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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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Related in: MedlinePlus

Group I (JE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fab's JE11 (group I) and LA5 (group IV) at 24 Å resolution. Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S5. Top left inset shows one of the class-averages used for building the map. EM density is shown in gray mesh. D8 monomer crystal structure is represented as a grey surface except for epitope footprints that follow the same color code as [9]: group I (JE11): red; group IV (LA5): orange. Actual Fab chains also follow this color code. B. Summary of JE11 (group I) contacts. D8 residues in red belong to the initial definition of group I epitope, assessed by DXMS. Salmon-colored residues complete the definition of group I conformational epitope. Black bold-contours highlight residues previously picked for mutation analysis [9]. C. Footprint of completed JE11 epitope. Red and salmon footprints evidence initial and additional epitope residues. Despite being juxtaposed to each other, group IV (LA5) and group I (JE11) footprints do not intersect. Black labels inform on residues resulting in a loss of MAb/Ag affinity upon mutation to alanine, while mutated residues in white did not lead to any relevant change in binding.
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ppat-1004495-g003: Group I (JE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fab's JE11 (group I) and LA5 (group IV) at 24 Å resolution. Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S5. Top left inset shows one of the class-averages used for building the map. EM density is shown in gray mesh. D8 monomer crystal structure is represented as a grey surface except for epitope footprints that follow the same color code as [9]: group I (JE11): red; group IV (LA5): orange. Actual Fab chains also follow this color code. B. Summary of JE11 (group I) contacts. D8 residues in red belong to the initial definition of group I epitope, assessed by DXMS. Salmon-colored residues complete the definition of group I conformational epitope. Black bold-contours highlight residues previously picked for mutation analysis [9]. C. Footprint of completed JE11 epitope. Red and salmon footprints evidence initial and additional epitope residues. Despite being juxtaposed to each other, group IV (LA5) and group I (JE11) footprints do not intersect. Black labels inform on residues resulting in a loss of MAb/Ag affinity upon mutation to alanine, while mutated residues in white did not lead to any relevant change in binding.

Mentions: Determining the full epitope for group II and III MAbs is necessary to explain the wide spread in CS-E blocking ability (10–80%) observed between group II and group III MAbs. To address this question, we used negative stain single particle D8:Fab EM reconstructions. To map the relative positions of the different antibodies on D8, we used Fabs from two different groups, simultaneously bound to D8. JE11-Fab (group I) was included as a reference in all subsequent ternary complexes because it does not cross-block binding of any other MAb groups [9]. We then reconstructed the three-dimensional arrangements by embedding individual atomic models within the low-resolution maps of the multivalent complexes obtained by EM. Docking was guided using previously determined experimental constraints: X-ray crystallography definition of group IV epitope, DXMS definition of group I epitope, and alanine-scan definitions of groups II and III epitopes [9]. Since we had previously determined the high-resolution crystal structure of D8/LA5 (group IV), we first reconstructed the D8/JE11/LA5 complex (Fig. 3). We then reconstructed the D8/JE11/CC7.1 complex, since a definition of the group II epitope was available (Fig. 4). Fitting of those reconstructed atomic models gave best-fit correlation values of 0.9016 and 0.9207, respectively. These two first processes served as an internal proof of concept, illustrating that our experimental epitope definitions agreed with the associated EM maps.


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 I (JE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fab's JE11 (group I) and LA5 (group IV) at 24 Å resolution. Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S5. Top left inset shows one of the class-averages used for building the map. EM density is shown in gray mesh. D8 monomer crystal structure is represented as a grey surface except for epitope footprints that follow the same color code as [9]: group I (JE11): red; group IV (LA5): orange. Actual Fab chains also follow this color code. B. Summary of JE11 (group I) contacts. D8 residues in red belong to the initial definition of group I epitope, assessed by DXMS. Salmon-colored residues complete the definition of group I conformational epitope. Black bold-contours highlight residues previously picked for mutation analysis [9]. C. Footprint of completed JE11 epitope. Red and salmon footprints evidence initial and additional epitope residues. Despite being juxtaposed to each other, group IV (LA5) and group I (JE11) footprints do not intersect. Black labels inform on residues resulting in a loss of MAb/Ag affinity upon mutation to alanine, while mutated residues in white did not lead to any relevant change in binding.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004495-g003: Group I (JE11) footprint.A. EM reconstruction of the D8 monomer in complex with Fab's JE11 (group I) and LA5 (group IV) at 24 Å resolution. Projection Matching and Fourier Shell Correlation (FSC) are shown in figure S5. Top left inset shows one of the class-averages used for building the map. EM density is shown in gray mesh. D8 monomer crystal structure is represented as a grey surface except for epitope footprints that follow the same color code as [9]: group I (JE11): red; group IV (LA5): orange. Actual Fab chains also follow this color code. B. Summary of JE11 (group I) contacts. D8 residues in red belong to the initial definition of group I epitope, assessed by DXMS. Salmon-colored residues complete the definition of group I conformational epitope. Black bold-contours highlight residues previously picked for mutation analysis [9]. C. Footprint of completed JE11 epitope. Red and salmon footprints evidence initial and additional epitope residues. Despite being juxtaposed to each other, group IV (LA5) and group I (JE11) footprints do not intersect. Black labels inform on residues resulting in a loss of MAb/Ag affinity upon mutation to alanine, while mutated residues in white did not lead to any relevant change in binding.
Mentions: Determining the full epitope for group II and III MAbs is necessary to explain the wide spread in CS-E blocking ability (10–80%) observed between group II and group III MAbs. To address this question, we used negative stain single particle D8:Fab EM reconstructions. To map the relative positions of the different antibodies on D8, we used Fabs from two different groups, simultaneously bound to D8. JE11-Fab (group I) was included as a reference in all subsequent ternary complexes because it does not cross-block binding of any other MAb groups [9]. We then reconstructed the three-dimensional arrangements by embedding individual atomic models within the low-resolution maps of the multivalent complexes obtained by EM. Docking was guided using previously determined experimental constraints: X-ray crystallography definition of group IV epitope, DXMS definition of group I epitope, and alanine-scan definitions of groups II and III epitopes [9]. Since we had previously determined the high-resolution crystal structure of D8/LA5 (group IV), we first reconstructed the D8/JE11/LA5 complex (Fig. 3). We then reconstructed the D8/JE11/CC7.1 complex, since a definition of the group II epitope was available (Fig. 4). Fitting of those reconstructed atomic models gave best-fit correlation values of 0.9016 and 0.9207, respectively. These two first processes served as an internal proof of concept, illustrating that our experimental epitope definitions agreed with the associated EM maps.

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