A Contribution to the Under-standing of their Operation

Deckblatt-Impressum Widmung Publikationshinweise Inhalt Verzeichnis der Abkürzungsverzeichnis Einleitung Literaturübersicht Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Abbildungsverzeichnis Tabellenverzeichnis Literatur Danksagung SelbständigkeitserklärungDie Mitglieder der CLCA-Familie stellen eine neu entdeckte Proteinfamilie dar, die funk-tionell mit einer Kalzium-aktivierten Chloridleitfähigkeit in verschiedenen Zelltypen im Zusammenhang steht. Die Vertreter mCLCA3 (Maus) und eCLCA1 (Pferd) spielen eine besondere Rolle bei Krankheiten mit gestörter sekretorischer Funktion. Insbesondere ihre Bedeutung bei der Becherzellmetaplasie, z.B. bei Asthma und chronischer-obstruktiver Bronchiolitis, ist von hoher biomedizinischer Relevanz. Funktionelle Analysen im hetero-logen Zellsystem zeigten in früheren Arbeiten, dass diese CLCA- Proteine eine der jener im Gewebe ähnlichen Chloridleitfähigkeit induzieren, die durch Kalzium als intrazellulärer Botenstoff gesteuert wird. Unklar war jedoch bisher, ob die CLCA-Proteine mit Hilfe von Transmembrandomänen einen eigenständigen Chloridkanal bilden oder ob sie Regulatoren eines anderen, bislang unbekannten Chloridkanals darstellen. Für die CLCA-Proteine wurde früher ein Protein-Strukturmodell mit vier oder fünf Transmembrandomänen erstellt. Ein solcherart strukturiertes Protein könnte die Funktion eines Kanals erfüllen. In neueren immunhistochemischen und elektronenimmunhistochemischen Untersuchungen konnte das mCLCA3-Protein jedoch in der extrazellulären Muzinschicht über den Enterozyten gefunden werden, so dass mCLCA3 offenbar durchaus zumindest zum Teil von der Zelle abgegeben werden kann. Diese Arbeit sollte die Fragestellung prüfen, ob mCLCA3 und eCLCA1 Transmembran-proteine darstellen und so möglicherweise echte Kanäle ausbilden können, oder aber ob sie sezernierte Proteine sind. Zur Klärung dieser Frage wurden umfangreiche computergestützte und proteinbiochemische Untersuchungen durchgeführt. Zuerst wurden mit Hilfe von unter-schiedlichen Computer-Algorithmen die Aminosäurensequenzen der Proteine nach möglichen Transmembrandomänen durchgemustert. Unter Anwendung der konfokalen Fluoreszenz-Mikroskopie wurde mCLCA3 in transfizierten Säugetierzellen intrazellulär in sekretorischen Vesikeln lokalisiert, eine Plasmamembranassoziation dagegen war nicht nachweisbar. Nach Expression von mCLCA3 in Säugerzelllinien wurde in pulse chase-Experimenten die Transportkinetik von mCLCA3 untersucht und mittels Westernblotanalyse wurde das Protein näher charakterisiert. Des Weiteren wurde das Glykosylierungsmuster der Proteine protein-biochemisch bestimmt. Die Ergebnisse dieser Arbeit zeigen, dass die für die CLCA-Proteine charakteristische Spaltung des Vorläuferproteins für mCLCA3 und eCLCA1 im endo-plasmatischen Retikulum stattfindet. Die mCLCA3- und eCLCA1-Spaltprodukte werden vollständig in den extrazellulären Raum als reife Glykoproteine sezerniert. Aufgrund des Fehlens von Transmembrandomänen können mCLCA3 und eCLCA1 daher keine eigenständigen Ionenkanäle bilden, somit kann eine echte Kanalfunktion für diese CLCA-Proteine ausgeschlossen werden. Als sekretorische Proteine sind sie aber offenbar durchaus in der Lage, indirekt eine Chloridleitfähigkeit zu induzieren. Außerdem könnten die CLCA-Proteine über eine Aktivierung von Signalwegen und regulatorischen Mechanismen die für Atemwegserkrankungen wie Asthma des Menschen und chronisch-obstruktive Bronchiolitis des Pferdes charakteristische Becherzellmetaplasie hervorrufen. Zukünftige Arbeiten sollten die Interaktionspartner und Funktionsmechanismen dieser CLCA-Proteine in Bezug auf die durch sie induzierte Chloridsekretion als auch auf die Becherzellmetaplasie und Muzin-synthese identifizieren.Members of the CLCA-family represent an emerging protein family and are among the most promising molecular candidates for the calcium-activated chloride conductivity observed in several tissues. In particular, the murine mCLCA3 and the equine eCLCA1 have been identified as clinically relevant molecules in diseases with secretory dysfunctions. Their induction of goblet cell metaplasia in diseases like asthma and recurrent airway obstruction is of particular biomedical relevance. Functional analyses have indicated that these CLCA-proteins evoke a calcium-activated chloride conductance when heterologously expressed. However, it is not yet clear whether the CLCA proteins form chloride channels per se or function as mediators of other, yet unkown chloride channels. A four or five integral transmembrane model has initially been proposed for CLCA proteins. Such a protein structure could potentially form a transmembrane conductive pathway for anions. However, recent immunohistochemical and immune electron-microscopical analyses detected the mCLCA3-protein in the extracellular mucous layer, consistent with a secreted protein. This thesis was designed to address the issue of whether mCLCA3 und eCLCA1 are integral transmembrane proteins and act as real anion channels or wether they are secretory proteins. In this study, systematic computer-based und biochemical analyses were conducted. First, the amino acid sequences of the two proteins were screened for potential transmembrane domains using several computer algorithms. Subsequently, confocal microscopy on mCLCA3-transfected cells revealed that the mCLCA3-protein was intracellularly localized in secretory vesicles without any association with the plasma membrane. In addition, the intracellular trafficking was investigated by pulse chase experiments in transfected and metabolically labeled mammalian cells. The locations of the proteins were then characterized by westernblot analyses and the glycosylation patterns of the proteins were determined. The results show that the characteristic cleavage of the eCLCA1 und mCLCA3 precursor proteins takes place in the endoplasmic reticulum. Both cleavage products of the mCLCA3-protein are released as glycosylated, mature proteins into the extracellular space. This was similarly confirmed for the eCLCA1-protein. Thus, due to the lack of any transmembrane domains, these proteins are unable to form anion channels on their own. These results strongly suggest that eCLCA1 und mCLCA3 are secretory glycoproteins rather than transmembrane molecules. As such they could interact with other chloride channel proteins to induce the chloride conductivity observed in several previous experiments. Moreover, they could act as signalling molecules to evoke goblet cell metaplasia characteristic for complex airway diseases including asthma and recurrent airway obstructions. Future studies will have to address the interaction partners and the mechanisms responsible for their indirect induction of chloride secretion, goblet cell metaplasia and mucin secretion

Grayanotoxin I Intoxication in Pet Pigs

Contaminated honey is a common cause of grayanotoxin intoxication in humans. Intoxication of animals, especially cattle, is usually due to ingestion of plants of the Ericaceae family, such as Rhododendron. Here, we report the ingestion of Pieris japonica as the cause of grayanotoxin I intoxication in 2 miniature pigs that were kept as pets. The pigs showed sudden onset of pale oral mucosa, tachycardia, tachypnea, hypersalivation, tremor, and ataxia that progressed to lateral recumbency. The pathological examination of one pig revealed no specific indications for intoxication except for the finding of plant material of Pieris japonica in the intestine. Grayanotoxin I was identified in the ingested plant, gastric content, blood, liver, bile, kidney, urine, lung, and skeletal muscle via HPLC-MS/MS. Grayanotoxin I should be considered as a differential etiological diagnosis in pigs with unspecific signs and discovery of ingested plant material as the only indication in the pathologic examination

Genomic, biochemical and expressional properties reveal strong conservation of the CLCA2 gene in birds and mammals

Recent studies have revealed the dynamic and complex evolution of CLCA1 gene homologues in and between mammals and birds with a particularly high diversity in mammals. In contrast, CLCA2 has only been found as a single copy gene in mammals, to date. Furthermore, CLCA2 has only been investigated in few mammalian species but not in birds. Here, we established core genomic, protein biochemical and expressional properties of CLCA2 in several bird species and compared them with mammalian CLCA2. Chicken, turkey, quail and ostrich CLCA2 were compared to their mammalian orthologues using in silico, biochemical and expressional analyses. CLCA2 was found highly conserved not only at the level of genomic and exon architecture but also in terms of the canonical CLCA2 protein domain organization. The putatively prototypical galline CLCA2 (gCLCA2) was cloned and immunoblotting as well as immunofluorescence analyses of heterologously expressed gCLCA2 revealed protein cleavage, glycosylation patterns and anchoring in the plasma membrane similar to those of most mammalian CLCA2 orthologues. Immunohistochemistry found highly conserved CLCA2 expression in epidermal keratinocytes in all birds and mammals investigated. Our results suggest a highly conserved and likely evolutionarily indispensable role of CLCA2 in keratinocyte function. Its high degree of conservation on the genomic, biochemical and expressional levels stands in contrast to the dynamic structural complexities and proposed functional diversifications between mammalian and avian CLCA1 homologues, insinuating a significant degree of negative selection of CLCA2 orthologues among birds and mammals. Finally, and again in contrast to CLCA1, the high conservation of CLCA2 makes it a strong candidate for studying basic properties of the functionally still widely unresolved CLCA gene family

mCLCA3 Modulates IL-17 and CXCL-1 Induction and Leukocyte Recruitment in Murine Staphylococcus aureus Pneumonia

The human hCLCA1 and its murine ortholog mCLCA3 (calcium-activated chloride channel regulators) are exclusively expressed in mucus cells and linked to inflammatory airway diseases with increased mucus production, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Both proteins have a known impact on the mucus cell metaplasia trait in these diseases. However, growing evidence points towards an additional role in innate immune responses. In the current study, we analyzed Staphylococcus aureus pneumonia, an established model to study pulmonary innate immunity, in mCLCA3-deficient and wild-type mice, focusing on the cellular and cytokine-driven innate inflammatory response. We compared clinical signs, bacterial clearance, leukocyte immigration and cytokine responses in the bronchoalveolar compartment, as well as pulmonary vascular permeability, histopathology, mucus cell number and mRNA expression levels of selected genes (mClca1 to 7, Muc5ac, Muc5b, Muc2, Cxcl-1, Cxcl-2, Il-17). Deficiency of mCLCA3 resulted in decreased neutrophilic infiltration into the bronchoalveolar space during bacterial infection. Only the cytokines IL-17 and the murine CXCL-8 homolog CXCL-1 were decreased on mRNA and protein levels during bacterial infection in mCLCA3-deficient mice compared to wild-type controls. However, no differences in clinical outcome, histopathology or mucus cell metaplasia were observed. We did not find evidence for regulation of any other CLCA homolog that would putatively compensate for the lack of mCLCA3. In conclusion, mCLCA3 appears to modulate leukocyte response via IL-17 and murine CXCL-8 homologs in acute Staphylococcus aureus pneumonia which is well in line with the proposed function of hCLCA1 as a signaling molecule acting on alveolar macrophages

Role of goblet cell protein CLCA1 in murine DSS colitis

Background The secreted goblet cell protein CLCA1 (chloride channel regulator, calcium-activated-1) is, in addition to its established role in epithelial chloride conductance regulation, thought to act as a multifunctional signaling protein, including cellular differentiation pathways and induction of mucus production. Specifically, CLCA1 has recently been shown to modulate early immune responses by regulation of cytokines. Here, we analyze the role of CLCA1, which is highly expressed and secreted by colon goblet cells, in the course of murine dextran sodium sulfate-induced colitis. Findings We compared Clca1-deficient and wild type mice under unchallenged and DSS-challenged conditions at various time points, including weight loss, colon weight-length- ratio and histological characterization of inflammation and regeneration. Expression levels of relevant cytokines, trefoil factor 3 and E-cadherin were assessed via quantitative PCR and cytometric bead arrays. Lack of CLCA1 was associated with a more than two-fold increased expression of Cxcl-1- and Il-17-mRNA during DSS colitis. However, no differences were found between Clca1-deficient and wild type mice under unchallenged or DSS-challenged conditions in terms of clinical findings, disease progression, colitis outcome, epithelial defects or regeneration. Conclusions CLCA1 is involved in the modulation of cytokine responses in the colon, albeit differently than what had been observed in the lungs. Obviously, the pathways involved depend on the type of challenge, time point or tissue environment

Interspecies diversity of chloride channel regulators, calcium-activated 3 genes

Members of the chloride channel regulators, calcium-activated (CLCA) family, have been implicated in diverse biomedical conditions, including chronic inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, the activation of macrophages, and the growth and metastatic spread of tumor cells. Several observations, however, could not be repeated across species boundaries and increasing evidence suggests that select CLCA genes are particularly prone to dynamic species-specific evolvements. Here, we systematically characterized structural and expressional differences of the CLCA3 gene across mammalian species, revealing a spectrum of gene duplications, e.g., in mice and cows, and of gene silencing via diverse chromosomal modifications in pigs and many primates, including humans. In contrast, expression of a canonical CLCA3 protein from a single functional gene seems to be evolutionarily retained in carnivores, rabbits, guinea pigs, and horses. As an accepted asthma model, we chose the cat to establish the tissue and cellular expression pattern of the CLCA3 protein which was primarily found in mucin-producing cells of the respiratory tract and in stratified epithelia of the esophagus. Our results suggest that, among developmental differences in other CLCA genes, the CLCA3 gene possesses a particularly high dynamic evolutionary diversity with pivotal consequences for humans and other primates that seem to lack a CLCA3 protein. Our data also help to explain previous contradictory results on CLCA3 obtained from different species and warrant caution in extrapolating data from animal models in conditions where CLCA3 may be involved

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype