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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.


Caspr2 recognition does not depend on glycosylation or tertiary structure(A) Cells were transfected to express Caspr2 in the presence of tunicamycin or vehicle (DMSO). Tunicamycin treatment resulted in intracellular localization of Caspr2, but the transfected cells were still recognized by patients' sera and a commercial antibody to Caspr2 (ab33994). Scale bar = 10 μm. (B) Lysates from cells transfected with empty vector, cells transfected with Caspr2 treated with vehicle (DMSO), Caspr2 cells treated with tunicamycin (Tun), and Caspr2 lysates treated with PNGase F were probed on Western blot with a commercial Caspr2 antibody and patient sera.
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Figure 2: Caspr2 recognition does not depend on glycosylation or tertiary structure(A) Cells were transfected to express Caspr2 in the presence of tunicamycin or vehicle (DMSO). Tunicamycin treatment resulted in intracellular localization of Caspr2, but the transfected cells were still recognized by patients' sera and a commercial antibody to Caspr2 (ab33994). Scale bar = 10 μm. (B) Lysates from cells transfected with empty vector, cells transfected with Caspr2 treated with vehicle (DMSO), Caspr2 cells treated with tunicamycin (Tun), and Caspr2 lysates treated with PNGase F were probed on Western blot with a commercial Caspr2 antibody and patient sera.

Mentions: Caspr2 has 12 potential N-linked glycosylation sites in its extracellular domain.16 In order to determine whether glycosylation of these sites is required for recognition by Caspr2 autoantibodies, we transfected cultured cells to express Caspr2 while these cells were treated with tunicamycin (figure 2). (In these experiments, human antibodies were applied after fixation and permeabilization since Caspr2 was no longer membrane-expressed.) Tunicamycin treatment prevented cell surface expression of Caspr2 and resulted in a shift of the Caspr2 band on Western blot, but it did not prevent antibody recognition. Protein extract from cells transfected to express Caspr2 was also deglycosylated with PNGase F, resulting in a shift in the band on Western blot (figure 2B). This method of deglycosylation likewise did not prevent recognition of Caspr2 by the autoantibodies, even in the denatured state (i.e., on the Western blot). We further tested a panel of 6 sera for the ability to recognize Caspr2 under denaturing conditions (on Western blot), and all samples were able to do so (figure e-1). Caspr2 autoantibodies therefore recognize the protein under denaturing conditions and glycosylation is not required for antibody recognition.


Caspr2 autoantibodies target multiple epitopes.

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

Caspr2 recognition does not depend on glycosylation or tertiary structure(A) Cells were transfected to express Caspr2 in the presence of tunicamycin or vehicle (DMSO). Tunicamycin treatment resulted in intracellular localization of Caspr2, but the transfected cells were still recognized by patients' sera and a commercial antibody to Caspr2 (ab33994). Scale bar = 10 μm. (B) Lysates from cells transfected with empty vector, cells transfected with Caspr2 treated with vehicle (DMSO), Caspr2 cells treated with tunicamycin (Tun), and Caspr2 lysates treated with PNGase F were probed on Western blot with a commercial Caspr2 antibody and patient sera.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Caspr2 recognition does not depend on glycosylation or tertiary structure(A) Cells were transfected to express Caspr2 in the presence of tunicamycin or vehicle (DMSO). Tunicamycin treatment resulted in intracellular localization of Caspr2, but the transfected cells were still recognized by patients' sera and a commercial antibody to Caspr2 (ab33994). Scale bar = 10 μm. (B) Lysates from cells transfected with empty vector, cells transfected with Caspr2 treated with vehicle (DMSO), Caspr2 cells treated with tunicamycin (Tun), and Caspr2 lysates treated with PNGase F were probed on Western blot with a commercial Caspr2 antibody and patient sera.
Mentions: Caspr2 has 12 potential N-linked glycosylation sites in its extracellular domain.16 In order to determine whether glycosylation of these sites is required for recognition by Caspr2 autoantibodies, we transfected cultured cells to express Caspr2 while these cells were treated with tunicamycin (figure 2). (In these experiments, human antibodies were applied after fixation and permeabilization since Caspr2 was no longer membrane-expressed.) Tunicamycin treatment prevented cell surface expression of Caspr2 and resulted in a shift of the Caspr2 band on Western blot, but it did not prevent antibody recognition. Protein extract from cells transfected to express Caspr2 was also deglycosylated with PNGase F, resulting in a shift in the band on Western blot (figure 2B). This method of deglycosylation likewise did not prevent recognition of Caspr2 by the autoantibodies, even in the denatured state (i.e., on the Western blot). We further tested a panel of 6 sera for the ability to recognize Caspr2 under denaturing conditions (on Western blot), and all samples were able to do so (figure e-1). Caspr2 autoantibodies therefore recognize the protein under denaturing conditions and glycosylation is not required for antibody recognition.

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.