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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.


Single-domain deletion constructs were recognized by patients' sera(A) Single-domain deletion constructs were generated as shown. (B) Each of these constructs was recognized by patients' sera on Western blot probed with a commercial antibody to Caspr2 (Caspr2) and patients' sera (sera 1).
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Figure 3: Single-domain deletion constructs were recognized by patients' sera(A) Single-domain deletion constructs were generated as shown. (B) Each of these constructs was recognized by patients' sera on Western blot probed with a commercial antibody to Caspr2 (Caspr2) and patients' sera (sera 1).

Mentions: Caspr2 has a very large extracellular domain, comprising more than 1,200 amino acids. The extracellular domain has previously been subdivided into 8 regions based on homology to various protein domains. We have followed a previously proposed definition of these domains (http://www.uniprot.org/uniprot/Q9UHC6#section_seq). Starting from the N-terminus of the protein and moving to the transmembrane region, these domains are Discoidin (Disc), LamininG (Lam1), LamininG (Lam2), Egf (Egf1), FibrinogenC (FibC), LamininG (Lam3), Egf (Egf2), and LamininG (Lam4). There is a single transmembrane domain and a relatively small intracellular region with a protein 4.1-binding domain. We generated a series of plasmids for expressing Caspr2 with deletions of each of the 8 extracellular subdomains (figure 3A). We named these constructs with a delta symbol followed by the domain deleted. ΔDisc, for instance, has the Disc domain deleted.


Caspr2 autoantibodies target multiple epitopes.

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

Single-domain deletion constructs were recognized by patients' sera(A) Single-domain deletion constructs were generated as shown. (B) Each of these constructs was recognized by patients' sera on Western blot probed with a commercial antibody to Caspr2 (Caspr2) and patients' sera (sera 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Single-domain deletion constructs were recognized by patients' sera(A) Single-domain deletion constructs were generated as shown. (B) Each of these constructs was recognized by patients' sera on Western blot probed with a commercial antibody to Caspr2 (Caspr2) and patients' sera (sera 1).
Mentions: Caspr2 has a very large extracellular domain, comprising more than 1,200 amino acids. The extracellular domain has previously been subdivided into 8 regions based on homology to various protein domains. We have followed a previously proposed definition of these domains (http://www.uniprot.org/uniprot/Q9UHC6#section_seq). Starting from the N-terminus of the protein and moving to the transmembrane region, these domains are Discoidin (Disc), LamininG (Lam1), LamininG (Lam2), Egf (Egf1), FibrinogenC (FibC), LamininG (Lam3), Egf (Egf2), and LamininG (Lam4). There is a single transmembrane domain and a relatively small intracellular region with a protein 4.1-binding domain. We generated a series of plasmids for expressing Caspr2 with deletions of each of the 8 extracellular subdomains (figure 3A). We named these constructs with a delta symbol followed by the domain deleted. ΔDisc, for instance, has the Disc domain deleted.

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.