Identification of binding sites for anti-aquaporin 4 antibodies in patients with neuromyelitis optica (1)
Anti-aquaporin 4 antibodies (AQP4-Ab) are specifically detected in patients with neuromyelitis optica (NMO).
The M23 sequence was used in this study. DNA sequences for human and mouse AQP4 show 95% homology and demonstrate only 15 amino acid differences. If AQP4-Ab recognizes the AQP4 extracellular domain, only 4 amino acids differences occur between human and mouse and 3 differences between human and rat.
Positively-stained cells observed in 10 fields (×100) were graded into 3 groups in accordance to their pattern of staining: 3, similar staining to the original w-hAQP4 assay; 2, linear staining at the cell margin, but weaker than 3; 1, limited staining without clear linear patterning, and 0, negative stain.
The staining intensity for 9 of the 10 sera was recovered to those of w-hAQP4 when the mouse AQP4 mutant A228E was used and was partly recovered with T149M mutant, but not with S62T/N64K mutant.
Only 3 of the sera showed a reduction in staining intensity with mutant E228A of hAQP4. In contrast, most of the sera except one did not show staining decrement with the mutant T62S/K64N and M149T compared with w-hAQP4.
The immunostaining of rat and mouse CNS tissues with NMO sera showed more intense staining with rat tissue than mouse tissue.
Solubilized AQP4 protein as an antigen for western blotting and fragmented recombinant proteins containing each of the AQP4 extracellular domains were tested to detect anti-AQP4 Abs. However, these assays failed to be recognized by NMO sera. Thus, AQP4 may be recognized as an antigen in tertiary preserved structures. AQP4 demon- strates three extracellular loops that connect 6 alpha helices that span the membrane. AQP4 is associated with several transmembrane proteins including α and β-dystroglycan, dystrophin isoform Dp71, and syntrophin. The major epitope of AQP4-Ab is located at the E-loop of human AQP4 around the position of amino acid #228.