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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

D8 oligomeric interface is secluded to the C-terminal region 235–262.A. From top to bottom: selected class averages of (i) unliganded D8 oligomer, (ii) oligomeric D8+JE11-Fab, and (iii) oligomeric D8+JE11-Fab+LA5-Fab. Despite the monodispersity of unliganded D8 oligomeric sample, particles showed a varying number of drupelets, because of their orientation on the EM grid. The class averages with the highest number of drupelets always show six drupelets surrounding a central one (red arrows). B. SEC profiles of recombinant D8 Δ235, Δ262, and Δ263 suggest that D8 oligomerises through the C-terminal domain. SEC markers as grey curve with MW given in kDa. C. Putative D8 hexameric model based on EM data and SEC-MALS using biochemical constraints relative to the dimer and oligomer interfaces. The black circle highlights the putative seventh and central drupelet, arising from all six SU C-terminal extremities, converging toward the IMV envelope. D. CS-E microarray indicating that D8 hexamer (0.33 µM) binds more effectively to CS-E compared to 6-times molar excess of D8 monomer (2 µM) E. GAG microarray obtained with D8 oligomer (0.1 µM). D8 oligomer binds CS-E with higher affinity than the monomer, and also weakly binds to other CS species but not to DS, HA, heparin and HS. Micro array binding experiment was performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).
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ppat-1004495-g007: D8 oligomeric interface is secluded to the C-terminal region 235–262.A. From top to bottom: selected class averages of (i) unliganded D8 oligomer, (ii) oligomeric D8+JE11-Fab, and (iii) oligomeric D8+JE11-Fab+LA5-Fab. Despite the monodispersity of unliganded D8 oligomeric sample, particles showed a varying number of drupelets, because of their orientation on the EM grid. The class averages with the highest number of drupelets always show six drupelets surrounding a central one (red arrows). B. SEC profiles of recombinant D8 Δ235, Δ262, and Δ263 suggest that D8 oligomerises through the C-terminal domain. SEC markers as grey curve with MW given in kDa. C. Putative D8 hexameric model based on EM data and SEC-MALS using biochemical constraints relative to the dimer and oligomer interfaces. The black circle highlights the putative seventh and central drupelet, arising from all six SU C-terminal extremities, converging toward the IMV envelope. D. CS-E microarray indicating that D8 hexamer (0.33 µM) binds more effectively to CS-E compared to 6-times molar excess of D8 monomer (2 µM) E. GAG microarray obtained with D8 oligomer (0.1 µM). D8 oligomer binds CS-E with higher affinity than the monomer, and also weakly binds to other CS species but not to DS, HA, heparin and HS. Micro array binding experiment was performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).

Mentions: For structural studies, we prepared monomeric D8 that either only contains the CAH domain (residues 1–234) or lacks the distal C-terminal cysteine (C262, residues 1–261). However, we have previously shown that the full D8 ectodomain forms a disulfide-linked dimer (through C262) that further associates non-covalently to form an oligomer [8]. Despite oligomeric D8 having an molecular weight that was considerably larger than the 158 kDa MW marker on SEC, we had previously described the D8 oligomeric state as tetrameric, based on D8 migration during native gel electrophoresis [8]. However, size exclusion chromatography with inline multi-angle light scattering (SEC-MALS), assigned an average MW of 228 kDa for the D8 oligomer compared to 42 kDa obtained for the D8 monomer, using two different SEC resins (Fig. S3). The SEC-MALS data indicate a hexameric D8 arrangement. EM class averages of the D8 oligomer revealed no more than 7 drupelets, with 6 drupelets surrounding a central drupelet that appears of lesser intensity (Figs. 7A, S3 and S4). While a D8 heptamer remained a possibility, our SEC data coupled to non-reducing SDS-PAGE suggested that the D8 oligomer is formed by en even number of D8 monomers. When subjected to SEC both D8 oligomers, either lacking (1–261) or containing (1–262) the C-terminal cysteine, eluted at the identical volume (Fig. 7). When the D8 oligomers were subjected to non-reducing SDS PAGE, no D8 monomer, but only disulfide-linked D8 dimers were observed [8]. Therefore, only an even numbered D8 oligomer, such as a hexamer appears possible, as an octamer would not be supported by the SEC-MALS data. We speculate that the 7th and central drupelet is not a result of heptameric D8 but rather formed by the convergence of the C-terminal extremities (235–262) of all six D8 subunits (SUs). We confirmed the monodispersity of the sample by size SEC-MALS (Fig. S3), which suggested that 2D class averages are of a single species, and most likely represent different orientations of the hexamer (Fig. S4A). However, obtaining three-dimensional maps of the D8 hexamer was problematic due to apparent conformational flexibility of D8. In fact, the CAH fold domains do not adopt a uniform arrangement in the different hexameric D8 class averages of figure 7A, but are instead placed haphazardly around the central drupelet, likely due to flexibility of the connecting linker to the C-terminal domain. The C-terminal domain was also disordered in the crystal structure of D8 [8]. Hence, multiple conformations of the D8 hexamer prevent the reconstruction of a three-dimensional model via EM. This flexibility also suggests that CAH fold domains do not participate in the oligomeric interface and correlates with an apparent higher MW for the D8 hexamer (MW of 228 kDa compared to theoretical 192 kDa).


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)

D8 oligomeric interface is secluded to the C-terminal region 235–262.A. From top to bottom: selected class averages of (i) unliganded D8 oligomer, (ii) oligomeric D8+JE11-Fab, and (iii) oligomeric D8+JE11-Fab+LA5-Fab. Despite the monodispersity of unliganded D8 oligomeric sample, particles showed a varying number of drupelets, because of their orientation on the EM grid. The class averages with the highest number of drupelets always show six drupelets surrounding a central one (red arrows). B. SEC profiles of recombinant D8 Δ235, Δ262, and Δ263 suggest that D8 oligomerises through the C-terminal domain. SEC markers as grey curve with MW given in kDa. C. Putative D8 hexameric model based on EM data and SEC-MALS using biochemical constraints relative to the dimer and oligomer interfaces. The black circle highlights the putative seventh and central drupelet, arising from all six SU C-terminal extremities, converging toward the IMV envelope. D. CS-E microarray indicating that D8 hexamer (0.33 µM) binds more effectively to CS-E compared to 6-times molar excess of D8 monomer (2 µM) E. GAG microarray obtained with D8 oligomer (0.1 µM). D8 oligomer binds CS-E with higher affinity than the monomer, and also weakly binds to other CS species but not to DS, HA, heparin and HS. Micro array binding experiment was performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004495-g007: D8 oligomeric interface is secluded to the C-terminal region 235–262.A. From top to bottom: selected class averages of (i) unliganded D8 oligomer, (ii) oligomeric D8+JE11-Fab, and (iii) oligomeric D8+JE11-Fab+LA5-Fab. Despite the monodispersity of unliganded D8 oligomeric sample, particles showed a varying number of drupelets, because of their orientation on the EM grid. The class averages with the highest number of drupelets always show six drupelets surrounding a central one (red arrows). B. SEC profiles of recombinant D8 Δ235, Δ262, and Δ263 suggest that D8 oligomerises through the C-terminal domain. SEC markers as grey curve with MW given in kDa. C. Putative D8 hexameric model based on EM data and SEC-MALS using biochemical constraints relative to the dimer and oligomer interfaces. The black circle highlights the putative seventh and central drupelet, arising from all six SU C-terminal extremities, converging toward the IMV envelope. D. CS-E microarray indicating that D8 hexamer (0.33 µM) binds more effectively to CS-E compared to 6-times molar excess of D8 monomer (2 µM) E. GAG microarray obtained with D8 oligomer (0.1 µM). D8 oligomer binds CS-E with higher affinity than the monomer, and also weakly binds to other CS species but not to DS, HA, heparin and HS. Micro array binding experiment was performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).
Mentions: For structural studies, we prepared monomeric D8 that either only contains the CAH domain (residues 1–234) or lacks the distal C-terminal cysteine (C262, residues 1–261). However, we have previously shown that the full D8 ectodomain forms a disulfide-linked dimer (through C262) that further associates non-covalently to form an oligomer [8]. Despite oligomeric D8 having an molecular weight that was considerably larger than the 158 kDa MW marker on SEC, we had previously described the D8 oligomeric state as tetrameric, based on D8 migration during native gel electrophoresis [8]. However, size exclusion chromatography with inline multi-angle light scattering (SEC-MALS), assigned an average MW of 228 kDa for the D8 oligomer compared to 42 kDa obtained for the D8 monomer, using two different SEC resins (Fig. S3). The SEC-MALS data indicate a hexameric D8 arrangement. EM class averages of the D8 oligomer revealed no more than 7 drupelets, with 6 drupelets surrounding a central drupelet that appears of lesser intensity (Figs. 7A, S3 and S4). While a D8 heptamer remained a possibility, our SEC data coupled to non-reducing SDS-PAGE suggested that the D8 oligomer is formed by en even number of D8 monomers. When subjected to SEC both D8 oligomers, either lacking (1–261) or containing (1–262) the C-terminal cysteine, eluted at the identical volume (Fig. 7). When the D8 oligomers were subjected to non-reducing SDS PAGE, no D8 monomer, but only disulfide-linked D8 dimers were observed [8]. Therefore, only an even numbered D8 oligomer, such as a hexamer appears possible, as an octamer would not be supported by the SEC-MALS data. We speculate that the 7th and central drupelet is not a result of heptameric D8 but rather formed by the convergence of the C-terminal extremities (235–262) of all six D8 subunits (SUs). We confirmed the monodispersity of the sample by size SEC-MALS (Fig. S3), which suggested that 2D class averages are of a single species, and most likely represent different orientations of the hexamer (Fig. S4A). However, obtaining three-dimensional maps of the D8 hexamer was problematic due to apparent conformational flexibility of D8. In fact, the CAH fold domains do not adopt a uniform arrangement in the different hexameric D8 class averages of figure 7A, but are instead placed haphazardly around the central drupelet, likely due to flexibility of the connecting linker to the C-terminal domain. The C-terminal domain was also disordered in the crystal structure of D8 [8]. Hence, multiple conformations of the D8 hexamer prevent the reconstruction of a three-dimensional model via EM. This flexibility also suggests that CAH fold domains do not participate in the oligomeric interface and correlates with an apparent higher MW for the D8 hexamer (MW of 228 kDa compared to theoretical 192 kDa).

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