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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 surface GAG-binding activity of α-wt and γ-wt.(A) Parental (K1) or GAG-mutant (pgsD677, pgsA745) CHO cells were incubated with the indicated concentration of wt (α-wt, γ-wt) or mutant (γ-m1, γ-m2) chemokines for 60 min at 4°C, and after extensive washing to remove free chemokine, were labelled with K15C mAb and a PE-goat anti-mouse Ig secondary antibody. Fixed cells were analyzed by flow cytometry. Values represent the mean fluorescence intensity±SD of three independent experiments performed in triplicate (B) Primary human-microvascular endothelial cells (HMVEC) were incubated with the indicated concentration of chemically synthesized (α-wt, α-m, γ-wt, γ-m1, γ-m2; left) or recombinant (γ-wtr, γ-K2427Sr, γ-m1r, right) chemokines and treated as in (A). Values represent the mean fluorescence intensity±SD of two independent experiments performed in triplicate. *p<0.05, **p<0.01, ***p<0.005 as compared to the binding obtained for the corresponding concentration of γ-wt (for γ-m1 and γ-m2) or γ-wtr (for γ-k2427Sr and γ-m1r) chemokines.
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pone-0002543-g003: Cell surface GAG-binding activity of α-wt and γ-wt.(A) Parental (K1) or GAG-mutant (pgsD677, pgsA745) CHO cells were incubated with the indicated concentration of wt (α-wt, γ-wt) or mutant (γ-m1, γ-m2) chemokines for 60 min at 4°C, and after extensive washing to remove free chemokine, were labelled with K15C mAb and a PE-goat anti-mouse Ig secondary antibody. Fixed cells were analyzed by flow cytometry. Values represent the mean fluorescence intensity±SD of three independent experiments performed in triplicate (B) Primary human-microvascular endothelial cells (HMVEC) were incubated with the indicated concentration of chemically synthesized (α-wt, α-m, γ-wt, γ-m1, γ-m2; left) or recombinant (γ-wtr, γ-K2427Sr, γ-m1r, right) chemokines and treated as in (A). Values represent the mean fluorescence intensity±SD of two independent experiments performed in triplicate. *p<0.05, **p<0.01, ***p<0.005 as compared to the binding obtained for the corresponding concentration of γ-wt (for γ-m1 and γ-m2) or γ-wtr (for γ-k2427Sr and γ-m1r) chemokines.

Mentions: Recognition of CXCL12 proteins by the K15C mAb is not masked by their interaction with GAG [23]. Using this mAb, we observed that the adsorption on the CXCR4 negative CHO-K1 cells was greatly increased for γ-wt as compared to α-wt (Figure 3A). Of note, and of particular biological relevance, we found that γ-wt also binds onto primary, human-microvascular endothelial cells (HMVEC) with the highest efficiency as compared to α-wt (Figure 3B). It is interesting to note that while the γ-m1 mutant protein retained the capacity to bind on CHO-K1 parental cells, this capacity was notably decreased in HMVEC, a divergence that could be accounted for by differences in the amount and nature of negatively charged structures that contribute to CXCL12 binding in both cell types. Interestingly, a recombinant CXCL12γ derivative carrying K24S and K27S substitutions (γ-K2427Sr) that invalidate the HS-binding consensus site located in the core of the protein [25] (Figure 3B) also exhibits a reduced capacity to bind on HMVEC cells as compared to the corresponding γ-wtr protein. In keeping with these results, the γ-m2 was virtually devoid of any binding capacity on both cell types. The specificity of CXCL12γ binding to GAG was assessed in mutant CHO-pgsD677 cells, derived from CHO-K1 cells, which lack both N-acetylglucosaminyltransferase and glucuronyltransferase activities and are deficient for HS synthesis, the binding of γ-m1 became undetectable, whereas a residual signal was still detectable for γ-wt (Figure 3A), and at a similar extent for the γ-K2427Sr (data not shown). Comparable phenomena were observed in CHO-pgsA745 cells, which lack any GAG synthesis due to a xylose-transferase mutation. The residual binding of γ-wt (or γ-K2427Sr) observed at high concentrations of the chemokine (250 nM), in the absence of any synthesized GAG, can be accounted for by the interaction of the C-ter domain with other negatively charged structures, like the abundant sulphate glycosphingolipids (sulphatides) that have been previously shown to interact at high concentrations with α-wt [32]. The enzymatic degradation of HS in CHO-K1 cells either by heparinase or heparitinase I confirmed the apparent selectiveness of the HS/γ-wt interaction at the cell surface, whereas degradation of chondroitin sulfate had no effect (Figure S1).


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 surface GAG-binding activity of α-wt and γ-wt.(A) Parental (K1) or GAG-mutant (pgsD677, pgsA745) CHO cells were incubated with the indicated concentration of wt (α-wt, γ-wt) or mutant (γ-m1, γ-m2) chemokines for 60 min at 4°C, and after extensive washing to remove free chemokine, were labelled with K15C mAb and a PE-goat anti-mouse Ig secondary antibody. Fixed cells were analyzed by flow cytometry. Values represent the mean fluorescence intensity±SD of three independent experiments performed in triplicate (B) Primary human-microvascular endothelial cells (HMVEC) were incubated with the indicated concentration of chemically synthesized (α-wt, α-m, γ-wt, γ-m1, γ-m2; left) or recombinant (γ-wtr, γ-K2427Sr, γ-m1r, right) chemokines and treated as in (A). Values represent the mean fluorescence intensity±SD of two independent experiments performed in triplicate. *p<0.05, **p<0.01, ***p<0.005 as compared to the binding obtained for the corresponding concentration of γ-wt (for γ-m1 and γ-m2) or γ-wtr (for γ-k2427Sr and γ-m1r) chemokines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481281&req=5

pone-0002543-g003: Cell surface GAG-binding activity of α-wt and γ-wt.(A) Parental (K1) or GAG-mutant (pgsD677, pgsA745) CHO cells were incubated with the indicated concentration of wt (α-wt, γ-wt) or mutant (γ-m1, γ-m2) chemokines for 60 min at 4°C, and after extensive washing to remove free chemokine, were labelled with K15C mAb and a PE-goat anti-mouse Ig secondary antibody. Fixed cells were analyzed by flow cytometry. Values represent the mean fluorescence intensity±SD of three independent experiments performed in triplicate (B) Primary human-microvascular endothelial cells (HMVEC) were incubated with the indicated concentration of chemically synthesized (α-wt, α-m, γ-wt, γ-m1, γ-m2; left) or recombinant (γ-wtr, γ-K2427Sr, γ-m1r, right) chemokines and treated as in (A). Values represent the mean fluorescence intensity±SD of two independent experiments performed in triplicate. *p<0.05, **p<0.01, ***p<0.005 as compared to the binding obtained for the corresponding concentration of γ-wt (for γ-m1 and γ-m2) or γ-wtr (for γ-k2427Sr and γ-m1r) chemokines.
Mentions: Recognition of CXCL12 proteins by the K15C mAb is not masked by their interaction with GAG [23]. Using this mAb, we observed that the adsorption on the CXCR4 negative CHO-K1 cells was greatly increased for γ-wt as compared to α-wt (Figure 3A). Of note, and of particular biological relevance, we found that γ-wt also binds onto primary, human-microvascular endothelial cells (HMVEC) with the highest efficiency as compared to α-wt (Figure 3B). It is interesting to note that while the γ-m1 mutant protein retained the capacity to bind on CHO-K1 parental cells, this capacity was notably decreased in HMVEC, a divergence that could be accounted for by differences in the amount and nature of negatively charged structures that contribute to CXCL12 binding in both cell types. Interestingly, a recombinant CXCL12γ derivative carrying K24S and K27S substitutions (γ-K2427Sr) that invalidate the HS-binding consensus site located in the core of the protein [25] (Figure 3B) also exhibits a reduced capacity to bind on HMVEC cells as compared to the corresponding γ-wtr protein. In keeping with these results, the γ-m2 was virtually devoid of any binding capacity on both cell types. The specificity of CXCL12γ binding to GAG was assessed in mutant CHO-pgsD677 cells, derived from CHO-K1 cells, which lack both N-acetylglucosaminyltransferase and glucuronyltransferase activities and are deficient for HS synthesis, the binding of γ-m1 became undetectable, whereas a residual signal was still detectable for γ-wt (Figure 3A), and at a similar extent for the γ-K2427Sr (data not shown). Comparable phenomena were observed in CHO-pgsA745 cells, which lack any GAG synthesis due to a xylose-transferase mutation. The residual binding of γ-wt (or γ-K2427Sr) observed at high concentrations of the chemokine (250 nM), in the absence of any synthesized GAG, can be accounted for by the interaction of the C-ter domain with other negatively charged structures, like the abundant sulphate glycosphingolipids (sulphatides) that have been previously shown to interact at high concentrations with α-wt [32]. The enzymatic degradation of HS in CHO-K1 cells either by heparinase or heparitinase I confirmed the apparent selectiveness of the HS/γ-wt interaction at the cell surface, whereas degradation of chondroitin sulfate had no effect (Figure S1).

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