BackgroundNeuromyelitis optica (NMO) is a demyelinating disease of the central nervous system (CNS) of putative autoimmune aetiology. Early discrimination between multiple sclerosis (MS) and NMO is important, as optimum treatment for both diseases may differ considerably. Recently, using indirect immunofluorescence analysis, a new serum autoantibody (NMO-IgG) has been detected in NMO patients. The binding sites of this autoantibody were reported to colocalize with aquaporin 4 (AQP4) water channels. Thus we hypothesized that AQP4 antibodies in fact characterize NMO patients.Methods and FindingsBased on these observations we cloned human water channel AQP4, expressed the protein in a eukaryotic transcription/translation system, and employed the recombinant AQP4 to establish a new radioimmunoprecipitation assay (RIPA). Indeed, application of this RIPA showed that antibodies against AQP4 exist in the majority of patients with NMO (n = 37; 21 positive) as well as in patients with isolated longitudinally extensive transverse myelitis (n = 6; six positive), corresponding to a sensitivity of 62.8% and a specificity of 98.3%. By contrast, AQP4 antibodies were virtually absent in 291 other participants, which included patients with MS (n = 144; four positive), patients with other inflammatory and noninflammatory neurological diseases (n = 73; one positive), patients with systemic autoimmune diseases (n = 45; 0 positive), and healthy participants (n = 29; 0 positive).ConclusionsIn the largest series reported so far to our knowledge, we quantified AQP4 antibodies in patients with NMO versus various other diseases, and showed that the aquaporin 4 water channel is a target antigen in a majority of patients with NMO. The newly developed assay represents a highly specific, observer-independent, and easily reproducible detection method facilitating clinically relevant discrimination between NMO, MS, and other inflammatory diseases.

Acid-secreting intercalated cells of the kidney collecting duct and tumor cells of renal oncocytoma express an anion exchanger that is immunologically related but not identical to the chloride-bicarbonate anion exchanger of erythrocytes (AE1). In this study, we have mapped the binding site of a monoclonal antibody against erythroid AE1 that does not react with either intercalated cells or oncocytoma. The epitope is located close to the NH2 terminus of AE1, indicating that AE1 in intercalated cells and oncocytoma differs in its NH2 terminus from erythroid AE1. This conclusion was supported by an antibody directed against residues 1-14 of erythroid AE1 that does not react with intercalated cells in oncocytoma. Polymerase chain reaction performed with mRNA from a human kidney revealed that the sequence containing the codons for Met-1 and Met-33 in erythroid mRNA is missing in the kidney transcript, whereas the sequence coding for Met-66 is present. DNA sequence data derived from cloning the 5' end of the human kidney AE1 mRNA clearly showed that the 5' untranslated region comprises part of intron 3, the complete exon 4 that is followed by exon 5 containing Met-66 as the site of translation initiation. Altogether, the results indicate that AE1 in the human kidney is an amino-terminally truncated form of erythroid AE1 that is restricted to the basolateral membrane domain of the acid-secreting intercalated cells of the collecting duct and is also expressed in oncocytoma.

The complete amino acid sequence of human antileukoprotease has been determined by direct sequencing of the inhibitory active protein isolated from seminal plasma (HUSI-I) and by sequence analysis of cDNA reverse-transcribed from mRNA prepared from cervical tissue. The inhibitor (Mr 11726) consists of 107 amino acid residues including 16 cysteines presumably forming disulfide bonds. The molecule comprises two consecutive domains which are homologous to each other, to the second domain of the basic protease inhibitor from Red Sea turtle (chelonianin) and to both domains of the whey proteins of rat and mouse. Both domains contain a pattern of cysteines known as the 'four-disulfide-core' that has also been found in wheat germ agglutinin and neurophysin.

Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.

Substitution by site-directed mutagenesis of any one of the histidine residues H721, H837, and H852 by glutamine, or of H752 by serine, inhibits Cl- flux mediated by band 3 expressed in Xenopus oocytes. Mutation of Lys 558 (K558N), the site of covalent binding of H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) in the outer membrane surface, in combination with any one of the His/Gln mutations leads to partial (H721Q; H837Q) or complete (H852Q) restoration of Cl- flux. In contrast, inhibition of Cl- flux by mutation of proline or lysine residues in the vicinity of His 837 at the inner membrane surface cannot be reversed by the second-site mutation K558N, indicating specificity of interaction between Lys 558 and His 837. The histidine-specific reagent diethyl pyrocarbonate (DEPC) is known to inhibit band 3-mediated anion exchange in red blood cells [Izuhara, K., Okubo, K., & Hamasaki, N. (1989) Biochemistry 28, 4725-4728]. It was also found to inhibit transport after expression in the oocyte of wild-type band 3, of the double mutants of the histidines listed above, and of the single mutant H752S. The effects on the wild type and the double mutants were indistinguishable, while the mutant H752S exhibited a considerably reduced sensitivity to inhibition, suggesting that His 752 is the most prominent site of action of DEPC. According to a hydrophobicity plot of band 3 and further independent evidence, Lys 558, the mutated histidines, and Glu 699, the mutation of which was also found to inhibit Cl- flux [Müller-Berger, S., Karbach, D., Kang, D., Aranibar, N., Wood, P. G., Rüterjans, H., & Passow, H. (1995) Biochemistry 34, 9325-9332], are most likely located in five different transmembrane helices. The interactions between Lys 558 and the various histidines suggest that these helices reside in close proximity. Together with the helix carrying Glu 699, they could form an access channel lined with an array of alternating histidine and glutamate residues. Together with a chloride ion bridging the gap between His 852 and His 837, they could have the potential to form, at low pH, a transmembrane chain of hydrogen bonds. The possible functional significance of such channel is discussed.