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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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Intraperitoneal recruitment of leukocytes induced by α-wt and γ-wt.BALB/c mice were intraperitoneally injected with identical volumes (300 µl) of either PBS (control) or a 30 nM solution of each chemokine. Cells that accumulated into the peritoneal cavity were recovered after 6 hr (A) or 15 hr (B) of treatment. Leukocyte subpopulations were characterized by flow cytometry using specific cell markers. Cell influx was calculated as the x-fold increase over values obtained in PBS-treated mice. PBS reference values were arbitrary set to one (dotted lines). In inset, total number of recovered peritoneal cells. Results are mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.005 as compared to PBS-treated mice.
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pone-0002543-g006: Intraperitoneal recruitment of leukocytes induced by α-wt and γ-wt.BALB/c mice were intraperitoneally injected with identical volumes (300 µl) of either PBS (control) or a 30 nM solution of each chemokine. Cells that accumulated into the peritoneal cavity were recovered after 6 hr (A) or 15 hr (B) of treatment. Leukocyte subpopulations were characterized by flow cytometry using specific cell markers. Cell influx was calculated as the x-fold increase over values obtained in PBS-treated mice. PBS reference values were arbitrary set to one (dotted lines). In inset, total number of recovered peritoneal cells. Results are mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.005 as compared to PBS-treated mice.

Mentions: The singular structural and functional features that distinguish γ-wt from α-wt prompted us to compare their respective capacities to promote haptotactic attraction of cells in vivo using chemokine concentrations in the range of these that consistently induce chemotaxis in vitro. To this purpose, we first evaluated the migration of leukocytes into the peritoneal cavity of BALB/c mice following administration of an endotoxin-free, 30 nM solution, of γ-wt or α-wt at 6 hours (hr) (Figure 6A) or 15 hr (Figure 6B) post-injection. After 6 hr of treatment, both α-wt and γ-wt induced a significant and equivalent increase of the absolute number of cells (fold increase 2.99±0.18 and 3±0.18, for α-wt and γ-wt respectively, as compared to control PBS injected animals), that was accounted for by the recruitment of myeloid cells, including both neutrophils and macrophages (Gr-1+CD11b+CD19-, 6.27±1.45 for α-wt and 8.72±4.95 for γ-wt). The situation was radically different at 15 hr post-injection as solely γ-wt promoted a sustained accumulation of leukocytes (fold increase 4.96±1.35 as compared to PBS-injected animals). At this time point, cell increase was basically accounted for by T lymphocytes (CD3+, 4.38±1.65) and B lymphocytes corresponding to B1 (CD19+CD11b+, 5.09±2.16) and B2 (CD19+CD11b-, 3.9±1) subpopulations. Importantly, both α-m and γ-m2, that totally lack HS-binding activity, failed to attract leukocytes either at 6 hr or 15 hr time points.


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)

Intraperitoneal recruitment of leukocytes induced by α-wt and γ-wt.BALB/c mice were intraperitoneally injected with identical volumes (300 µl) of either PBS (control) or a 30 nM solution of each chemokine. Cells that accumulated into the peritoneal cavity were recovered after 6 hr (A) or 15 hr (B) of treatment. Leukocyte subpopulations were characterized by flow cytometry using specific cell markers. Cell influx was calculated as the x-fold increase over values obtained in PBS-treated mice. PBS reference values were arbitrary set to one (dotted lines). In inset, total number of recovered peritoneal cells. Results are mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.005 as compared to PBS-treated mice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481281&req=5

pone-0002543-g006: Intraperitoneal recruitment of leukocytes induced by α-wt and γ-wt.BALB/c mice were intraperitoneally injected with identical volumes (300 µl) of either PBS (control) or a 30 nM solution of each chemokine. Cells that accumulated into the peritoneal cavity were recovered after 6 hr (A) or 15 hr (B) of treatment. Leukocyte subpopulations were characterized by flow cytometry using specific cell markers. Cell influx was calculated as the x-fold increase over values obtained in PBS-treated mice. PBS reference values were arbitrary set to one (dotted lines). In inset, total number of recovered peritoneal cells. Results are mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.005 as compared to PBS-treated mice.
Mentions: The singular structural and functional features that distinguish γ-wt from α-wt prompted us to compare their respective capacities to promote haptotactic attraction of cells in vivo using chemokine concentrations in the range of these that consistently induce chemotaxis in vitro. To this purpose, we first evaluated the migration of leukocytes into the peritoneal cavity of BALB/c mice following administration of an endotoxin-free, 30 nM solution, of γ-wt or α-wt at 6 hours (hr) (Figure 6A) or 15 hr (Figure 6B) post-injection. After 6 hr of treatment, both α-wt and γ-wt induced a significant and equivalent increase of the absolute number of cells (fold increase 2.99±0.18 and 3±0.18, for α-wt and γ-wt respectively, as compared to control PBS injected animals), that was accounted for by the recruitment of myeloid cells, including both neutrophils and macrophages (Gr-1+CD11b+CD19-, 6.27±1.45 for α-wt and 8.72±4.95 for γ-wt). The situation was radically different at 15 hr post-injection as solely γ-wt promoted a sustained accumulation of leukocytes (fold increase 4.96±1.35 as compared to PBS-injected animals). At this time point, cell increase was basically accounted for by T lymphocytes (CD3+, 4.38±1.65) and B lymphocytes corresponding to B1 (CD19+CD11b+, 5.09±2.16) and B2 (CD19+CD11b-, 3.9±1) subpopulations. Importantly, both α-m and γ-m2, that totally lack HS-binding activity, failed to attract leukocytes either at 6 hr or 15 hr time points.

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