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The gammaherpesvirus chemokine binding protein can inhibit the interaction of chemokines with glycosaminoglycans¹

Department of Medicine and Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK

⁴Correspondence: Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Level 5, Box 157, Hills Road, Cambridge CB2 2QQ, UK. E-mail: aa258{at}mole.bio.cam.ac.uk

SPECIFIC AIMS

Recent evidence indicates that the interaction of chemokines with glycosaminoglycans (GAGs) is critical for their in vivo bioactivity. We sought to determine whether this interaction was targeted by the murine gammaherpesvirus-68 (MHV-68) encoded chemokine binding protein, vCKBP-3 (M3), to neutralize chemokine activity and interrupt established chemokine gradients.

PRINCIPAL FINDINGS

1. vCKBP-3 prevents chemokines from binding to heparin
We first wished to determine whether vCKBP-3 could inhibit chemokines from binding to the GAG, heparin. Using a Flashplate-based assay where heparin-BSA is coated to a plate and then ¹²⁵I-chemokine is added, we showed that vCKBP-3 is able to inhibit many chemokines from binding to heparin (Fig. 1 ) depending upon whether the chemokine can bind vCKBP-3. We confirmed this result using a heparin column and applying ¹²⁵I-labeled chemokine in the presence or absence of vCKBP-3.

View larger version (46K): [in this window] [in a new window]   Figure 1. vCKBP-3 prevents the chemokine-GAG interaction. Basic Flashplates were coated with heparin-BSA or BSA alone. 400 pM of 125I-chemokines were then added in the presence or absence of vCKBP-3 and counts were read after an O/N incubation at 4°C. The effect of 1 µg/mL of vCKBP-3 on CXCL8, CXCL1, CXCL10, CXCL12, CCL5, and CCL2 is shown (A–F). The plates were read after O/N incubation at 4°C. Background counts (125I-chemokine on BSA-coated wells) were subtracted from each point. The mean ± SD for triplicate samples is shown.

2. Chemokine binding to cell surface GAGs is prevented by vCKBP-3
In vivo, it is thought that chemokines are presented to the leukocytes on GAG molecules present on cell surfaces within the basement membrane and on the surface of endothelial cells. We used CHO-K1 cells, which are rich in GAGs, and CHO-745 cells, which are deficient in xylosyl transferase and result in under-expression of GAGs, to examine the effect of vCKBP-3 on chemokine binding to complex cell surface GAGs. Using two separate assays we were able to show that vCKBP-3 could prevent chemokines from binding to cell surface GAGs.

3. GAG-bound chemokines can be displaced by vCKBP-3
Disruption of established chemokine gradients would be a desirable feature of a vCKBP. Using the Flashplate assay described above we were able to show that vCKBP-3 was able to displace GAG-bound chemokine (Fig. 2 ). The kinetics of this displacement were chemokine specific, most probably reflecting the relative positions of the vCKBP-3 and GAG binding sites within individual chemokines.

View larger version (21K): [in this window] [in a new window]   Figure 2. vCKBP-3 displaces CXCL8 and CCL5 from heparin. Basic Flashplates were coated with heparin-BSA or BSA alone and 400 pM 125I-CCL5 (A) or 125I-CXCL8 (B) were added and incubated overnight at 4°C. vCKBP-3 was then added and plates read at the indicated times. Background counts (125I-chemokine on BSA-coated wells) were subtracted from each point. The mean ± SD for triplicate samples is shown.

CONCLUSIONS AND SIGNIFICANCE

Viruses have evolved multiple immune evasion strategies in order to manipulate the host for their own survival and perpetuation. vCKBP-3 is encoded by MHV–68. It has been shown to inhibit both chemokine receptor binding and calcium flux induced by CXCL8, CCL5, CCL3, CCL2 and CX3CL1. Recently, we and others have identified the structural requirements of CXCL8 and CCL2 for vCKBP-3 binding. Both sets of data concur that vCKBP-3 binds to the receptor binding N-loop of both chemokines. These data explain how vCKBP-3 is able to prevent chemokines from binding to their G-protein-coupled receptors (GPCRs) and inhibit calcium flux. Neither study examined the effect of vCKBP-3 on chemokine-GAG binding.

Prevention of GAG binding would be a desirable immune evasion strategy. The binding of chemokines to GAGs is a prerequisite for their uptake, transcytosis to the apical side of the endothelial cell, and appropriate solid phase presentation to the passing leukocyte. This strategy has been proposed to be used by vCKBP-1 (encoded by myxoma virus) to inhibit in vivo chemokine activity. We have shown that vCKBP-3 is able to prevent chemokine from binding to heparin and cell surface GAGs. This activity depends upon its ability to bind chemokine, implying that it works by interacting with chemokine and not GAG.

It was important to determine if the ability of vCKBP-3 to block the chemokine-GAG interaction also enabled it to displace GAG-bound chemokines. This property of vCKBP-3 would allow it to displace chemokines that have already bound to the endothelial cell surface, thereby disrupting established chemokine gradients. We found that vCKBP-3 could completely displace GAG-bound CXCL8 and CCL5. However, there was a large difference in the kinetics of this displacement. This difference is unlikely to be due to different affinities of CXCL8 and CCL5 for vCKBP-3 but rather to the different regions used by CCL5 and CXCL8 to bind to heparin. Previous work has shown that residues Lys²⁰, Arg⁶⁰, and Arg⁶⁷ in CXCL8 and residues Arg⁴⁴, Lys⁴⁵, and Arg⁴⁷ in CCL5 are responsible for heparin binding. However, when the residues Arg⁴⁴, Lys⁴⁵, and Arg⁴⁷ in CCL5 were mutated to alanine, mutant proteins retained some ability to bind to heparin, indicating that other residues in CCL5 also participate in GAG binding. It is possible that these GAG binding residues within CCL5 also participate in vCKBP-3 binding. We predict that CCL5 also binds to vCKBP-3 via its N-loop and that there is some overlap in the GAG and vCKBP-3 binding domains. The residues of CXCL8 important for GAG binding lie within the C terminus (Arg⁶⁰ Lys⁶⁴, Lys⁶⁷, and Arg⁶⁸) and the proximal loop (Lys²⁰) of CXCL8. Since vCKBP-3 does not require the C terminus of CXCL8 to bind vCKBP-3, it could prevent GAG binding by either masking Lys²⁰ and/or induce conformational changes within the molecule that affect its GAG binding capacity. We have not been able to exclude a role for Lys²⁰ in vCKBP-3 binding. However, mutation of the nearby His¹⁸ to leucine did not result in a significant drop in vCKBP-3 affinity. Furthermore, although mutation of Lys²⁰ to alanine causes a reduction in heparin binding it dose not abrogate it. Therefore, even if vCKBP-3 did mask Lys²⁰ when it bound to CXCL8, this would be unlikely to prevent CXCL8 from binding to heparin.

vCKBP-3 appears to mimic receptor binding to CXCL8 and CCL2, thereby masking the sites that bind to the receptor within the N terminus. By mimicking receptor binding, vCKBP-3 is able to simultaneously inhibit GPCR and GAG binding. It is conceivable that upon receptor binding, conformational changes are induced within the CXCL8 molecule that abrogate GAG binding, thereby allowing the chemokine to be removed from the cell surface and internalized.

vCKBP-3 appears to act at two distinct levels to inhibit chemokine activity (Fig. 3 ). It prevents the chemokine from engaging its GPCR and prevents and abrogates GAG binding. vCKBP-1 can also prevent chemokines from interacting with GAGs, but is not thought to have an effect on GPCR binding. Since GAG binding is now thought to be a prerequisite for in vivo activity of chemokines, it would appear that inhibition of only GAG or GPCR binding would be sufficient for in vivo neutralization of chemokine activity by vCKBP-3. Why then does vCKBP-3 maintain the capacity to inhibit both chemokine functions? It is possible that its ability to inhibit GAG binding is a consequence of its binding to the chemokine. By binding to the N-loop, vCKBP-3 appears to be able to abrogate the chemokine’s ability to bind to heparin. This property gives vCKBP-3 the additional and desirable function of disrupting established chemokine gradients. This scavenger-like property would allow vCKBP-3 to displace chemokines and remove them from the site of infection making it a more effective inhibitor of chemokine activity.

View larger version (70K): [in this window] [in a new window]   Figure 3. Proposed mechanism of vCKBP-3 action. In the absence of vCKBP-3 chemokine is presented to the passing leukocyte on the surface of the endothelial cell allowing the leukocyte to respond via its G-protein-coupled receptor and migrate to the source of infection (A). In the presence of vCKBP-3, the passing leukocyte does not respond to the chemokine because vCKBP-3 blocks chemokine from binding to the G-protein-coupled receptor, and also prevents establishment of chemokine gradients on the endothelial cell surface and within the basement membrane (B). In addition, any chemokine that is presented on the endothelial cell is displaced and removed by vCKBP-3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0485fje;

² Present address: Nextgen Sciences Ltd., Alconbury North Airfield, Alconbury, Huntingdon, Cambridgeshire, UK.

³ Present address: Babraham Institute, Babraham, Cambridge CB2 4AT, UK.

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