The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel.

Blood calcium concentration is maintained within a narrow range despite large variations in dietary input and body demand. The Transient Receptor Potential ion channel TRPV5 has been implicated in this process. We report here that TRPV5 is stimulated by the mammalian hormone klotho. Klotho, a beta-glucuronidase, hydrolyzes extracellular sugar residues on TRPV5, entrapping the channel in the plasma membrane. This maintains durable calcium channel activity and membrane calcium permeability in kidney. Thus, klotho activates a cell surface channel by hydrolysis of its extracellular N-linked oligosaccharides.

The epithelial calcium channel, ECaC, is activated by hyperpolarization and regulated by cytosolic calcium.

The recently cloned epithelial Ca(2+) channel, ECaC, which is expressed in the apical membrane of 1,25-dihydroxyvitamin D(3)-responsible epithelia, was characterized in Xenopus laevis oocytes by measuring the Ca(2+)-activated Cl(-) current which is a sensitive read-out of the Ca(2+) influx. ECaC-expressing oocytes responded to a voltage ramp with a maximal inward current of -2.1 +/- 0.3 microA at a holding potential of -99 +/- 1 mV. The inward current decreased progressively at less negative potentials and at +50 mV a small Ca(2+)-induced outward current was observed. The Ca(2+) influx-evoked current at a hyperpolarizing pulse to -100 mV displayed a fast activation followed by a rapid but partial inactivation. Loading of the oocytes with the Ca(2+) chelator BAPTA delayed the activation and blocked the inactivation of ECaC. When a series of brief hyperpolarizing pulses were given a significant decline in the peak response and subsequent plateau phase was observed. In conclusion, the distinct electrophysiological features of ECaC are hyperpolarization-dependent activation, Ca(2+)-dependent regulation of channel conductance and desensitization during repetitive stimulation.

Molecular identification of the apical Ca2+ channel in 1, 25-dihydroxyvitamin D3-responsive epithelia.

In mammals, the extracellular calcium concentration is maintained within a narrow range despite large variations in daily dietary input and body demand. The small intestine and kidney constitute the influx pathways into the extracellular Ca2+ pool and, therefore, play a primary role in Ca2+ homeostasis. We identified an apical Ca2+ influx channel, which is expressed in proximal small intestine, the distal part of the nephron and placenta. This novel epithelial Ca2+ channel (ECaC) of 730 amino acids contains six putative membrane-spanning domains with an additional hydrophobic stretch predicted to be the pore region. ECaC resembles the recently cloned capsaicin receptor and the transient receptor potential-related ion channels with respect to its predicted topology but shares less than 30% sequence homology with these channels. In kidney, ECaC is abundantly present in the apical membrane of Ca2+ transporting cells and colocalizes with 1,25-dihydroxyvitamin D3-dependent calbindin-D28K. ECaC expression in Xenopus oocytes confers Ca2+ influx with properties identical to those observed in distal renal cells. Thus, ECaC has the expected properties for being the gatekeeper of 1,25-dihydroxyvitamin D3-dependent active transepithelial Ca2+ transport.

Characterization of murine monoclonal antibodies against 60-kD Ro/SS-A and La/SS-B autoantigens.

Small cytoplasmic ribonucleoproteins (scRNPs) are important autoantigens in patients with systemic lupus erythematosus and Sjögren's syndrome. MoAbs against these proteins were made by immunization of BALB/c mice with purified human recombinant 60-kD Ro/SS-A or 50-kD La/SS-B proteins. Five stable hybridoma cell lines were obtained, of which four secreted anti-Ro/SS-A antibodies (clones 1D8, 1D11, 2G10 and 6G8) and one produced anti-La/SS-B antibodies (clone 7F6). The MoAbs were further characterized using four different immunoassays: immunofluorescence, immunoblotting, RNA precipitation combined with Northern blotting, and recombinant protein precipitation. All four MoAbs against Ro/SS-A recognized the native protein and one of them (2G10) recognized also intact scRNP particles. Interestingly, hY3-RNA was reproducibly not efficiently precipitated by MoAb 2G10. Epitope mapping using deletion mutants of the 60-kD Ro/SS-A antigen showed that MoAb 1D8 recognized the C-terminal part of this protein, while 1D11 and 2G10 recognized distinct epitopes in the region between the RNP motif and the putative zinc finger domain. The epitopes recognized by these MoAbs are highly conserved among species, and the epitope recognized by MoAb 2G10 may be identical to an autoepitope recognized by sera of patients. This is the first report describing the isolation and characterization of MoAbs of the IgG class against the 60-kD Ro/SS-A and La/SS-B autoantigens obtained by immunization with purified human recombinant proteins. These MoAbs can be of great use in studying the cellular processes in which scRNPs are involved, and may help to determine why these scRNPs become autoantigenic in autoimmune diseases.

The 56K autoantigen is identical to human annexin XI.

Anti-56K autoantibodies are present in sera from patients with various autoimmune diseases, predominantly in sera from patients with rheumatoid arthritis, systemic lupus erythematosus, or Sjögren's syndrome. To clarify the molecular structure of this autoantigen, we isolated a 2.0-kilobase pair cDNA clone considered to encode the full-length 56K autoantigen. The longest open reading frame encodes a 505-amino acid polypeptide, with a predicted molecular mass of 54.4 kDa. The in vitro translated protein is recognized by all anti-56K positive patient sera tested. Antibodies affinity-purified using the bacterially expressed recombinant protein recognized the 56K autoantigen in a HeLa cell extract. cDNA sequencing revealed that the 56K cDNA shares a high degree of homology in both nucleotide (87%) and amino acid sequence (92.5%) with bovine annexin XI, indicating that the 56K cDNA encodes the human homologue of annexin XI, a member of the Ca(2+)-dependent phospholipid binding protein family. Anti-56K autoantibody exhibits both a cytoplasmic and a nuclear staining in immunofluorescence experiments. Patients' sera recognize preferentially the N-terminal region of the protein, which is specific for 56K/annexin XI and not shared by other annexins, indicating that the autoimmune response to 56K/annexin XI in these patients is specific for this annexin family member.

Structural analysis of a variant clone of Snyder-Theilen feline sarcoma virus.

A variant clone of Snyder-Theilen feline sarcoma virus (ST-FeSV) encoding a polyprotein with a molecular weight of approximately 104 kDa (P104) was compared to the P85 encoding prototype clone of ST-FeSV. Analysis of chimeric genes constructed with the viral oncogenes of the two clones indicated that the variant clone coded for a larger polyprotein than the prototype clone because of genetic differences in its 3' portion. Comparative DNA sequence analysis revealed that one nucleotide just upstream of the termination condon TGA in the prototype proviral DNA was deleted from the variant clone resulting in a 468-bp larger open reading frame. Furthermore, it appeared that the U3 regions of the long terminal repeats (LTRs) of the variant clone contained an insertion of 71 bp as compared to the LTRs of the prototype clone. In addition, both clones differed also from each other with respect to genetic sequences deleted from their env gene regions.