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Structure and function of A41, a vaccinia virus chemokine binding protein.

Bahar MW, Kenyon JC, Putz MM, Abrescia NG, Pease JE, Wise EL, Stuart DI, Smith GL, Grimes JM - PLoS Pathog. (2008)

Bottom Line: Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors.Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding.The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

View Article: PubMed Central - PubMed

Affiliation: The Division of Structural Biology and The Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

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Inhibition of Binding of Chemokines to A41 in the Presence of Heparin Sodium Salt(A) A41VOTE was immobilised on a sensor chip and increasing concentrations (0, 31.25, 62.5, 125, 250, 500 ng/ml) of heparin sodium salt (low molecular weight, MW 4,000–6,000, Sigma) were mixed with hCCL28 and passed across the sensor chip. Chemokine binding to A41 is shown by sensorgrams expressed as RU. (B,C). Heparin inhibits binding of human CCL21, CCL25, CCL26 and CCL28 to A41VOTE (B) and A41E. coli (C) (filled symbols depict activity with chemokine only). (D) Binding of human CCL25 to A41VOTE in the presence of various sulphated GAGs (heparin, heparan sulphate [heparan s], chondroitin sulphate B [chond s B, = dermatan], chondroitin sulphate C [chond s C], hyaluronic acid [hyalur ac] and dextran sulphate [dextran s]) at 125 ng/ml (grey bars) and 250 ng/ml (white bars). Data are expressed as the percentage binding compared to CCL25 alone.
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ppat-0040005-g002: Inhibition of Binding of Chemokines to A41 in the Presence of Heparin Sodium Salt(A) A41VOTE was immobilised on a sensor chip and increasing concentrations (0, 31.25, 62.5, 125, 250, 500 ng/ml) of heparin sodium salt (low molecular weight, MW 4,000–6,000, Sigma) were mixed with hCCL28 and passed across the sensor chip. Chemokine binding to A41 is shown by sensorgrams expressed as RU. (B,C). Heparin inhibits binding of human CCL21, CCL25, CCL26 and CCL28 to A41VOTE (B) and A41E. coli (C) (filled symbols depict activity with chemokine only). (D) Binding of human CCL25 to A41VOTE in the presence of various sulphated GAGs (heparin, heparan sulphate [heparan s], chondroitin sulphate B [chond s B, = dermatan], chondroitin sulphate C [chond s C], hyaluronic acid [hyalur ac] and dextran sulphate [dextran s]) at 125 ng/ml (grey bars) and 250 ng/ml (white bars). Data are expressed as the percentage binding compared to CCL25 alone.

Mentions: The failure of A41 to inhibit chemokine-induced chemotaxis and the Kd values for its interaction with chemokines (Table 2), suggest that A41 might function by blocking the interaction of chemokines with GAGs. This was investigated by immobilising A41 on a sensor chip, passing hCCL28 across the chip alone or in the presence of increasing concentrations of heparin (sodium salt, MW 4,000–6,000) and measuring hCCL28 binding by BIACORE. The interaction of A41 and hCCL28 was inhibited in a dose-dependent manner and complete inhibition was achieved with 500 ng/ml heparin (Figure 2A). Similarly, heparin inhibited the binding of hCCL21, hCCL25, hCCL26 and hCCL28 to immobilised A41 in a dose-dependent manner when A41 was produced in either mammalian cells (Figure 2B) or E. coli (Figure 2C). The concentration of heparin used to achieve 50% inhibition was ∼100 ng/ml (∼20 nM) and this was lower than used by other investigators to achieve disruption of the M-T7 chemokine binding protein from MYXV with RANTES [15] or the interaction of chemokines with the endothelial cell surface [26]. Apart from heparin, other sulphated GAGs were also tested for their ability to inhibit binding of chemokines to A41. Heparin and dextran sulphate inhibited the hCCL25-A41 interaction, but heparan sulphate, chondroitin sulphate B, chondroitin sulphate C and hyaluronic acid did not (Figure 2D). Similar results were obtained with A41 produced from E. coli or mammalian cells and with hCCL21, hCCL26 and hCCL28 (data not shown). Notably each GAG able to inhibit the hCCL25-A41 interaction was more highly charged than those that did not inhibit.


Structure and function of A41, a vaccinia virus chemokine binding protein.

Bahar MW, Kenyon JC, Putz MM, Abrescia NG, Pease JE, Wise EL, Stuart DI, Smith GL, Grimes JM - PLoS Pathog. (2008)

Inhibition of Binding of Chemokines to A41 in the Presence of Heparin Sodium Salt(A) A41VOTE was immobilised on a sensor chip and increasing concentrations (0, 31.25, 62.5, 125, 250, 500 ng/ml) of heparin sodium salt (low molecular weight, MW 4,000–6,000, Sigma) were mixed with hCCL28 and passed across the sensor chip. Chemokine binding to A41 is shown by sensorgrams expressed as RU. (B,C). Heparin inhibits binding of human CCL21, CCL25, CCL26 and CCL28 to A41VOTE (B) and A41E. coli (C) (filled symbols depict activity with chemokine only). (D) Binding of human CCL25 to A41VOTE in the presence of various sulphated GAGs (heparin, heparan sulphate [heparan s], chondroitin sulphate B [chond s B, = dermatan], chondroitin sulphate C [chond s C], hyaluronic acid [hyalur ac] and dextran sulphate [dextran s]) at 125 ng/ml (grey bars) and 250 ng/ml (white bars). Data are expressed as the percentage binding compared to CCL25 alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211551&req=5

ppat-0040005-g002: Inhibition of Binding of Chemokines to A41 in the Presence of Heparin Sodium Salt(A) A41VOTE was immobilised on a sensor chip and increasing concentrations (0, 31.25, 62.5, 125, 250, 500 ng/ml) of heparin sodium salt (low molecular weight, MW 4,000–6,000, Sigma) were mixed with hCCL28 and passed across the sensor chip. Chemokine binding to A41 is shown by sensorgrams expressed as RU. (B,C). Heparin inhibits binding of human CCL21, CCL25, CCL26 and CCL28 to A41VOTE (B) and A41E. coli (C) (filled symbols depict activity with chemokine only). (D) Binding of human CCL25 to A41VOTE in the presence of various sulphated GAGs (heparin, heparan sulphate [heparan s], chondroitin sulphate B [chond s B, = dermatan], chondroitin sulphate C [chond s C], hyaluronic acid [hyalur ac] and dextran sulphate [dextran s]) at 125 ng/ml (grey bars) and 250 ng/ml (white bars). Data are expressed as the percentage binding compared to CCL25 alone.
Mentions: The failure of A41 to inhibit chemokine-induced chemotaxis and the Kd values for its interaction with chemokines (Table 2), suggest that A41 might function by blocking the interaction of chemokines with GAGs. This was investigated by immobilising A41 on a sensor chip, passing hCCL28 across the chip alone or in the presence of increasing concentrations of heparin (sodium salt, MW 4,000–6,000) and measuring hCCL28 binding by BIACORE. The interaction of A41 and hCCL28 was inhibited in a dose-dependent manner and complete inhibition was achieved with 500 ng/ml heparin (Figure 2A). Similarly, heparin inhibited the binding of hCCL21, hCCL25, hCCL26 and hCCL28 to immobilised A41 in a dose-dependent manner when A41 was produced in either mammalian cells (Figure 2B) or E. coli (Figure 2C). The concentration of heparin used to achieve 50% inhibition was ∼100 ng/ml (∼20 nM) and this was lower than used by other investigators to achieve disruption of the M-T7 chemokine binding protein from MYXV with RANTES [15] or the interaction of chemokines with the endothelial cell surface [26]. Apart from heparin, other sulphated GAGs were also tested for their ability to inhibit binding of chemokines to A41. Heparin and dextran sulphate inhibited the hCCL25-A41 interaction, but heparan sulphate, chondroitin sulphate B, chondroitin sulphate C and hyaluronic acid did not (Figure 2D). Similar results were obtained with A41 produced from E. coli or mammalian cells and with hCCL21, hCCL26 and hCCL28 (data not shown). Notably each GAG able to inhibit the hCCL25-A41 interaction was more highly charged than those that did not inhibit.

Bottom Line: Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors.Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding.The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

View Article: PubMed Central - PubMed

Affiliation: The Division of Structural Biology and The Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

Show MeSH
Related in: MedlinePlus