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Murine anti-vaccinia virus D8 antibodies target different epitopes and differ in their ability to block D8 binding to CS-E.

Matho MH, de Val N, Miller GM, Brown J, Schlossman A, Meng X, Crotty S, Peters B, Xiang Y, Hsieh-Wilson LC, Ward AB, Zajonc DM - PLoS Pathog. (2014)

Bottom Line: The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV).Using EM, we identified the binding site for each antibody specificity group on D8.Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Imunology (LIAI), La Jolla, California, United States of America.

ABSTRACT
The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV). Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E). CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 4' and 6' of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV) fully abrogated CS-E binding, while MAbs of a second group (group III) displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E.

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Mapping of the CS-E binding site on vaccinia D8 ectodomain.A. Docking of CS-E dodecasaccharide to D8. Framed regions highlight regularly spaced, positively charged residue pairs K41/R220, R44/K108, and K48/K98, which are predicted to interact with negatively charged sulfate moieties of CS-E. B. Mapping of CS-E binding site. Mutation R220A led to a ∼50% decrease in CS-E binding compared to wt, while CS-E binding to the double mutants R220/R44 and R220/K48 was almost fully abrogated, corroborating the CS-E docking model. Data were averaged from three experiments.
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ppat-1004495-g002: Mapping of the CS-E binding site on vaccinia D8 ectodomain.A. Docking of CS-E dodecasaccharide to D8. Framed regions highlight regularly spaced, positively charged residue pairs K41/R220, R44/K108, and K48/K98, which are predicted to interact with negatively charged sulfate moieties of CS-E. B. Mapping of CS-E binding site. Mutation R220A led to a ∼50% decrease in CS-E binding compared to wt, while CS-E binding to the double mutants R220/R44 and R220/K48 was almost fully abrogated, corroborating the CS-E docking model. Data were averaged from three experiments.

Mentions: These CS-E binding data led us to refine our previous docking results by using a dodecasaccharide fragment of CS-E, instead of the previously used CS-A [8]. In the docked model, each sulfate group is found in the vicinity of the charged D8 residue pairs K48/K98, R44/K108, and K41/R220, delineating the crevice, which corroborates the high specificity of CS-E over CS-A that was used for docking prior to our knowledge of the exact ligand (Fig. 2A). In effect, CS-A bears only one sulfate group, on 4′-hydroxyl of GalNAc, while CS-E has an additional sulfate on the 6′-hydroxyl group. Hence, docking data converged with the experimental definition of the ligand, since it pointed to the aforementioned positively charged residual pairs that are probably necessary to form salt bridges with both sulfate moieties. Alanine substitutions of residues lining the crevice led to moderate (R220A) or severe (R220A/R44A, and R220A/K48A) reductions in CS-E binding (Fig. 2B). This suggests that D8 binding to CS-E is likely mediated by a network of electrostatic interactions that form pairs on opposing sides of the entire D8 crevice, involving residues K41, R44, K48, K98 and K108 (Fig. 2A). In the D8/LA5-Fab complex [MAb of group IV, pdb code 4ETQ], we observed that only two of these residue pairs (K41/R220 and R44/K108) are part of the epitope. However, LA5 fully blocks CS-E binding of D8, even without K48/K98 coverage.


Murine anti-vaccinia virus D8 antibodies target different epitopes and differ in their ability to block D8 binding to CS-E.

Matho MH, de Val N, Miller GM, Brown J, Schlossman A, Meng X, Crotty S, Peters B, Xiang Y, Hsieh-Wilson LC, Ward AB, Zajonc DM - PLoS Pathog. (2014)

Mapping of the CS-E binding site on vaccinia D8 ectodomain.A. Docking of CS-E dodecasaccharide to D8. Framed regions highlight regularly spaced, positively charged residue pairs K41/R220, R44/K108, and K48/K98, which are predicted to interact with negatively charged sulfate moieties of CS-E. B. Mapping of CS-E binding site. Mutation R220A led to a ∼50% decrease in CS-E binding compared to wt, while CS-E binding to the double mutants R220/R44 and R220/K48 was almost fully abrogated, corroborating the CS-E docking model. Data were averaged from three experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256255&req=5

ppat-1004495-g002: Mapping of the CS-E binding site on vaccinia D8 ectodomain.A. Docking of CS-E dodecasaccharide to D8. Framed regions highlight regularly spaced, positively charged residue pairs K41/R220, R44/K108, and K48/K98, which are predicted to interact with negatively charged sulfate moieties of CS-E. B. Mapping of CS-E binding site. Mutation R220A led to a ∼50% decrease in CS-E binding compared to wt, while CS-E binding to the double mutants R220/R44 and R220/K48 was almost fully abrogated, corroborating the CS-E docking model. Data were averaged from three experiments.
Mentions: These CS-E binding data led us to refine our previous docking results by using a dodecasaccharide fragment of CS-E, instead of the previously used CS-A [8]. In the docked model, each sulfate group is found in the vicinity of the charged D8 residue pairs K48/K98, R44/K108, and K41/R220, delineating the crevice, which corroborates the high specificity of CS-E over CS-A that was used for docking prior to our knowledge of the exact ligand (Fig. 2A). In effect, CS-A bears only one sulfate group, on 4′-hydroxyl of GalNAc, while CS-E has an additional sulfate on the 6′-hydroxyl group. Hence, docking data converged with the experimental definition of the ligand, since it pointed to the aforementioned positively charged residual pairs that are probably necessary to form salt bridges with both sulfate moieties. Alanine substitutions of residues lining the crevice led to moderate (R220A) or severe (R220A/R44A, and R220A/K48A) reductions in CS-E binding (Fig. 2B). This suggests that D8 binding to CS-E is likely mediated by a network of electrostatic interactions that form pairs on opposing sides of the entire D8 crevice, involving residues K41, R44, K48, K98 and K108 (Fig. 2A). In the D8/LA5-Fab complex [MAb of group IV, pdb code 4ETQ], we observed that only two of these residue pairs (K41/R220 and R44/K108) are part of the epitope. However, LA5 fully blocks CS-E binding of D8, even without K48/K98 coverage.

Bottom Line: The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV).Using EM, we identified the binding site for each antibody specificity group on D8.Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Imunology (LIAI), La Jolla, California, United States of America.

ABSTRACT
The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV). Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E). CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 4' and 6' of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV) fully abrogated CS-E binding, while MAbs of a second group (group III) displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E.

Show MeSH
Related in: MedlinePlus