Identification and characterisation of the BPI/LBP/PLUNC-like gene repertoire in chickens reveals the absence of a LBP gene.

Palate, lung and nasal epithelial clone (PLUNC) proteins are structural homologues to the innate defence molecules LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). PLUNCs make up the largest portion of the wider BPI/LBP/PLUNC-like protein family and are amongst the most rapidly evolving mammalian genes. In this study we systematically identified and characterised BPI/LBP/PLUNC-like protein-encoding genes in the chicken genome. We identified eleven complete genes (and a pseudogene). Five of them are clustered on a >50 kb locus on chromosome 20, immediately adjacent to BPI. In addition to BPI, we have identified presumptive orthologues LPLUNCs 2, 3, 4 and 6, and BPIL-2. We find no evidence for the existence of single domain containing proteins in birds. Strikingly our analysis also suggests that there is no LBP orthologue in chicken. This observation may in part account for the relative resistance to LPS toxicity observed in birds. Our results indicate significant differences between the avian and mammalian repertoires of BPI/LBP/PLUNC-like genes at the genomic and transcriptional levels and provide a framework for further functional analyses of this gene family in chickens.

Human LPLUNC1 is a secreted product of goblet cells and minor glands of the respiratory and upper aerodigestive tracts.

Long PLUNC1 (LPLUNC1, C20orf114) is a member of a family of poorly described proteins (PLUNCS) expressed in the upper respiratory tract and oral cavity, which may function in host defence. Although it is one of the most highly expressed genes in the upper airways and has been identified in sputum and nasal secretions by proteomic studies, localisation of LPLUNC1 protein has not yet been described. We developed affinity purified antibodies and localised the protein in tissues of the human respiratory tract, oro- and nasopharynx. We have complemented these studies with analysis of LPLUNC1 expression in primary human lung cell cultures and used Western blotting to study the protein in cell culture secretions and in BAL. LPLUNC1 is a product of a population of goblet cells in the airway epithelium and nasal passages and is also present in airway submucosal glands and minor glands of the oral and nasal cavities. The protein is not expressed in peripheral lung epithelial cells. LPLUNC1 is present in bronchoalveolar lavage fluid as two glycosylated isoforms and primary airway epithelial cells produce identical proteins as they undergo mucociliary differentiation. Our results suggest that LPLUNC1 is an abundant, secreted product of goblet cells and minor mucosal glands of the respiratory tract and oral cavity and suggest that the protein functions in the complex milieu that protects the mucosal surfaces in these locations.

Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein.

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response.

Differential epithelial expression of the putative innate immune molecule SPLUNC1 in cystic fibrosis.

INTRODUCTION: Short PLUNC1 (SPLUNC1) is the founding member of a family of proteins (PLUNCS) expressed in the upper respiratory tract and oral cavity, which may function in host defence. It is one of the most highly expressed genes in the upper airways and the protein has been detected in sputum and nasal secretions. The biology of the PLUNC family is poorly understood but in keeping with the putative function of the protein as an immune defence protein, a number of RNA and protein studies have indicated that SPLUNC1 is increased in inflammatory/infectious conditions such as Cystic Fibrosis (CF), COPD and allergic rhinitis. METHODS: We used immunohistochemistry to localise SPLUNC1 in lung tissue from patients with CF and a range of other lung diseases. We used a range of additional markers for distinct cell types to try to establish the exact site of secretion of SPLUNC1. We have complemented these studies with a molecular analysis of SPLUNC1 gene expression in primary human lung cell cultures and isolated inflammatory cell populations. RESULTS: In CF, expression of SPLUNC1 is significantly elevated in diseased airways and positive staining was noted in some of the inflammatory infiltrates. The epithelium of small airways of CF lung exhibit significantly increased SPLUNC1 staining compared to similar sized airways in non-CF lungs where staining is absent. Strong staining was also seen in mucous plugs in the airways, these included many inflammatory cells. No alveolar epithelial staining was noted in CF tissue. Airway epithelial staining did not co-localise with MUC5AC suggesting that the protein was not produced by goblet cells. Using serial sections stained with neutrophil elastase and CD68 we could not demonstrate co-localisation of SPLUNC1 with either neutrophils or macrophages/monocytes, indicating that these cells were not a source of SPLUNC1 in the airways of CF lungs. No change in staining pattern was noted in the small airways or lung parenchyma of other lung diseases studied including, COPD, emphysema or pneumonia where significant NE and CD68 staining was noted. Cultures of primary tracheobronchial epithelial cells were analysed by RT-PCR and showed that pro-inflammatory mediators did not induce expression of SPLUNC1. We have also shown that SPLUNC1 gene expression was not seen in isolated human mononuclear cells, macrophages or neutrophils. CONCLUSION: These studies show that SPLUNC1 is specifically and significantly increased in the small airways of lungs from patients with CF. They further suggest that it is the airway epithelium that is responsible for the increased levels of SPLUNC1 in CF and not inflammatory cells; this could be a defensive response to the infectious component of the disease.

Expression of pro-apoptotic Bfk isoforms reduces during malignant transformation in the human gastrointestinal tract.

Reduced expression of pro-apoptotic Bcl-2 family proteins has been described in many gastrointestinal cancers, and may play a role in tumourigenesis. The human homologue of the pro-apoptotic Bcl-2 protein, Bfk, is predominantly expressed in tissues of the gastrointestinal tract. In colon, four alternatively spliced isoforms were identified; of which two are pro-apoptotic when overexpressed. In the transition from normal tissue to tumour, pro-apoptotic Bfk isoform expression is substantially reduced in up to 80% of tumours isolated from the human gastrointestinal tract (8/10 colonic tumours and 26/37 of all gastrointestinal tumours) compared to 3/117 tumours from outside the gastrointestinal tract. These data suggest that pro-apoptotic isoforms of Bfk may help to protect against the development of human gastrointestinal malignancy.

Three novel Bid proteins generated by alternative splicing of the human Bid gene.

Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. Bid(S) contains the N-terminal regulatory domains of Bid without the BH3 domain; Bid(EL) corresponds to full-length Bid with additional N-terminal sequence; and Bid(ES) contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies Bid(EL) induces apoptosis, whereas Bid(S) abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. Bid(ES) induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli.