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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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Group III (EE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and EE11 (group III) at 22 Å resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) is shown in figure S7. Epitope footprints follow the same color code as [9]: group I (JE11): red; group III (EE11): blue. Actual Fab chains follow the same color code. B. Summary of EE11 (group III) contacts. D8 residues colored in blue belong to the initial definition of group III epitope, assessed by alanine scanning. Cyan residues complete the definition of group III epitope. C. Footprint of completed EE11 epitope. The initial definition obtained by alanine scanning and PMA is depicted in blue and the current definition deduced from the EM particle reconstruction is in cyan. Group IV (LA5) footprint in orange does intersect with group III (EE11) epitope at residues R44 and K108 (orange/cyan or orange/blue stripes).
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ppat-1004495-g005: Group III (EE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and EE11 (group III) at 22 Å resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) is shown in figure S7. Epitope footprints follow the same color code as [9]: group I (JE11): red; group III (EE11): blue. Actual Fab chains follow the same color code. B. Summary of EE11 (group III) contacts. D8 residues colored in blue belong to the initial definition of group III epitope, assessed by alanine scanning. Cyan residues complete the definition of group III epitope. C. Footprint of completed EE11 epitope. The initial definition obtained by alanine scanning and PMA is depicted in blue and the current definition deduced from the EM particle reconstruction is in cyan. Group IV (LA5) footprint in orange does intersect with group III (EE11) epitope at residues R44 and K108 (orange/cyan or orange/blue stripes).

Mentions: We used EE11-Fab to build a group III ternary complex for which we did not have a full definition (Fig. 5). Model-to-map correlation for this complex was 0.9115. The total EE11:D8 BSA was 1710 Å2, which is larger than group I and II epitopes but smaller than group IV (2434 Å2). A total of twenty-six D8 residues interact with EE11 MAb. Six D8 residues interact with the light chain (LC) and twenty-four with the heavy chain (HC) (Fig. 5B, C). Novel D8 contacts are E30, T34, T35, R44, N46, F47, K48, G49, G50, Y51, N59, E60, V62, L63, S64 and additional peptide 58 residues K98, K99, K100 and S102, with residues involved in CS-E binding indicated in bold. Group IV (LA5) footprint (in orange) intersects with the group III (EE11) epitope at residues R44 and K108 (Fig. 5C).


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)

Group III (EE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and EE11 (group III) at 22 Å resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) is shown in figure S7. Epitope footprints follow the same color code as [9]: group I (JE11): red; group III (EE11): blue. Actual Fab chains follow the same color code. B. Summary of EE11 (group III) contacts. D8 residues colored in blue belong to the initial definition of group III epitope, assessed by alanine scanning. Cyan residues complete the definition of group III epitope. C. Footprint of completed EE11 epitope. The initial definition obtained by alanine scanning and PMA is depicted in blue and the current definition deduced from the EM particle reconstruction is in cyan. Group IV (LA5) footprint in orange does intersect with group III (EE11) epitope at residues R44 and K108 (orange/cyan or orange/blue stripes).
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ppat-1004495-g005: Group III (EE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fabs JE11 (group I) and EE11 (group III) at 22 Å resolution. See figure legend 3 for general description. Projection Matching and Fourier Shell Correlation (FSC) is shown in figure S7. Epitope footprints follow the same color code as [9]: group I (JE11): red; group III (EE11): blue. Actual Fab chains follow the same color code. B. Summary of EE11 (group III) contacts. D8 residues colored in blue belong to the initial definition of group III epitope, assessed by alanine scanning. Cyan residues complete the definition of group III epitope. C. Footprint of completed EE11 epitope. The initial definition obtained by alanine scanning and PMA is depicted in blue and the current definition deduced from the EM particle reconstruction is in cyan. Group IV (LA5) footprint in orange does intersect with group III (EE11) epitope at residues R44 and K108 (orange/cyan or orange/blue stripes).
Mentions: We used EE11-Fab to build a group III ternary complex for which we did not have a full definition (Fig. 5). Model-to-map correlation for this complex was 0.9115. The total EE11:D8 BSA was 1710 Å2, which is larger than group I and II epitopes but smaller than group IV (2434 Å2). A total of twenty-six D8 residues interact with EE11 MAb. Six D8 residues interact with the light chain (LC) and twenty-four with the heavy chain (HC) (Fig. 5B, C). Novel D8 contacts are E30, T34, T35, R44, N46, F47, K48, G49, G50, Y51, N59, E60, V62, L63, S64 and additional peptide 58 residues K98, K99, K100 and S102, with residues involved in CS-E binding indicated in bold. Group IV (LA5) footprint (in orange) intersects with the group III (EE11) epitope at residues R44 and K108 (Fig. 5C).

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