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Proteomics of the upper airways: studies on a new lipopolysaccharide-binding protein, PLUNC
Linköping University, Department of Molecular and Clinical Medicine, Occupational and Environmental Medicine. Linköping University, Faculty of Health Sciences.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is now significant interest in identifying, quantifying and characterizing the human proteome, and new powerful techniques (proteomics) have evolved to deal with this giant task. In the present study, proteomics have been applied for the first time to map the proteins of the upper airways. The protein contents of human nasal fluid (NLF) and saliva were analysed using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and the proteins were identified by peptide mass fingerprinting using matrix assisted laser desorptioniionization time of night mass spectrometry (MALDI-TOF MS) or by amino acid sequencing using electrospray ionization tandem mass spectrometry (ESI- MS/MS). More than 100 proteins were identified and protein maps of nasal fluid and saliva were thus established. Of particular interest was the identification of a new lipopolysaccharide (LPS)-binding protein, PLUNC (palate lung and nasal epithelial clone), which was shown to be the only protein in NLF that binds to LPS. PLUNC was characterized as multiple isoforms (Mr/p1: 27/5.1, 26/5.2, 25/5.3, 27.5/5.1, 27/5.2, 26/5.3, 25.1/5.5 and 24.8/5.4), and several of these isoforms were demonstrated to be sialylated. Notably, decreased levels of PLUNC were found in NLF of (i) smokers, (ii) epoxy workers with airway irritation, and (iii) patients with seasonal allergic rhinitis (SAR) during allergy season. In addition, the levels of von Ebner's gland protein, α1-antitrypsin, cystatin S, Clara cell protein 16 and lipocortin-1 were altered, either in smokers or SAR patients or both. One previously unidentified NLF protein was found in SAR patients during allergy season but not before season: this protein was identified as eosinophil lysophospholipase. Many of these proteins were post-translationally modified by glycosylation (PLUNC, α1-antitrypsin, von Ebner's gland protein), phosphorylation (cystatin S), acetylation (eosinophil lysophospholipase), or truncation (lipocortin-1). Altogether, these findings illustrate the potential use of proteomics for identifying new markers of upper airway inflammation and for revealing structural details of such markers. The findings also indicate that allergic inflammation in the nasal mucosa is associated with decreased nasal fluid levels of the endogenous proteinase inhibitors, cystatin S and von Ebner's gland protein, and of the new irritation marker, PLUNC. Further studies are required to explore the possibility that PLUNC plays an important part in microbial  recognition and that this function is impaired after exposure to airway irritants and during upper airway inflammation.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2005. , 63 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 927
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-31423Local ID: 17204ISBN: 91-85497-64-9 (print)OAI: oai:DiVA.org:liu-31423DiVA: diva2:252246
Public defence
2005-12-16, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-02Bibliographically approved
List of papers
1. Newly identified proteins in human nasal lavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting
Open this publication in new window or tab >>Newly identified proteins in human nasal lavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting
2002 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 2, no 1, 112-120 p.Article in journal (Refereed) Published
Abstract [en]

Human nasal lavage fluids (NLFs) were analyzed with two-dimensional gel electrophoresis (2-DE) and proteins were identified with peptide mass fingerprinting using matrix-assisted laser desoption/ionization time of flight mass spectrometry. In some cases, the identification was verified by analysis of post-source decay fragmentation spectra. Many of the identified proteins were new forms or fragments of previously found proteins (e.g. albumin, lactoferrin, cystatin, calgranulin, von Ebners gland protein and palate lung nasal epithelium clone), while others were proteins that have previously been indicated by 2-DE image matching or immunoblots (e.g. apolipoprotein AI, lysozyme C, and Clara cell secretory protein). Some new proteins, not shown before in 2-DE patterns of NLF were also found, e.g. mammaglobin B, 2-microglobulin and immunoglobulin J chain. Of the identified NLF proteins many appear to be involved in inflammatory and immune responses. A study was therefore conducted to investigate if the levels of these proteins were changed in smokers compared to nonsmokers. It was found that NLF from smokers contained decreased levels of Clara cell secretory protein, and increased proportions of a truncated variant of lipocortin-1, three acidic forms of α1-antitrypsin, and one phosphorylated form of cystatin S. Furthermore, NLF from smokers contained increased proportions of a new variant of palate lung nasal epithelium clone (PLUNC), a recently identified airway irritation marker. The results demonstrate that 2-DE of NLF may be used to assess alterations of proteins or post-translationally modified proteins in smokers. Clara cell secretory protein (CC 16, CC 10) and lipocortin-1 are two anti-inflammatory, phospholipase A2 inhibitory proteins, and α1-antitrypsin and cystatin S are two proteinase inhibitors. Changed levels of these proteins may therefore be of importance to the airway inflammation caused by smoking. The results also support the notion that PLUNC is involved in inflammatory responses in the upper airways.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26518 (URN)10.1002/1615-9861(200201)2:1<112::AID-PROT112>3.0.CO;2-N (DOI)11076 (Local ID)11076 (Archive number)11076 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Mapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting
Open this publication in new window or tab >>Mapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting
2003 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 3, no 6, 1003-1015 p.Article in journal (Refereed) Published
Abstract [en]

Human saliva contains a large number of proteins that can be used for diagnosis and are of great potential in clinical and epidemiological research. The aim of this work was to map the proteins in saliva by two-dimensional gel electrophoresis (2-DE), and to identify abundant proteins by peptide mass fingerprinting using trypsin cleavage and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. One hundred proteins were identified representing 20 different identities according to accession numbers. Abundant proteins expressed in different forms were: α-amylase, immunoglobulin A, prolactin-inducible protein, zinc-α2-glycoprotein and cystatins (S, SA, D and SN). Other proteins found were interleukin-1 receptor antagonist, von Ebner’s gland protein (lipocalin-1) and calgranulin A and B (S100A8 and A9). Furthermore, apolipoprotein A-I, β2-microglobulin, glutathione S-transferase P and fatty acid-binding protein were also identified. Our results show that human saliva contains a large number of proteins that are involved in inflammatory and immune responses. The 2-DE protein map constructed opens the possibility to investigate protein changes associated with disease processes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26469 (URN)10.1002/pmic.200300426 (DOI)11020 (Local ID)11020 (Archive number)11020 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. PLUNC (palate, lung and nasal epithelial clone) proteins in human nasal lavage fluid
Open this publication in new window or tab >>PLUNC (palate, lung and nasal epithelial clone) proteins in human nasal lavage fluid
Show others...
2003 (English)In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 31, no 4, 810-814 p.Article in journal (Refereed) Published
Abstract [en]

PLUNC (palate, lung and nasal epithelial clone) is a newly discovered gene that is expressed in the upper respiratory tract and is suggested to be of importance in host defence against bacteria. We have identified two forms of the PLUNC protein in human nasal lavage fluid (NLF) using two-dimensional gel electrophoresis (2-DE) and MS. The apparent molecular masses and isoelectric points of these forms are 24.8 kDa/pI 5.4 and 25.1 kDa/pI 5.5. Notably, the 24.8 kDa/pI 5.4 form of PLUNC is an abundant protein in the 2-DE protein patterns of NLF from healthy subjects. Decreased levels of PLUNC were found in NLF from smokers and workers exposed to reactive epoxy chemicals, indicating that long-term exposure to airway irritants impairs the production of PLUNC in the upper respiratory tract. We have also investigated the presence of lipopolysaccharide (LPS)-binding proteins in NLF. Five proteins were found to adsorb to a LPS-coated surface; two of these proteins correspond to the two PLUNC forms, as judged by 2-DE pattern matching. For comparison, human saliva was found to contain a set of LPS-binding proteins with similar 2-DE spot positions (the same pIs but somewhat lower apparent molecular masses of 20 kDa). These results indicate that PLUNC may be a new marker of airway inflammation and may play a part in the innate immune response, and that human saliva contains yet other members of the family of LPS-binding proteins.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26470 (URN)10.1042/BST0310810 (DOI)11021 (Local ID)11021 (Archive number)11021 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. PLUNC in human nasal lavage fluid: multiple isoforms that bind to lipopolysaccharide
Open this publication in new window or tab >>PLUNC in human nasal lavage fluid: multiple isoforms that bind to lipopolysaccharide
2004 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1699, no 1-2, 57-63 p.Article in journal (Refereed) Published
Abstract [en]

Here, we demonstrate the presence of multiple isoforms of palate lung nasal epithelial clone (PLUNC) in human nasal lavage fluid (NLF). Eight isoforms were separated by two-dimensional gel electrophoresis (2-DE), and peptide mapping of the proteins was performed using MALDI-TOF MS (matrix assisted laser desorption/ionization time of flight mass spectrometry) of tryptic and asparginase cleavages. The identification was verified by amino acid sequencing after analysis of collision-induced dissociation (CID) fragmentation spectra with nanoelectrospray MS/MS. One isoform showed an electrophoretic mobility shift after N-glycosidase treatment, indicating that at least one of the PLUNC isoforms is glycosylated. We also demonstrate that PLUNC in NLF binds to lipopolysaccharide (LPS) in vitro; indeed, out of all proteins present in NLF only the PLUNC isoforms were found to adsorb to an LPS-coated surface. These results show that PLUNC is expressed as multiple LPS-binding isoforms in human NLF. The possibility that PLUNC may play a role in the innate immune response of the upper airways is inferred.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-22261 (URN)10.1016/j.bbapap.2004.01.001 (DOI)1434 (Local ID)1434 (Archive number)1434 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
5. Comparative proteomics of nasal fluid in seasonal allergic rhinitis
Open this publication in new window or tab >>Comparative proteomics of nasal fluid in seasonal allergic rhinitis
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2006 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 2, 330-338 p.Article in journal (Refereed) Published
Abstract [en]

A comparative proteomic approach was applied to examine nasal lavage fluid (NLF) from patients with seasonal allergic rhinitis (SAR, n = 6) and healthy subjects (controls, n = 5). NLF samples were taken both before allergy (pollen) season and during season, and proteins were analyzed by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after tryptic cleavage. Twenty proteins were selected and quantified. During allergy season, the levels of six sialylated isoforms of PLUNC (palate lung nasal epithelial clone) were lower in SAR patients than controls, as were the levels of six isoforms of von Ebner's gland protein (VEGP), including a previously undescribed form with N-linked glycosylation, and of cystatin S. PLUNC is a new innate immunity protein and VEGP and cystatin S are two endogenous proteinase inhibitors. By contrast, the levels of an acidic form of alpha-1-antitrypsin were higher in SAR patients than controls. One previously unidentified NLF protein was found in all samples from the SAR patients during allergy season but not in any sample before allergy season:  this protein was identified as eosinophil lysophospholipase (Charcot-Leyden crystal protein/galactin 10). MS/MS analysis of the N-terminus of the protein showed removal of Met and acetylation of Ser. Altogether, these findings illustrate the potential use of proteomics for identifying protein changes associated with allergic rhinitis and for revealing post-translational modifications of such new potential markers of allergic inflammation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-32843 (URN)10.1021/pr050341h (DOI)18783 (Local ID)18783 (Archive number)18783 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved

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