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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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D8 binds to CS-E and anti-D8 MAbs display different levels of competition with CS-E.A. GAG microarray performed with monomeric D8 antigen. B. MAb/CS-E cross-blocking experiments using representatives of all four antibody specificity groups and oligomeric D8. C. MAb/CS-E cross-blocking of group III MAbs D. Summary of CS-E cross-blocking abilities of various MAbs. Group III MAbs are characterized by large variations in cross-blocking ability. Microarray binding experiments were 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-g001: D8 binds to CS-E and anti-D8 MAbs display different levels of competition with CS-E.A. GAG microarray performed with monomeric D8 antigen. B. MAb/CS-E cross-blocking experiments using representatives of all four antibody specificity groups and oligomeric D8. C. MAb/CS-E cross-blocking of group III MAbs D. Summary of CS-E cross-blocking abilities of various MAbs. Group III MAbs are characterized by large variations in cross-blocking ability. Microarray binding experiments were performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).

Mentions: To broadly assess D8-GAG interactions, we employed microarrays containing immobilized chondroitin sulfate polysaccharides enriched in specific sulfation motifs (CS-A, -C, -D, and –E), dermatan sulfate (DS), hyaluronic acid (HA), heparin and heparan sulfate (HS). We observed weak binding of monomeric D8 to chondroitin sulfate, a CS preparation containing a mix of sulfation motifs (CS-A, -C, -D, and –E). In contrast, D8 displayed strong concentration-dependent binding to CS-E. Binding to heparan sulfate or chondroitin sulfate with different sulfation patterns was not observed (Fig. 1A).


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 binds to CS-E and anti-D8 MAbs display different levels of competition with CS-E.A. GAG microarray performed with monomeric D8 antigen. B. MAb/CS-E cross-blocking experiments using representatives of all four antibody specificity groups and oligomeric D8. C. MAb/CS-E cross-blocking of group III MAbs D. Summary of CS-E cross-blocking abilities of various MAbs. Group III MAbs are characterized by large variations in cross-blocking ability. Microarray binding experiments were 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

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

ppat-1004495-g001: D8 binds to CS-E and anti-D8 MAbs display different levels of competition with CS-E.A. GAG microarray performed with monomeric D8 antigen. B. MAb/CS-E cross-blocking experiments using representatives of all four antibody specificity groups and oligomeric D8. C. MAb/CS-E cross-blocking of group III MAbs D. Summary of CS-E cross-blocking abilities of various MAbs. Group III MAbs are characterized by large variations in cross-blocking ability. Microarray binding experiments were performed in triplicate, and the data represent the average of 10 spots per concentration averaged from the three experiments (±SEM, error bars).
Mentions: To broadly assess D8-GAG interactions, we employed microarrays containing immobilized chondroitin sulfate polysaccharides enriched in specific sulfation motifs (CS-A, -C, -D, and –E), dermatan sulfate (DS), hyaluronic acid (HA), heparin and heparan sulfate (HS). We observed weak binding of monomeric D8 to chondroitin sulfate, a CS preparation containing a mix of sulfation motifs (CS-A, -C, -D, and –E). In contrast, D8 displayed strong concentration-dependent binding to CS-E. Binding to heparan sulfate or chondroitin sulfate with different sulfation patterns was not observed (Fig. 1A).

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