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Caspr2 autoantibodies target multiple epitopes.

Olsen AL, Lai Y, Dalmau J, Scherer SS, Lancaster E - Neurol Neuroimmunol Neuroinflamm (2015)

Bottom Line: To better understand the mechanisms of autoantibodies to the axonal protein contactin-associated protein-like 2 (Caspr2) by studying their target epitopes.All deletion constructs were recognized by patients' sera, although reactivity was significantly reduced with deletion of the discoidin-like subdomain and strongly reduced or abolished with larger deletions of multiple N-terminal subdomains.Reactivity for some epitopes is not dependent on glycosylation or native protein structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology (A.L.O., Y.L., J.D., S.S.S., E.L.), The University of Pennsylvania, Philadelphia; and ICREA-IDIBAPS (J.D.), Hospital Unit, University of Barcelona, Spain.

ABSTRACT

Objective: To better understand the mechanisms of autoantibodies to the axonal protein contactin-associated protein-like 2 (Caspr2) by studying their target epitopes.

Methods: A plasmid for expressing Caspr2 was modified so that the various extracellular subdomains were deleted individually and in groups. Cultured cells were transfected to express these constructs and assayed by immunofluorescence staining with a commercial Caspr2 antibody and a panel of patient sera known to react with Caspr2. Western blotting was also performed. The role of glycosylation in immunogenicity was tested with tunicamycin and PNGase F treatment.

Results: Patient antibodies bound to the extracellular domain of Caspr2. Neither native protein structure nor glycosylation was required for immunoreactivity. Caspr2 constructs with single or multidomain deletions were expressed on the plasma membrane. All deletion constructs were recognized by patients' sera, although reactivity was significantly reduced with deletion of the discoidin-like subdomain and strongly reduced or abolished with larger deletions of multiple N-terminal subdomains. Caspr2 with all subdomains deleted except the discoidin-like domain was still recognized by the antibodies.

Conclusion: Caspr2 autoantibodies recognize multiple target epitopes in the extracellular domain of Caspr2, including one in the discoidin-like domain. Reactivity for some epitopes is not dependent on glycosylation or native protein structure.

No MeSH data available.


Some single-domain deletion constructs showed decreased labeling by patients' sera(A) Cultured cells were transfected to express wild-type and single-domain deletion constructs. All constructs were expressed on the cell membrane and all were recognized by a panel of patients' sera. Scale bar = 10 μm. (B) The relative staining in the human and commercial channels was quantified for the various deletion constructs. Only ΔDisc differed significantly from full-length Caspr2 using this method.
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Figure 4: Some single-domain deletion constructs showed decreased labeling by patients' sera(A) Cultured cells were transfected to express wild-type and single-domain deletion constructs. All constructs were expressed on the cell membrane and all were recognized by a panel of patients' sera. Scale bar = 10 μm. (B) The relative staining in the human and commercial channels was quantified for the various deletion constructs. Only ΔDisc differed significantly from full-length Caspr2 using this method.

Mentions: The single subdomain deletion constructs were all expressed when transfected into cultured HEK293 T cells. It is interesting that patients' sera reacted with each of these constructs on Western blot (figure 3B) and also by immunofluorescence (figure 4). All 10 tested patient sera were able to immunostain living cells expressing each of these single-domain deletion constructs, confirming that they were all expressed on the cell membrane (figure 4). This pattern of findings showed that no single extracellular subdomain was absolutely necessary for antibody recognition and that multiple epitopes must exist in the extracellular domain.


Caspr2 autoantibodies target multiple epitopes.

Olsen AL, Lai Y, Dalmau J, Scherer SS, Lancaster E - Neurol Neuroimmunol Neuroinflamm (2015)

Some single-domain deletion constructs showed decreased labeling by patients' sera(A) Cultured cells were transfected to express wild-type and single-domain deletion constructs. All constructs were expressed on the cell membrane and all were recognized by a panel of patients' sera. Scale bar = 10 μm. (B) The relative staining in the human and commercial channels was quantified for the various deletion constructs. Only ΔDisc differed significantly from full-length Caspr2 using this method.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496632&req=5

Figure 4: Some single-domain deletion constructs showed decreased labeling by patients' sera(A) Cultured cells were transfected to express wild-type and single-domain deletion constructs. All constructs were expressed on the cell membrane and all were recognized by a panel of patients' sera. Scale bar = 10 μm. (B) The relative staining in the human and commercial channels was quantified for the various deletion constructs. Only ΔDisc differed significantly from full-length Caspr2 using this method.
Mentions: The single subdomain deletion constructs were all expressed when transfected into cultured HEK293 T cells. It is interesting that patients' sera reacted with each of these constructs on Western blot (figure 3B) and also by immunofluorescence (figure 4). All 10 tested patient sera were able to immunostain living cells expressing each of these single-domain deletion constructs, confirming that they were all expressed on the cell membrane (figure 4). This pattern of findings showed that no single extracellular subdomain was absolutely necessary for antibody recognition and that multiple epitopes must exist in the extracellular domain.

Bottom Line: To better understand the mechanisms of autoantibodies to the axonal protein contactin-associated protein-like 2 (Caspr2) by studying their target epitopes.All deletion constructs were recognized by patients' sera, although reactivity was significantly reduced with deletion of the discoidin-like subdomain and strongly reduced or abolished with larger deletions of multiple N-terminal subdomains.Reactivity for some epitopes is not dependent on glycosylation or native protein structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology (A.L.O., Y.L., J.D., S.S.S., E.L.), The University of Pennsylvania, Philadelphia; and ICREA-IDIBAPS (J.D.), Hospital Unit, University of Barcelona, Spain.

ABSTRACT

Objective: To better understand the mechanisms of autoantibodies to the axonal protein contactin-associated protein-like 2 (Caspr2) by studying their target epitopes.

Methods: A plasmid for expressing Caspr2 was modified so that the various extracellular subdomains were deleted individually and in groups. Cultured cells were transfected to express these constructs and assayed by immunofluorescence staining with a commercial Caspr2 antibody and a panel of patient sera known to react with Caspr2. Western blotting was also performed. The role of glycosylation in immunogenicity was tested with tunicamycin and PNGase F treatment.

Results: Patient antibodies bound to the extracellular domain of Caspr2. Neither native protein structure nor glycosylation was required for immunoreactivity. Caspr2 constructs with single or multidomain deletions were expressed on the plasma membrane. All deletion constructs were recognized by patients' sera, although reactivity was significantly reduced with deletion of the discoidin-like subdomain and strongly reduced or abolished with larger deletions of multiple N-terminal subdomains. Caspr2 with all subdomains deleted except the discoidin-like domain was still recognized by the antibodies.

Conclusion: Caspr2 autoantibodies recognize multiple target epitopes in the extracellular domain of Caspr2, including one in the discoidin-like domain. Reactivity for some epitopes is not dependent on glycosylation or native protein structure.

No MeSH data available.