Table 1.
Laboratory and clinical data for anti-AQP1 seropositive patients.
Figure 1.
Measurement of anti-AQP1 antibodies in the sera of different patient groups and healthy controls by RIPA.
The serum samples are (left to right) from patients with suspected NMOsd without (AQP4-neg) or with (AQP4-pos) anti-AQP4 antibodies, MS, other neuroimmune diseases (131/142 with MG), or healthy controls. The numbers in parenthesis below the group are the number of anti-AQP1 antibody-positive sera and the total number of test sera for each group of patients. The dashed and solid horizontal lines denote the cut-off values for ambiguous and positive titers. The left and right y axis show, respectively, the precipitated cpm and the estimated antibody titer.
Figure 2.
Liver powder removes anti-AQP1 antibodies.
Two exclusively anti-AQP1-positive and two exclusively anti-AQP4-positive serum samples were pretreated with guinea pig liver powder, then the supernatants were tested by RIPA using indirectly radiolabeled AQP1 (left panel) or AQP4 (right panel). Key: +, pretreated serum; -, untreated serum.
Figure 3.
Anti-AQP1 specificity of the identified autoantibodies.
(A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using 125I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.
Figure 4.
Detection of binding of anti-AQP1 antibody to denatured AQP1 by Western blotting.
Yeast-expressed AQP1 was electrophoresed and transferred onto nitrocellulose membranes, which were then incubated with the test sera (see Methods). Lanes 1-3: Three sera from healthy controls at dilutions 1/250, 1/60, and 1/30, respectively; lane 4: commercial rabbit anti-AQP1 antibody; lanes 5-8: representative anti-AQP1-positive sera with titers of 132, 25, 10.5, and 5.8 nM, at dilutions of 1/500, 1/250, 1/30, and 1/30, respectively. None of the test anti-AQP1 sera bound to the control protein MuSK (not shown).
Figure 5..
Detection of anti-AQP1 antibodies by an ELISA with immobilized purified human yeast-expressed AQP1.
Sera previously tested by RIPA for AQP1 antibodies were tested for binding to immobilized AQP1 by ELISA. First column contains 31 sera found positive by the RIPA (including a double-positive anti-AQP1/AQP4, empty square, and 3 sera from anti-AQP1-positive MS patients, empty circles). The following 3 columns contain 5 anti-AQP4-positive/anti-AQP1-negative sera, 30 sera from anti-AQP1-negative MS patients and 44 sera from healthy controls). The dashed horizontal line denotes the cut-off (O.D.450: 0.36) for positive values.
Figure 6.
Anti-AQP1 antibodies and anti-AQP4 antibodies do not bind to the other antigen.
(A and B). Search for cross-reactivity of anti-AQP1 and anti-AQP4 antibodies with AQP4 and AQP1, respectively. Three (A) or five (B) double-positive serum samples were incubated with immobilized AQP1 (A) or AQP4 (B) to immunoadsorb the corresponding antibodies, then were tested by RIPA for binding to 125I-labeled AQP1 or AQP4 (white bars) in parallel with the untreated sera (black bars). HC, healthy control. (C). Lack of correlation of the amount of radiolabeled AQP1 precipitated by anti-AQP1 antibodies (y axis) or AQP4 (x axis) precipitated anti-AQP4 antibodies by double-positive sera in identical regular RIPAs performed using the two labeled antigens.
Figure 7.
Ig class and IgG subclass of the anti-AQP1 antibodies.
The Ig class and IgG subclass of the anti-AQP1 antibodies from 7 positive human sera was tested by RIPA using anti-class/subclass second antibodies. Sera no. 1-4 are from patients 11-14 in Table 1 and sera no. 5-7 are from the remaining anti-AQP1 antibody-positive sera.
Figure 8.
Determination of the percentage of antibodies directed against the extracellular domain of membrane-embedded AQP1.
Four anti-AQP1-positive sera were left untreated or were preincubated with increasing numbers of AQP1-GFP- (filled symbols) or EGFP-transfected (shown only for serum 3; empty triangle) HEK293 cells treated with secretin to increase surface expression of AQP1; the untreated and treated samples were tested in the usual RIPA for anti-AQP1 antibodies. The sera were also treated with AQP4-transfected HEK293 cells (shown only for serum 3; open circle). Serum 4 was also treated with an extract of AQP1-transfected HEK293 cells (open square).
Figure 9.
Anti-AQP1 antibody binding to synthetic peptides corresponding to AQP1 extracellular and cytoplasmic domains.
A. Synthetic peptides corresponding to human AQP1 extracellular loops A (residues 36–49), C (116–137), and E (183–188, 199–210) or to the intracellular N-terminus (1–22), loop B (81–100), and the C-terminus (248–270) are shown (from Uniprot/Swissprot entry P29972, for the human AQP1). Extracellular residues are shown in bold and underlined. Loop-E is formed by two segments separated by a 14 residue gap; in “pept-Loop-E”, 10 of these 14 residues (between TG and SE) were omitted (marked by:..). In the first 4 peptides, an extra tyrosine residue was added to the N-terminus for future radioiodination studies. Below the AQP1 peptide sequences are shown the corresponding AQP4 sequences; (*), identical amino acid residues; (:) and (.), homologous residues. B. Peptide mapping with anti-AQP1 positive sera. 22 sera from patients with known clinical characteristics (17 NMOsd and 5 MS) were tested for binding to 6 synthetic peptides corresponding to the 3 extracellular loops (loops A, C and E) and to the 3 cytoplasmic segments (N-terminal, Loop-B and C-terminal) by an ELISA assay. Positive values are considered those above O.D.450 0.45 (see Methods). It is shown that most NMOsd sera bind to the Loop-A peptide.