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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

Summary of D8 murine epitome and CS-E binding site.A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues for all VACV anti-D8 murine MAbs of the four epitope groups. Resolution depends on the method used for a specific assessment. Newly-defined epitope residues are highlighted in bold italic. Residues in red are important for both CS-E, and group III and IV MAb binding.
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ppat-1004495-g006: Summary of D8 murine epitome and CS-E binding site.A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues for all VACV anti-D8 murine MAbs of the four epitope groups. Resolution depends on the method used for a specific assessment. Newly-defined epitope residues are highlighted in bold italic. Residues in red are important for both CS-E, and group III and IV MAb binding.

Mentions: The resolution of the EM maps obtained with negative staining provides an accurate epitope definition of group III but not at atomic resolution. In order to validate this newly defined interface, we picked three seemingly critical residues for alanine scanning mutagenesis, two of which are involved in CS-E binding (E30, R44, K48). A 3-fold decrease in affinity was observed for D8 E30A (Fig. S2). However, this was likely due to suboptimal folding of D8 E30A, as the control antibody (JE11) also showed reduced binding (9-fold). In both cases, only the association phase was affected. This suggested that E30 does not contribute greatly to EE11 binding. We observed an almost 10-fold decrease in EE11 affinity with D8 R220A/R44A and R220A/K48A mutants compared to either wild-type (wt) D8 or R220A D8, suggesting both R44 and K48 are important residues for EE11 binding (Fig. S2). Both mutants bound normally to control antibodies. Figure 6 summarizes the details of and the techniques used to identify the complete murine D8 epitome.


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)

Summary of D8 murine epitome and CS-E binding site.A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues for all VACV anti-D8 murine MAbs of the four epitope groups. Resolution depends on the method used for a specific assessment. Newly-defined epitope residues are highlighted in bold italic. Residues in red are important for both CS-E, and group III and IV MAb binding.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004495-g006: Summary of D8 murine epitome and CS-E binding site.A. Updated footprint of groups I (red), II (green), III (blue) and IV (orange) are represented. The yellow line reminds the CS-E path between positively charged residue pairs (black frames). B. Summary of D8 epitope residues for all VACV anti-D8 murine MAbs of the four epitope groups. Resolution depends on the method used for a specific assessment. Newly-defined epitope residues are highlighted in bold italic. Residues in red are important for both CS-E, and group III and IV MAb binding.
Mentions: The resolution of the EM maps obtained with negative staining provides an accurate epitope definition of group III but not at atomic resolution. In order to validate this newly defined interface, we picked three seemingly critical residues for alanine scanning mutagenesis, two of which are involved in CS-E binding (E30, R44, K48). A 3-fold decrease in affinity was observed for D8 E30A (Fig. S2). However, this was likely due to suboptimal folding of D8 E30A, as the control antibody (JE11) also showed reduced binding (9-fold). In both cases, only the association phase was affected. This suggested that E30 does not contribute greatly to EE11 binding. We observed an almost 10-fold decrease in EE11 affinity with D8 R220A/R44A and R220A/K48A mutants compared to either wild-type (wt) D8 or R220A D8, suggesting both R44 and K48 are important residues for EE11 binding (Fig. S2). Both mutants bound normally to control antibodies. Figure 6 summarizes the details of and the techniques used to identify the complete murine D8 epitome.

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