Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands

Ning, Fangkun; Wang, Chao; Berry, Karin Zemski; Kandasamy, Pitchaimani; Liu, Haolin; Murphy, Robert C.; Voelker, Dennis R.; Nho, Chu Won; Pan, Choel-Ho; Dai, Shaodong; Niu, Liwen; Chu, Hang; Zhang, Gongyi

doi:10.1096/fj.14-259291

Cited by 19 publications

(21 citation statements)

References 52 publications

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“…13,16–21 The protein was also reported to associate with lipopolysaccharides (LPS) from E. coli and P. aeruginosa , albeit through unknown mechanisms. 7,18,22–25 To date, while SPLUNC1’s structural mechanism in regulating ENaC is relatively well understood, the structural basis of SPLUNC1’s actions as a surfactant and antibacterial factor have remained less clear.…”

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confidence: 99%

“…6,25 The 2013 report focused on the role the body of SPLUNC1 played in presenting the S18 peptide, which is disordered in all reported structures, for ENaC inhibition in both normal and CF tissues in vitro. By contrast, the 2014 report highlighted the ability of SPLUNC1 to associate in vitro with lipids of established pulmonary functions, specifically dipalmitoylphosphatidylcholine (DPPC).…”

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confidence: 99%

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Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1

et al. 2016

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SPLUNC1 is an abundantly secreted innate immune protein in the mammalian respiratory tract that exerts bacteriostatic and antibiofilm effects, binds to lipopolysaccharide (LPS), and acts as a fluid-spreading surfactant. Here, we unravel the structural elements essential for the surfactant and antimicrobial functions of human SPLUNC1 (Short Palate Lung Nasal Epithelial Clone 1). A unique α-helix (α4) that extends from the body of SPLUNC1 is required for the bacteriostatic, surfactant, and LPS-binding activities of this protein. Indeed, we find that mutation of just four leucine residues within this helical motif to alanine is sufficient to significantly reduce the fluid spreading abilities of SPLUNC1, as well as its bacteriostatic actions against the Gram-negative pathogens Burkholderia cenocepacia and Pseudomonas aeruginosa. Conformational flexibility in the body of the SPLUNC1 is also involved in the bacteriostatic, surfactant, and LPS-binding functions of the protein as revealed by disulfide mutants introduced into SPLUNC1. In addition, SPLUNC1 exerts antibiofilm effects against Gram-negative bacteria, although α4 is not involved in this activity. Interestingly, though, the introduction of surface electrostatic mutations away from α4 based on the unique dolphin SPLUNC1 sequence, and confirmed by crystal structure, are shown to impart antibiofilm activity against Staphylococcus aureus, the first SPLUNC1-depenent effect against a Gram-positive bacterium reported to date. Together, these data pinpoint SPLUNC1 structural motifs required for the antimicrobial and surfactant actions of this protective human protein.

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Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1

et al. 2016

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“…SPLUNC1 was early shown to bind LPS and studies that followed showed SPLUNC1's ability to decrease the growth of several Gramnegative bacteria, such as Mycoplasma pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae [155][156][158][159][160]. A later study indicated that other types of lipids active in innate immunity, instead of LPS, were the true assigned target for SPLUNC1 [157]. Nevertheless, the extent of the truncation of SPLUNC1 in nasopharyngeal aspirate, about one third of the protein, should have a substantial impact on both the function and structure of the protein.…”

Section: Results and Discussion Paper Imentioning

confidence: 99%

“…A later study finally verified the structure of SPLUNC1 and could show a cavity, similar to the cavity of BPI, for the binding of lipids. However, SPLUNC1 was shown not to bind lipopolysaccharide, but instead other types of lipids, such as sphingomyelin, phosphatidylcholine and dipalmitoylphosphatidylcholine, also important in the innate immune system [157]. Over time its antimicrobial function has been supported by several studies.…”

Section: Splunc1mentioning

confidence: 99%

Upper Airway Mucosal Inflammation : Proteomic Studies after Exposure to Irritants and Microbial Agents

Fornander¹

2015

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People are, in their daily lives, exposed to a number of airborne foreign compounds that do not normally affect the body. However, depending on the nature of these compounds, dose and duration of exposure, various airway symptoms may arise. Early symptoms are often manifested as upper airway mucosal inflammation which generates changes in protein composition in the airway lining fluid.This thesis aims at identifying, understanding mechanisms and characterizing protein alterations in the upper airway mucosa that can be used as potential new biomarkers for inflammation in the mucosa. The protein composition in the mucosa was studied by sampling of nasal lavage fluid that was further analyzed with a proteomic approach using two-dimensional gel electrophoresis and mass spectrometry. Additionally, by studying factors on site through environmental examination, health questionnaires and biological analyses, we have tried to understand the background to these protein alterations and their impact on health.Respiratory symptoms from the upper airways are common among people who are exposed to irritative and microbial agents. This thesis have focused on personnel in swimming pool facilities exposed to trichloramine, metal industry workers exposed to metalworking fluids, employees working in damp and moldy buildings and infants diagnosed with respiratory syncytial virus infection. The common denominator in these four studies is that the subjects experience upper airway mucosal inflammation, which is manifested as cough, rhinitis, phlegm etc. In the three occupational studies, the symptoms were work related. Notably, a high prevalence of perceived mucosal symptoms was shown despite the relatively low levels of airborne irritants revealed by the environmental examination. Protein profiling verified an ongoing inflammatory response by identification of several proteins that displayed altered levels. Interestingly, innate immune proteins dominated and four protein alterations occurred in most of the studies; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin. Similarly, these proteins were also found in nasal fluid from children with virus infection and in addition a truncated form of SPLUNC1 and two other S100 proteins (S100A7-like 2 and S100A16), not previously found in nasal secretion, were identified.Altogether, the results indicate the potential use of a proteomic approach for identifying new biomarkers for the upper respiratory tract at an early stage in the disease process after exposure to irritant and microbial agents. The results indicate an effect on the innate immunity system and the proteins; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin are especially promising new biomarkers. Moreover, further studies of these proteins may help us to understand the molecular mechanisms involved in irritant-induced airway inflammation. POPULÄRVETENSKAPLIG SAMMANFATTNING FÖRÄNDRAD PROTEINSAMMANSÄTTNING I DE ÖVRE LUFTVÄGARNA EFTER EXPONERING FÖR SLEMHINNEIRRITERANDE ÄMNENVarje dag utsätts vi för partikla...

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“…Sterol carrier protein 2 and cholesteryl ester transfer protein assume this role for cholesterol and cholesteryl esters, respectively [82] . In addition, in the airways, the highly hydrophobic protein short palate lung epithelial clone protein 1 binds certain phospholipids and sphingolipids [83,84] and may possibly also function as a cholesteryl ester carrier. However, much research is still needed to dissect the focused delivery of AMLs to the microbial target.…”

Section: Transporters Of Amlsmentioning

confidence: 99%

Antimicrobial lipids: Emerging effector molecules of innate host defense

Porter

Daniel²,

Alvarez³

et al. 2015

WJI

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The antimicrobial properties of host-derived derived lipids have become increasingly recognized and evidence is mounting that antimicrobial lipids (AMLs), like antimicrobial peptides, are effector molecules of the innate immune system and are regulated by its conserved pathways. This review, with primary focus on the human body, provides some background on the biochemistry of lipids, summarizes their biological functions, expands on their antimicrobial properties and site-specific composition, presents modes of synergism with antimicrobial peptides, and highlights the more recent reports on the regulation of AML production as well as bacterial resistance mechanisms. Based on extant data a concept of innate epithelial defense is proposed where epithelial cells, in response to microbial products and proinflammatory cytokines and through activation of conserved innate signaling pathways, increase their lipid uptake and up-regulate transcription of enzymes involved in antimicrobial lipid biosynthesis, and induce transcription of antimicrobial peptides as well as cytokines and chemokines. The subsequently secreted antimicrobial peptides and lipids then attack and eliminate the invader, assisted by or in synergism with other antimicrobial molecules delivered by other defense cells that have been recruited to the site of infection, in most of the cases. This review invites

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Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands

Cited by 19 publications

References 52 publications

Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1

Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1

Upper Airway Mucosal Inflammation : Proteomic Studies after Exposure to Irritants and Microbial Agents

Antimicrobial lipids: Emerging effector molecules of innate host defense

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