Fine specificity of antibodies against AQP4: Epitope mapping reveals intracellular epitopes

Abstract

The autoantibody to aquaporin-4 (AQP4) is a marker and a pathogenetic factor in Neuromyelitis Optica (NMO) (Devic's syndrome). Our aim was to identify B-cell antigenic linear epitopes of the AQP4 protein and investigate similarities with other molecules. To this end, we screened sera from 21 patients positive for anti-AQP4 antibodies (study group), from 23 SLE and 23 pSS patients without neurologic involvement (disease controls) and from 28 healthy individuals (normal controls). Eleven peptides, spanning the entire intracellular and extracellular domains of the AQP4 molecule, were synthesized, and all sera were screened for anti-peptide antibodies by ELISA. Specificity was evaluated by homologous inhibition assays. NMO positive sera exhibited reactivity against 3 different peptides spanning the sequences aa1-22 (AQPpep1) (42.9% of patients), aa88-113 (AQPpep4) (33%) and aa252-275 (AQPpep8) (23.8%). All epitopes were localized in the intracellular domains of AQP4. Homologous inhibition rates were ranging from 71.1% to 84.3%. A 73% sequence homology was observed between AQPpep8' aa257-271, a 15-mer peptide part of the AQPpep8 aa252-275, and the aa219-233 domain of the Tax1-HTLV-1 binding protein (TAX1BP1), a host protein associated with replication of the Human T-Lymphotropic Virus 1 (HTLV-1). Antibodies against the AQP4 and the TAX1BP1 15-mer peptides were detected in 26.3% (N = 5) and 31.6% (N = 6) of NMO positive sera (r(s) = 0.81, P < 0.0001). Healthy controls did not react with these peptides, while homologous and cross-inhibition assays confirmed binding specificity. This first epitope mapping for AQP4 reveals that a significant proportion of anti-AQP4 antibodies target linear epitopes localized in the intracellular domains of the channel. One of the epitopes displays high similarity with a portion of TAX1BP1 protein.

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients

“…6). Another research group performed an epitope mapping using peptides spanning all intracellular and extracellular (but not transmembrane) parts of AQP4 (63,64). They found that ϳ20% of NMO patients and ϳ10% of MS patients have increased serum IgG reactivities against an intracellular region (AQP4 252-275), which overlaps with the peptide filtered in our study.…”

Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

“…The latter point is supported by the fact that infection of oligodendrocytes and astrocytes during PML has been proposed to be LSTc-independent (53). Antibodies against intracellular and nuclear epitopes are, however, well documented in autoimmune diseases (54), and a recent study showed that the intracellular antibody receptor tripartite motif–containing protein 21 (TRIM21), a ubiquitously expressed E3 ubiquitin ligase, provides a mechanism to protect mice from lethal adenoviral infection through intracellular antibody recognition (55). Hence, JCPyV-specific antibodies may contribute to both extracellular and intracellular mechanisms in PML.…”

Reactivity to AQP4 epitopes in relapsing–remitting multiple sclerosis

“…In NMO patients, this T-cell epitope resides at amino acids 281-300 adjacent to the most prominent B-cell linear epitope which resides at amino acids 252-275 (Kampylafka et al, 2011). Because both of these epitopes are intracellular and were not found in any other disease tested, the results suggest that in NMO, contrary to the common belief that only the extracellular epitopes are important for disease progression, some extracellular domains may be also relevant, at least in pointing out antigenic similarities between NMO and RRMS patients.…”

“…The suggestion that there may be clinical intermediate types within the spectrum of NMO and MS subsets, prompted us to investigate whether we could detect signs of NMO autoimmunity in typical MS patients. A sensitive ELISA method, recently developed by our group (Kampylafka et al, 2011) to describe the dominant B-cell linear epitopes in NMO-AQP4 positive patients, was employed.…”