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4, lane 2, lower panel). were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by Brompheniramine immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in Brompheniramine the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4. strong class=”kwd-title” Keywords: Aquaporin-4, Astrocyte, Autoimmune disease, Endocytosis, Monoclonal antibody, Neuromyelitis optica Graphical abstract Open in a separate window 1.?Introduction Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system generally characterized by a disease-specific autoantibody called NMO-IgG [1]. A target of NMO-IgG is a water channel, aquaporin-4 (AQP4) [2], and binding of NMO-IgG to AQP4 expressed in astrocytic end-feet followed by complement-dependent and cell-mediated disruption of astrocytes causes demyelination in NMO [3], [4], [5], [6], [7]. NMO-IgG-induced endocytosis of AQP4 accompanied by excitatory amino acid transporter 2 (EAAT2), which leads to disruption of glutamate homeostasis and excitotoxicity of oligodendrocytes, was also proposed [7], [8]. AQP4 has two dominant isoformsM1 and M23both of which are simultaneously expressed and functions as a homo/heterotetramer randomly incorporating these two isoforms [32], [33]. AQP4 has a unique feature, namely formation of orthogonal arrays Brompheniramine of particles (OAPs), in which AQP4 tetramers are orthogonally arranged [9], [10]. M23 lacks the initial 22 amino acids of the N-terminal cytoplasmic domain of M1 due to the difference in transcriptional start sites [11]. This 22-amino-acid domain interferes with formation of the OAPs [12]; therefore, M1 homotetramers form few arrays, while M23 homotetramers form much larger arrays than do endogenously expressed AQP4 homo/heterotetramers. The epitopes for NMO-IgG in AQP4 are located TM4SF18 in the extracellular domains (ECDs), consisting of three loops connecting the six transmembrane domains. In most cases, NMO-IgG preferentially binds to cells expressing M23 rather than to those expressing M1 alone, implying that OAP-formation of AQP4 contributes to recognition by NMO-IgG since the primary sequences of ECDs of M1 and M23 are identical [13], [14], [15], [16], [17], [18]. Multiple groups have reported that NMO-IgG only recognizes native AQP4 integrated into a lipid bilayer, suggesting that the epitope of NMO-IgG is formed by more than one loop in a conformational structure [13], [16], [23]. But some NMO-IgG can recognize denatured AQP4 as well as linear peptides corresponding to one of the extracellular loops Brompheniramine [34]. NMO-IgG-induced endocytosis and/or degradation of AQP4 have been observed in some cell lines, including HEK293, CHO, and U87-MG, ectopically expressing AQP4. However, while a group demonstrated that NMO-IgG only induced endocytosis of M1, the other group reported that recombinant NMO-IgGs cloned from cerebrospinal fluid plasmablasts of an NMO patient induced endocytosis of both isoform [8], [19], [20], [21]. In addition to AQP4 over-expressed in a stable cell line, NMO-IgGCinduced endocytosis of endogenous AQP4 in a primary culture of rat, mouse, and human astrocytes was also reported [8], [20], [22]. On the other hand, one of the recombinant monoclonal NMO-IgGs, rAb-53 induced little endocytosis of endogenous mouse AQP4 (mAQP4) in primary cultured astrocytes [15], [21], [23], despite the fact that it did endocytosis of ectopically expressed AQP4 in a stable cell line [21]. These conflicting findings.