Limits...
The Anti-inflammatory Protein TSG-6 Regulates Chemokine Function by Inhibiting Chemokine/Glycosaminoglycan Interactions.

Dyer DP, Salanga CL, Johns SC, Valdambrini E, Fuster MM, Milner CM, Day AJ, Handel TM - J. Biol. Chem. (2016)

Bottom Line: We also show that the Link_TSG6-binding sites on chemokines overlap with chemokine GAG-binding sites, and that the affinities of Link_TSG6 for these chemokines (KD values 1-85 nm) broadly correlate with chemokine-GAG affinities.Link_TSG6 also inhibits chemokine presentation on endothelial cells not only through a direct interaction with chemokines but also by binding and therefore masking the availability of GAGs.Along with previous work, these findings suggest that TSG-6 functions as a pluripotent regulator of chemokines by modulating chemokine/GAG interactions, which may be a major mechanism by which TSG-6 produces its anti-inflammatory effects in vivo.

View Article: PubMed Central - PubMed

Affiliation: From the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093-0684, the Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, Scotland, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Correlation between chemokine affinity for Link_TSG6 and HS. Kinetic affinity estimates for each chemokine binding to immobilized HS (56, 57) or Link_TSG6 are plotted, as calculated using SPR. Data shown are from individual measurements representative of two independent experiments. CXCL8 is excluded from this figure as previous studies did not enable evaluation of a robust affinity estimate for the CXCL8/HS interaction (57).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4933465&req=5

Figure 3: Correlation between chemokine affinity for Link_TSG6 and HS. Kinetic affinity estimates for each chemokine binding to immobilized HS (56, 57) or Link_TSG6 are plotted, as calculated using SPR. Data shown are from individual measurements representative of two independent experiments. CXCL8 is excluded from this figure as previous studies did not enable evaluation of a robust affinity estimate for the CXCL8/HS interaction (57).

Mentions: Previous studies revealed that the GAG-binding region of CXCL8 is involved in its interaction with Link_TSG6 (30), leading us to hypothesize that GAG-binding domains of the chemokines identified above will also be important for Link_TSG6-chemokine complex formation. To this end, we tested whether well characterized GAG-binding deficient chemokine mutants, known to have significantly impaired interactions with heparin or HS, also show a reduced affinity for Link_TSG6. As demonstrated by the sensorgrams in Fig. 2 and the calculated affinities in Table 1, this turned out to be the case. Binding to Link_TSG6 was effectively abolished for the R18A/K19A mutant of CCL2 (59), the R44A/K45A/R47A mutant of CCL5 (60), and a C-terminal deletion mutant of CCL21 (53). Although interaction with Link_TSG6 could be detected with the CCL7 mutant K18A/K19A/K22A (GAG-binding (56)), there was insufficient signal to calculate a reliable dissociation constant, indicative of a weak interaction. The CXCL12 GAG-binding deficient mutant K24S/H25S/K27S (61) could still bind Link_TSG6 suggesting additional epitopes contribute to the interaction; nevertheless, the affinity was ∼55-fold lower than WT CXCL12. Similarly, the CCL27 K25A mutant displayed an ∼9-fold reduction in affinity for Link_TSG6, and the retention of residual binding is unsurprising as a single point mutation would not be expected to completely eliminate GAG or protein binding. Finally, we could detect only a weak interaction between Link_TSG6 and CCL3 (15 mm (30)), similar to the weak-to-no interaction reported for this chemokine with GAGs (57, 62). Recently, we published apparent affinities determined by SPR for the interaction of HS with CXCL4, CXCL8, CXCL11, CXCL12, CCL2, CCL5, CCL7 (56, 57), and CCL27. A plot of these apparent affinities against Link_TSG6-chemokine affinities demonstrates that there is a positive correlation (Fig. 3). Taken together, these data suggest that the chemokine-binding sites for Link_TSG6 and GAGs overlap, similar to the situation with CXCL8 (30).


The Anti-inflammatory Protein TSG-6 Regulates Chemokine Function by Inhibiting Chemokine/Glycosaminoglycan Interactions.

Dyer DP, Salanga CL, Johns SC, Valdambrini E, Fuster MM, Milner CM, Day AJ, Handel TM - J. Biol. Chem. (2016)

Correlation between chemokine affinity for Link_TSG6 and HS. Kinetic affinity estimates for each chemokine binding to immobilized HS (56, 57) or Link_TSG6 are plotted, as calculated using SPR. Data shown are from individual measurements representative of two independent experiments. CXCL8 is excluded from this figure as previous studies did not enable evaluation of a robust affinity estimate for the CXCL8/HS interaction (57).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4933465&req=5

Figure 3: Correlation between chemokine affinity for Link_TSG6 and HS. Kinetic affinity estimates for each chemokine binding to immobilized HS (56, 57) or Link_TSG6 are plotted, as calculated using SPR. Data shown are from individual measurements representative of two independent experiments. CXCL8 is excluded from this figure as previous studies did not enable evaluation of a robust affinity estimate for the CXCL8/HS interaction (57).
Mentions: Previous studies revealed that the GAG-binding region of CXCL8 is involved in its interaction with Link_TSG6 (30), leading us to hypothesize that GAG-binding domains of the chemokines identified above will also be important for Link_TSG6-chemokine complex formation. To this end, we tested whether well characterized GAG-binding deficient chemokine mutants, known to have significantly impaired interactions with heparin or HS, also show a reduced affinity for Link_TSG6. As demonstrated by the sensorgrams in Fig. 2 and the calculated affinities in Table 1, this turned out to be the case. Binding to Link_TSG6 was effectively abolished for the R18A/K19A mutant of CCL2 (59), the R44A/K45A/R47A mutant of CCL5 (60), and a C-terminal deletion mutant of CCL21 (53). Although interaction with Link_TSG6 could be detected with the CCL7 mutant K18A/K19A/K22A (GAG-binding (56)), there was insufficient signal to calculate a reliable dissociation constant, indicative of a weak interaction. The CXCL12 GAG-binding deficient mutant K24S/H25S/K27S (61) could still bind Link_TSG6 suggesting additional epitopes contribute to the interaction; nevertheless, the affinity was ∼55-fold lower than WT CXCL12. Similarly, the CCL27 K25A mutant displayed an ∼9-fold reduction in affinity for Link_TSG6, and the retention of residual binding is unsurprising as a single point mutation would not be expected to completely eliminate GAG or protein binding. Finally, we could detect only a weak interaction between Link_TSG6 and CCL3 (15 mm (30)), similar to the weak-to-no interaction reported for this chemokine with GAGs (57, 62). Recently, we published apparent affinities determined by SPR for the interaction of HS with CXCL4, CXCL8, CXCL11, CXCL12, CCL2, CCL5, CCL7 (56, 57), and CCL27. A plot of these apparent affinities against Link_TSG6-chemokine affinities demonstrates that there is a positive correlation (Fig. 3). Taken together, these data suggest that the chemokine-binding sites for Link_TSG6 and GAGs overlap, similar to the situation with CXCL8 (30).

Bottom Line: We also show that the Link_TSG6-binding sites on chemokines overlap with chemokine GAG-binding sites, and that the affinities of Link_TSG6 for these chemokines (KD values 1-85 nm) broadly correlate with chemokine-GAG affinities.Link_TSG6 also inhibits chemokine presentation on endothelial cells not only through a direct interaction with chemokines but also by binding and therefore masking the availability of GAGs.Along with previous work, these findings suggest that TSG-6 functions as a pluripotent regulator of chemokines by modulating chemokine/GAG interactions, which may be a major mechanism by which TSG-6 produces its anti-inflammatory effects in vivo.

View Article: PubMed Central - PubMed

Affiliation: From the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093-0684, the Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, Scotland, United Kingdom.

No MeSH data available.


Related in: MedlinePlus