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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.


Patients' sera recognize surface epitopes of Caspr2HEK293 T cells were transiently transfected to express Caspr2, stained with a series of patient sera live, and then stained with a commercial antibody to Caspr2 after permeabilization and fixation. Nuclei of transfected and untransfected cells were stained with DAPI (blue). Surface epitopes of Caspr2 were recognized by all 10 patients' sera (4 representative samples [S1–S4] are shown). Scale bar = 10 μm.
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Figure 1: Patients' sera recognize surface epitopes of Caspr2HEK293 T cells were transiently transfected to express Caspr2, stained with a series of patient sera live, and then stained with a commercial antibody to Caspr2 after permeabilization and fixation. Nuclei of transfected and untransfected cells were stained with DAPI (blue). Surface epitopes of Caspr2 were recognized by all 10 patients' sera (4 representative samples [S1–S4] are shown). Scale bar = 10 μm.

Mentions: We studied a panel of 10 sera from patients with Caspr2 antibodies identified using methods previously described.7 In order to confirm that patients' Caspr2 antibodies consistently recognize a surface epitope of Caspr2, HEK293 T cells were transfected to express wild-type Caspr2 and immunostained live with patient sera (1:500). The cells were then fixed, permeabilized, and treated with a rabbit Caspr2 antibody targeting an intracellular epitope, followed by fluorescent anti-human and anti-rabbit secondary antibodies (4 representative cells are shown in figure 1). All 10 patients' sera recognized a surface epitope on transfected cells but not on untransfected cells.


Caspr2 autoantibodies target multiple epitopes.

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

Patients' sera recognize surface epitopes of Caspr2HEK293 T cells were transiently transfected to express Caspr2, stained with a series of patient sera live, and then stained with a commercial antibody to Caspr2 after permeabilization and fixation. Nuclei of transfected and untransfected cells were stained with DAPI (blue). Surface epitopes of Caspr2 were recognized by all 10 patients' sera (4 representative samples [S1–S4] are shown). Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Patients' sera recognize surface epitopes of Caspr2HEK293 T cells were transiently transfected to express Caspr2, stained with a series of patient sera live, and then stained with a commercial antibody to Caspr2 after permeabilization and fixation. Nuclei of transfected and untransfected cells were stained with DAPI (blue). Surface epitopes of Caspr2 were recognized by all 10 patients' sera (4 representative samples [S1–S4] are shown). Scale bar = 10 μm.
Mentions: We studied a panel of 10 sera from patients with Caspr2 antibodies identified using methods previously described.7 In order to confirm that patients' Caspr2 antibodies consistently recognize a surface epitope of Caspr2, HEK293 T cells were transfected to express wild-type Caspr2 and immunostained live with patient sera (1:500). The cells were then fixed, permeabilized, and treated with a rabbit Caspr2 antibody targeting an intracellular epitope, followed by fluorescent anti-human and anti-rabbit secondary antibodies (4 representative cells are shown in figure 1). All 10 patients' sera recognized a surface epitope on transfected cells but not on untransfected cells.

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.