of migrated cells (±SD) into the Matrigel after 6 (p = 0.0459) or ten days (p = 0.0009) from implantation. (B) Immunofluorescent detection of PECAM-1/CD31+ endothelial cells in Matrigel neovessels with DAPI nuclear counter-labelling. Original magnifications ×100 (A) and ×600 (B).
Mentions: CXCL12α has the capacity to promote de novo formation of vessels, a property related to the ability of this chemokine to regulate both the traffic and survival of stem and progenitor cells [19], [35]. Thus, we compared the ability of γ-wt and α-wt to attract endothelial progenitors and initiate the angiogenic process. To this purpose, Matrigel plugs loaded with an endotoxin-free, 10 nM solution of either γ-wt or α-wt were implanted subcutaneously in BALB/c mice. Whereas virtually no infiltrating cells were detectable in control PBS Matrigel plugs (data not shown), γ-wt induced a more robust response (3-fold increase, p = 0.0009 Figure 7A) than α-wt regarding the total number of cells attracted at day 10 post-implantation. Vessel-like cellular tubes within Matrigel implants were particularly abundant in γ-wt-loaded implants. These vessel-like structures were mainly composed of endothelial cells expressing CD31/Platelet endothelial cell adhesion molecule (PECAM-1) (Figure 7B), a molecule that defines endothelial cells. Similar results were observed 6 days post-implantation, the minimal time-point required to observe angiogenesis using this technique [36] (p = 0.0459, Figure 7A table). Of note, both α-m and γ-m2 display a reduced capacity to promote cell infiltration and angiogenesis in Matrigel implants, demonstrating the importance of GAG binding for this process.

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