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• W2071601995 abstract "Proteins of the CLCA gene family have been proposed to mediate calcium-activated chloride currents. In this study, we used detailed bioinformatics analysis and found that no transmembrane domains are predicted in hCLCA1 or mCLCA3 (Gob-5). Further analysis suggested that they are globular proteins containing domains that are likely to be involved in protein-protein interactions. In support of the bioinformatics analysis, biochemical studies showed that hCLCA1 and mCLCA3, when expressed in HEK293 cells, could be removed from the cell surface and could be detected in the extracellular medium, even after short incubation times. The accumulation in the medium was shown to be brefeldin A-sensitive, demonstrating that hCLCA1 is constitutively secreted. The N-terminal cleavage products of hCLCA1 and mCLCA3 could be detected in bronchoalveolar lavage fluid taken from asthmatic subjects and ovalbumin-challenged mice, demonstrating release from cells in a physiological setting. We conclude that hCLCA1 and mCLCA3 are non-integral membrane proteins and therefore cannot be chloride channels in their own right. Proteins of the CLCA gene family have been proposed to mediate calcium-activated chloride currents. In this study, we used detailed bioinformatics analysis and found that no transmembrane domains are predicted in hCLCA1 or mCLCA3 (Gob-5). Further analysis suggested that they are globular proteins containing domains that are likely to be involved in protein-protein interactions. In support of the bioinformatics analysis, biochemical studies showed that hCLCA1 and mCLCA3, when expressed in HEK293 cells, could be removed from the cell surface and could be detected in the extracellular medium, even after short incubation times. The accumulation in the medium was shown to be brefeldin A-sensitive, demonstrating that hCLCA1 is constitutively secreted. The N-terminal cleavage products of hCLCA1 and mCLCA3 could be detected in bronchoalveolar lavage fluid taken from asthmatic subjects and ovalbumin-challenged mice, demonstrating release from cells in a physiological setting. We conclude that hCLCA1 and mCLCA3 are non-integral membrane proteins and therefore cannot be chloride channels in their own right. Many cells are known to possess calcium-dependent chloride channel activities. These include epithelial cells and smooth muscle cells (1Atherton H. Mesher J. Poll C.T. Danahay H. Naunyn-Schmiedeberg's Arch. Pharmacol. 2003; 367: 214-217Crossref PubMed Scopus (19) Google Scholar, 2Large W.A. Wang Q. Am. J. Physiol. 1996; 271: C435-C454Crossref PubMed Google Scholar). Although we know little about the identity of these channels, members of the CLCA gene family have been suggested to be candidate calcium-sensitive chloride channels (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar, 4Cunningham S.A. Awayda M.S. Bubien J.K. Ismailov I.I. Arrate M.P. Berdiev B.K. Benos D.J. Fuller C.M. J. Biol. Chem. 1995; 270: 31016-31026Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 5Gandhi R. Elble R.C. Gruber A.D. Schreur K.D. Ji H.L. Fuller C.M. Pauli B.U. J. Biol. Chem. 1998; 273: 32096-32101Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Although normally expressed in the gastrointestinal tract, up-regulation of the human calcium-activated chloride channel hCLCA1 has been linked to disease states such as asthma and cystic fibrosis (6Hoshino M. Morita S. Iwashita H. Nagi T. Nakanishi A. Ashida Y. Nishimura O. Fujisawa Y. Fujino M. Am. J. Respir. Crit. Care Med. 2002; 165: 1132-1136Crossref PubMed Scopus (125) Google Scholar, 7Toda M. Tulic M.K. Levitt R.C. Hamid Q. J. Allergy Clin. Immunol. 2002; 109: 246-250Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 8Hauber H.P. Manoukian J.J. Nguyen L.H.P. Sobol S.E. Levitt R.C. Holroyd K.J. McElvaney N.G. Griffin S. Hamid Q. Laryngoscope. 2003; 113: 1037-1042Crossref PubMed Scopus (36) Google Scholar, 9Hauber H.P. Tsicopoulos A. Wallaert B. Griffin S. McElvaney N.G. Daigneault P. Mueller Z. Olivenstein R. Holroyd K.J. Levitt R.C. Hamid Q. Eur. Respir. J. 2004; 23: 846-850Crossref PubMed Scopus (36) Google Scholar). This up-regulation was observed in bronchial epithelial cells and goblet cells. In the mouse, the ortholog of hCLCA1 (mCLCA3) exhibits a similar expression profile to hCLCA1 and, in addition, has been localized to mucin granules (10Leverkoehne I. Gruber A.D. J. Histochem. Cytochem. 2002; 50: 829-838Crossref PubMed Scopus (105) Google Scholar). The expression of mCLCA3 is up-regulated in mouse lung in response to ovalbumin challenge or upon challenge with more complex allergens such as Aspergillus fumigatus, systems that are utilized to model aspects of human asthma (11Nakanishi A. Morita S. Iwashita H. Sagiya Y. Ashida Y. Shirafuji H. Fujisawa Y. Nishimura O. Fujino M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5175-5180Crossref PubMed Scopus (282) Google Scholar, 12Zhou Y. Dong Q. Louahed J. Dragwa C. Savio D. Huang M. Weiss C. Tomer Y. McLane M.P. Nicolaides N.C. Levitt R.C. Am. J. Respir. Cell Mol. Biol. 2001; 25: 486-491Crossref PubMed Scopus (124) Google Scholar). Evidence for the CLCA family as calcium-sensitive chloride channels comes from heterologous expression of a number of CLCA isoforms in a range of cellular systems, which resulted in generation of membrane currents activated with high Ca2+ concentrations or with ionomycin. These currents were blocked with chloride channel blockers such as niflumic acid and were performed in Cl- selective conditions (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar, 4Cunningham S.A. Awayda M.S. Bubien J.K. Ismailov I.I. Arrate M.P. Berdiev B.K. Benos D.J. Fuller C.M. J. Biol. Chem. 1995; 270: 31016-31026Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Even in light of this evidence, questions still remain as to whether members of the CLCA family are themselves responsible for the chloride channel activity, or whether they are regulating the activity of an as yet unidentified ion channel. Calcium-sensitive chloride channels have been measured in cells in which expression of CLCA isoforms could not be detected (13Papassotiriou J. Eggermont J. Droogmans G. Nilius B. Pflügers Arch. 2001; 442: 273-279Crossref PubMed Scopus (24) Google Scholar). CLCA family members have also been linked to functions other than that of ion channels. For example, mCLCA3 has been suggested to control mucus production (10Leverkoehne I. Gruber A.D. J. Histochem. Cytochem. 2002; 50: 829-838Crossref PubMed Scopus (105) Google Scholar), and hCLCA2, which is expressed in pulmonary endothelial cells, has been shown to mediate binding of tumor cells via its interaction with β4 integrin (14Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Furthermore, at least one CLCA isoform (hCLCA3) has been demonstrated to be a secreted protein (15Gruber A.D. Schreur K.D. Ji H.L. Fuller C.M. Pauli B.U. Am. J. Physiol. Cell Physiol. 1999; 45: C1261-C1270Crossref Google Scholar). hCLCA3 is a truncated isoform with no predicted transmembrane domains. CLCA family members are proteolytically cleaved proteins, and there is controversy over which region of hCLCA1 is responsible for the proposed channel activity (16Ji H.-L. DuVall M.D. Patton H.K. Satterfield C.L. Fuller C.M. Benos D.J. Am. J. Physiol. Cell Physiol. 1998; 274: C455-C464Crossref PubMed Google Scholar, 17Ran S. Fuller C.M. Arrate M.P. Latorre R. Benos D.J. J. Biol. Chem. 1992; 267: 20630-20637Abstract Full Text PDF PubMed Google Scholar). Hydrophobicity analysis of hCLCA1 has suggested the presence of four transmembrane domains. This observation was supported by studies using epitope-tagged hCLCA1 to identify the intracellular and extracellular domains of the protein (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar). To date, no studies have confirmed these observations with untagged hCLCA1 or within a physiological setting. Contrary to previous suggestions, we report here that hCLCA1 and mCLCA3 do not contain any transmembrane domains and that hCLCA1 is secreted into the extracellular medium when overexpressed. In support of this finding, we were able to identify hCLCA1 and mCLCA3 in the bronchoalveolar lavage (BAL) 1The abbreviations used are: BAL, bronchoalveolar lavage; BFA, brefeldin A; FnIII, fibronectin type III; GFP, green fluorescent protein; HEK, human embryonic kidney; HRP, horseradish peroxidase; OVA, ovalbumin; PBS, phosphate-buffered saline; PNS, post-nuclear supernatant; VWA, von Willebrand factor, type A. 1The abbreviations used are: BAL, bronchoalveolar lavage; BFA, brefeldin A; FnIII, fibronectin type III; GFP, green fluorescent protein; HEK, human embryonic kidney; HRP, horseradish peroxidase; OVA, ovalbumin; PBS, phosphate-buffered saline; PNS, post-nuclear supernatant; VWA, von Willebrand factor, type A.of asthmatics and of ovalbumin-challenged mice, respectively. These results suggest that the ion channel activity associated with these proteins is likely to be due to a regulatory function and that hCLCA1 could not possess endogenous, calcium-sensitive chloride channel activity. Bioinformatics Analysis—Transmembrane analysis was carried out using the programs TMHMM (18Krogh A. Larsson B. von Heijne G. Sonnhammer E. L.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (8747) Google Scholar), HMMTOP (19Tusnády G.E. Simon I. Bioinformatics. 2001; 17: 849-850Crossref PubMed Scopus (1530) Google Scholar), TMAP (20Persson B. Argos P. J. Mol. Biol. 1994; 237: 182-192Crossref PubMed Scopus (421) Google Scholar), SOSUI (21Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics. 1998; 14: 378-379Crossref PubMed Scopus (1540) Google Scholar), and DAS (22Cserzo M. Wallin E. Simon I. von Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar). The von Willebrand factor type A (VWA) domain prediction and analysis was carried out using InterProScan (23Zdobnov E.M. Apweiler R. Bioinformatics. 2001; 17: 847-848Crossref PubMed Scopus (2097) Google Scholar), SMART (24Letunic I. Goodstadt L. Dickens N.J. Doerks T. Schultz J. Mott R. Ciccarelli F. Copley R.R. Ponting C.P. Bork P. Nucleic Acids Res. 2002; 30: 242-244Crossref PubMed Scopus (565) Google Scholar), Pfam (25Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar), and PROSITE (26Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J.A. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; 30: 235-238Crossref PubMed Scopus (898) Google Scholar) data bases. The fibronectin type III (FnIII) domain was identified using BLAST (27Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar) and also when searched against the domain data bases Pfam and SMART, using InterProScan. The FnIII domain assignment was confirmed by generating a Hidden Markov Model for this region from a CLCA family protein multiple sequence alignment and searching it against the SCOP Superfamily Hidden Markov Model data base (28Gough J. Karplus K. Hughey R. Chothia C. J. Mol. Biol. 2001; 313: 903-919Crossref PubMed Scopus (912) Google Scholar) using HHsearch (29Soding J. Bioinformatics. 2005; 21: 951-960Crossref PubMed Scopus (1802) Google Scholar). The N-terminal signal sequence was predicted using SignalP (30Bendtsen J.D. Nielsen H. von Heijne G. Brunak S. J. Mol. Biol. 2004; 340: 783-795Crossref PubMed Scopus (5586) Google Scholar). Antibodies and Reagents—A48 anti-hCLCA1 antibody is a rabbit polyclonal antibody raised against the peptide sequence ((C)VNAARRRVIPQQS(C)), corresponding to amino acid residues 681-693 in hCLCA1. The A6637 anti-mCLCA3 rabbit polyclonal antibody was raised against the peptide sequence (SGLRTAFTVIKKKYPTDGS), corresponding to amino acid residues 388-406 in mCLCA3 (produced by Invitrogen). Both antibodies were affinity purified. Horseradish peroxidase (HRP)-conjugated anti-V5 mouse monoclonal antibody (Invitrogen) was used to detect the V5 protein tag. Mouse monoclonal antibody 9E10 was used to detect the Myc protein tag (Calbiochem; Merck Biosciences Ltd., Nottingham, UK). Secondary antibodies used for Western blots were HRP-conjugated goat anti-rabbit IgG (Calbiochem) and HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich). Secondary antibodies used for immunofluorescence were goat anti-rabbit IgG-AlexaFluor 568, goat anti-mouse IgG-AlexaFluor 568, and goat anti-mouse IgG-AlexaFluor 488 (all from Molecular Probes). Secondary antibody used for immunogold labeling was goat anti-rabbit IgG conjugated to 5 nm of colloidal gold (British BioCell International, Cardiff, UK). Plasmid Constructs—Myc-tagged hCLCA1 (Myc-hCLCA1) was generated as described by Gruber et al. (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar) and was a gift from Dr. Bendicht Pauli. The construct used for these studies had the Myc tag placed between amino acids 366 and 367. hCLCA1-V5-His construct (pcDNA3.1-D-V5-His-TOPO-hCLCA1) was generated using full-length hCLCA1 (PubMed accession number AF127036) with a short Kozak sequence inserted prior to the initiator methionine. The mCLCA3 construct (pcDNA3.1-V5-His-TOPO-mCLCA3) was generated using full-length mCLCA3 (PubMed accession number AB017156). Full-length mCLCA3 was amplified from in-house mouse lung cDNA templates with Pfu turbo hot start and cloned into pcDNA3.1 using TOPO technology (Invitrogen). To construct the pCIN5-hCLCA1 vector, full-length hCLCA1 from pcDNA3.1-D-V5-His-TOPO-hCLCA1 was subcloned using a NotI restriction enzyme site inserted into pcDNA3.1-D-V5-His-TOPO-hCLCA1 by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene). The GFP construct (pEGFP-N1) was purchased from BD Biosciences, and pcDNA3.1+ and pCIN5_p1 vectors were from Invitrogen. For generation of the HEK293 clone stably expressing hCLCA1, pCIN5-hCLCA1 was transfected into HEK293 cells (American Type Culture Collection, Manassas, VA), using Lipofectamine 2000 (Invitrogen). Neomycin-resistant colonies of cells were subsequently isolated by ring cloning and expanded under Geneticin (Invitrogen) selection. Cell Lines and Transfections—HEK293 cells were grown in Dulbecco's modified Eagle's medium (Sigma), supplemented with 10% heat-inactivated fetal bovine serum; 2% penicillin, streptomycin, and glutamine; and 1% non-essential amino acids (all from Invitrogen). HEK293 cells stably expressing hCLCA1 (HEK-hCLCA1) were cultured in the presence of 0.8 mg/ml Geneticin. HEK293 cells were transiently transfected with hCLCA1-V5-His, Myc-tagged hCLCA1, mCLCA3, or GFP using Lipofectamine 2000, following the manufacturer's instructions (Invitrogen). Cells were used 24 h after transfection. Cell Fractionation—HEK-hCLCA1 cells were detached from a T75 flask using Versene (Invitrogen). 1 × 107 cells were transferred to 1 ml of homogenizing buffer (0.25 m sucrose, 1 mm EGTA, 10 mm Hepes, 2 mm MgCl2, and 1 mm ATP) (Sigma) containing HALT protease inhibitor mixture (Pierce; Perbio, Cramlington, UK). Cells were disrupted by passing them 15 times through a Balch-Rothman ball-bearing homogenizer, (31Balch W.E. Dunphy W.G. Braell W.A. Rothman J.E. Cell. 1984; 39: 405-416Abstract Full Text PDF PubMed Scopus (477) Google Scholar), with a clearance of 11 nm. Cell disruption was confirmed by mixing cells with trypan blue followed by examination on a light microscope. The disrupted cell suspension was spun at 2500 rpm for 10 min at 4 °C to pellet nuclei and any intact cells. The post-nuclear supernatant (PNS), in 1.5 ml of homogenizing buffer, was then spun at 40,000 × g for 30 min at 4 °C in a Beckman TL-100 ultracentrifuge (Beckman Coulter Inc.). The pellet from this spin contained membranes and any intact organelles. The supernatant contained cytosolic proteins. The membrane/organelle pellet was subjected to Triton X-114 phase separation (32Brusca J.S. Radolf J.D. Methods Enzymol. 1994; 228: 182-193Crossref PubMed Scopus (132) Google Scholar) to separate integral membrane proteins from hydrophilic proteins. Samples of PNS, cytosol, membranes, and Triton X-114 aqueous and detergent phases (10% of total) were taken for Western blotting. Supernatant samples were trichloroacetic acid-precipitated, by addition of 10% trichloroacetic acid, at 4 °C for 1 h and then spun at 14,000 rpm, in a microfuge at 4 °C for 10 min. The Triton X-114 detergent phase sample was acetone-precipitated, by adding a 10× volume of acetone, at 20 °C for 1 h before centrifugation to collect the precipitate, as described above. All samples were resuspended in sample buffer (Invitrogen). PNS (5 μg) was loaded on the gel, with the equivalent volume of other samples loaded, to enable comparison between lanes. Membrane Stripping—HEK-hCLCA1 cells were detached from a T75 flask (∼1 × 107 cells) with Versene, washed twice with Hanks' balanced salt solution (Invitrogen), and split into three equal volumes. Cells were pelleted and resuspended in 200 μl of either PBS, pH 7.4; pH 2.5 acid wash (0.9% NaCl, adjusted to pH 2.5 with acetic acid); or pH 11 alkaline wash (0.1 m sodium carbonate, pH 11) (33Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1374) Google Scholar) for 20 min at 4 °C. Cells were removed by spinning at 1500 rpm for 5 min, and the supernatant was then spun at 40,000 × g for 30 min at 4 °C in a Beckman TL-100 ultracentrifuge to ensure removal of all cellular membranes. 150 μl of supernatants were trichloroacetic acid-precipitated and resuspended in 150 μl of PBS. 15 μg of protein from the pH11 wash and the equivalent volume of the other washes were loaded per lane on the gel. For the corresponding immunofluorescence, cells grown on poly-l-lysine-coated coverslips were washed twice in Hanks' balanced salt solution, followed by a wash in either PBS or pH 11 alkaline wash for 20 min at 4 °C. For the electron microscopic immunogold labeling, cells grown on poly-l-lysine-coated, 6-well plates were treated as described above. For both immunocytochemistry procedures, cells were then washed five times with 1 ml of PBS containing 1% bovine serum albumin and then immunolabeled as described below. Time Course of hCLCA1 Release and Brefeldin A Treatment—HEK-hCLCA1 cells, grown on 6-well plates, were washed five times with 1 ml of serum-free medium, followed by incubations of 1-4 h in the last wash. Medium was taken, and cells were lysed in 100 μl of radioimmune precipitation assay buffer (Upstate, Dundee, UK) containing HALT protease inhibitor mixture for Western blot analysis. Medium samples were spun at 1500 rpm for 5 min to remove any cells, supernatants were trichloroacetic acid-precipitated, and pellets were resuspended in 100 μl of PBS to enable equivalent loading of lanes on the gel. For brefeldin A (BFA) treatment, the protocol was carried out as described above, in the presence or absence of 2 μg/ml BFA for 4 h, followed by three washes and a 4-h incubation in the absence of BFA. Western Blot Analysis—HEK293 cells were lysed in radioimmune precipitation assay buffer containing HALT protease inhibitor mixture. Lung tissue from naïve or ovalbumin (OVA)-challenged mice was also lysed in radioimmune precipitation assay buffer after chopping into 0.2-mm cubes on a McIlwain tissue chopper (Campden Instruments, Loughborough, UK). Samples were run on NuPage 4-12% polyacrylamide bis-Tris gels (Invitrogen). Proteins were electrophoretically transferred to nitrocellulose membranes (Invitrogen). Membranes were blocked in 5% nonfat milk and in Tris-buffered saline with 1% Tween 20 and probed with affinity-purified antibodies. A48 antibody was used at 0.5 μg/ml, 9E10 and A6637 antibodies were used at 1 μg/ml, and anti-V5-HRP was used at the manufacturer's recommended dilution. Following incubation with the appropriate HRP-conjugated secondary antibody, bands were visualized using ECL detection reagents (Amersham Biosciences). Immunofluorescence Microscopy—For surface labeling, cells grown on poly-l-lysine-coated coverslips were incubated in PBS/1% bovine serum albumin/0.1% sodium azide for 10 min and then incubated with 1 μg/ml primary antibody in the same buffer for 30 min. Cells were washed twice and then incubated with Alexa-conjugated secondary antibody for 30 min, washed three times, and fixed with 2% paraformaldehyde (Pioneer Research Chemicals Ltd., Colchester, UK). Coverslips were mounted in Citifluor (Agar Scientific Ltd., Stansted, UK), on glass slides and examined on a Leica TCS-4D confocal microscope (Leica, Mannheim, Germany). For internal labeling, cells were fixed with 2% paraformaldehyde, quenched in 15 mm glycine, and permeabilized with 0.1% saponin in PBS/1% bovine serum albumin. Cells were incubated with primary antibody, followed by secondary antibody in permeabilization buffer, for 1 h each, with washing between incubations. After extensive washing over a 45-min period, cells were mounted and examined as described above. Electron Microscopy—For surface immunogold electron microscopy, cells grown on poly-l-lysine-coated, 6-well plates were incubated as described for surface immunofluorescence, except that 5 nm of gold-conjugated goat anti-rabbit secondary antibody was used in place of the Alexa-conjugated secondary antibody. After the final wash, cells were fixed with 2% paraformaldehyde/1.5% glutaraldehyde (Agar Scientific Ltd.), post-fixed in 1% osmium tetroxide (Agar Scientific Ltd.)/1.5% potassium ferricyanide, and treated with tannic acid (34Simionescu N. Simionescu M. J. Cell Biol. 1976; 70: 608-621Crossref PubMed Scopus (475) Google Scholar) before dehydration and embedding as described by Hopkins and Trowbridge, (35Hopkins C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Crossref PubMed Scopus (446) Google Scholar). Ultrathin sections were cut on a Reichert-Jung Ultracut E ultramicrotome (Leica, Vienna, Austria), stained with lead citrate, and viewed in a Hitachi H-7500 transmission electron microscope (Hitachi Ltd., Tokyo, Japan). Human BAL Fluid Samples—BAL fluid was obtained at fiber optic bronchoscopy carried out at Guy's Hospital London according to American Thoracic Society guidelines. Subjects included asthmatics defined according to American Thoracic Society criteria and normal controls. The study was approved by the Ethics Committee of Guy's Hospital. Informed, written consent was obtained from the subjects prior to participation. Collected BAL was filtered through 100-μm filters, and the filtrate was spun at 1500 rpm for 5 min to pellet cells. The supernatant (BAL fluid) was aliquoted and snap-frozen on dry ice. Mouse OVA Challenge Model and BAL Samples—BALB/c mice were either naïve or sensitized to a suspension of 10 μg of OVA adjoined to 2 mg of aluminum hydroxide by intraperitoneal administration on two occasions (days 0 and 14). From day 24, animals were challenged on consecutive days with three daily intranasal instillations of OVA (50 μg) in saline. 24 h later, animals were sacrificed. The lungs were lavaged post-mortem using repeat instillations (5 × 1 ml) of lavage fluid (10 mm EDTA and 0.1% bovine serum albumin in PBS), pooled to form the BAL. BAL was spun at 1500 rpm for 5 min to pellet cells, and the supernatant (BAL fluid) was frozen. Bioinformatics Analysis Predicts No Transmembrane Regions for hCLCA1 and mCLCA3—Proteins of the CLCA family have been described as integral membrane proteins with either four or five transmembrane domains (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar, 15Gruber A.D. Schreur K.D. Ji H.L. Fuller C.M. Pauli B.U. Am. J. Physiol. Cell Physiol. 1999; 45: C1261-C1270Crossref Google Scholar, 36Gruber A.D. Fuller C.M. Elble R.C. Benos D.J. Pauli B.U. Curr. Genomics. 2000; 1: 201-222Crossref Scopus (20) Google Scholar, 37Pauli B.U. Abdel-Ghany M. Cheng H.C. Gruber A.D. Archibald H.A. Elble R.C. Clin. Exp. Pharmacol. Physiol. 2000; 27: 901-905Crossref PubMed Scopus (87) Google Scholar). Our own analysis, using five different programs, did not predict either hCLCA1 or mCLCA3 to possess any α-helical transmembrane domains. The CLCA family was further analyzed to determine the potential domain structure. A VWA domain is predicted in the central region of the protein. The majority of well-characterized VWA domains are found in cell adhesion and extracellular matrix proteins (38Tuckwell D. Biochem. Soc. Trans. 1999; 27: 835-840Crossref PubMed Scopus (51) Google Scholar) and thought to be involved in protein-protein interactions, frequently involving divalent cations. However, more distantly related VWA domains have also been found in intracellular proteins, many being components of multi-protein complexes. Approximately half of all VWA domains contain a MIDAS (metal ion-dependent adhesion site) motif (39Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (792) Google Scholar), which contains key residues required for metal-ion binding. These key residues are all conserved in the VWA domains of hCLCA1 and mCLCA3, suggesting metal-ion binding of this motif in these proteins. The second transmembrane domain previously described (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar, 15Gruber A.D. Schreur K.D. Ji H.L. Fuller C.M. Pauli B.U. Am. J. Physiol. Cell Physiol. 1999; 45: C1261-C1270Crossref Google Scholar, 36Gruber A.D. Fuller C.M. Elble R.C. Benos D.J. Pauli B.U. Curr. Genomics. 2000; 1: 201-222Crossref Scopus (20) Google Scholar, 37Pauli B.U. Abdel-Ghany M. Cheng H.C. Gruber A.D. Archibald H.A. Elble R.C. Clin. Exp. Pharmacol. Physiol. 2000; 27: 901-905Crossref PubMed Scopus (87) Google Scholar) for CLCA is located within the globular VWA domain, which thus supports the results of the transmembrane prediction algorithms that this is in fact not a transmembrane domain. An ∼100-residue region toward the C terminus of the CLCA family was predicted to be an FnIII domain. FnIII domains are found in a variety of proteins, the majority of which are involved in cell surface binding in some manner or are receptor protein tyrosine kinases or cytokine receptors (40Bork P. Doolittle R.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8990-8994Crossref PubMed Scopus (194) Google Scholar). The remaining regions of the CLCA family do not appear to show obvious similarities to any functionally annotated domains, although the region located between the VWA and FnIII domains, which is predicted to have an all-β secondary structure composition, does show homology to a protein (UniProt:Q8PU63) in an archaebacterium, Methanosarcina mazei. This protein also possesses the VWA and FnIII domains, but N-terminal of the VWA domain is a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain, which appears to be absent in the CLCA family. This region, situated between the N-terminal signal sequence and the VWA domain, instead contains eight cysteine residues conserved across the CLCA family and is predicted to have an α/β composition (Cys-containing domain). The very C-terminal region of the CLCA family does not appear to be conserved in sequence between family members. This extensive analysis predicts that the CLCA proteins have a globular domain structure, a summary of which is shown in Fig. 1, for hCLCA1 and mCLCA3. hCLCA1 Is Strongly Associated with Cell Membranes but Can Be Removed from the Cell Surface—We examined the nature of the association of hCLCA1 with cell membranes, using biochemical methods and Western blot analysis (Fig. 2A). Following the disruption of HEK-hCLCA1 cells, the nuclei were pelleted. The PNS was found to contain both full-length protein (125 kDa) and the N-terminal cleavage product of hCLCA1. In our studies, the N-terminal cleavage product, previously reported to be 90 kDa (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar), appears to migrate at 83 kDa. The PNS was centrifuged to pellet cell membranes, including intact organelles. Full-length and processed hCLCA1 were present in the membrane pellet, whereas no hCLCA1 was detected in the supernatant, representative of the cytosol. The membrane/organelle pellet was subjected to phase separation with Triton X-114 detergent (32Brusca J.S. Radolf J.D. Methods Enzymol. 1994; 228: 182-193Crossref PubMed Scopus (132) Google Scholar). Full-length hCLCA1 partitioned into the aqueous phase, as did a significant proportion of the N-terminal cleavage product. This observation suggested that hCLCA1 is not an integral membrane protein. The A48 antibody, raised against an epitope corresponding to amino acid residues 681-693, recognizes the N-terminal cleavage product. Several possible cleavage sites have been proposed for hCLCA1 (3Gruber A.D. Elble R.C. Ji H.L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (205) Google Scholar). One of these sites, situated between amino acids 660 and 661, can now be ruled out because the A48 epitope is downstream of that position. Immunofluorescent labeling of hCLCA1 on non-permeabilized HEK-hCLCA1 cells revealed that hCLCA1 was expressed at the" @default.
• W2071601995 created "2016-06-24" @default.
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• W2071601995 date "2005-07-01" @default.
• W2071601995 modified "2023-09-30" @default.
• W2071601995 title "hCLCA1 and mCLCA3 Are Secreted Non-integral Membrane Proteins and Therefore Are Not Ion Channels" @default.
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• W2071601995 doi "https://doi.org/10.1074/jbc.m504654200" @default.
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