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Validation of antibody reagents for mucin analysis in chronic inflammatory airway diseases.

ABSTRACT: In chronic inflammatory airway diseases, mucins display disease-related alterations in quantity, composition and glycosylation. This opens the possibility to diagnose and monitor inflammatory airway disorders and their exacerbation based on mucin properties. For such an approach to be reasonably versatile and diagnostically meaningful, the mucin of interest must be captured in a reliable, patient-independent way. To identify appropriate mucin-specific reagents, we tested anti-mucin antibodies on mucin-content-standardized, human bronchoalveolar lavage fluid samples in immunoblot assays. All commercially available monoclonal antibodies against the major airway mucin MUC5AC were screened, except for those with known specificity for carbohydrates, as glycosylation patterns are not mucin-specific. Our results indicated considerable inter-patient and inter-antibody variability in mucin recognition for all antibodies and samples tested. The best results in terms of signal strength and reproducibility were obtained with antibodies Mg-31, O.N.457 and 45M1. Additional epitope mapping experiments revealed that only one of the antibodies with superior binding to MUC5AC recognized linear peptide epitopes on the protein backbone.

SUBMITTER: Krause T

PROVIDER: S-EPMC5297535 | biostudies-literature | 2017 Feb/Mar

REPOSITORIES: biostudies-literature

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Validation of antibody reagents for mucin analysis in chronic inflammatory airway diseases.

Krause Thorsten T Röckendorf Niels N Gaede Karoline I KI Ramaker Katrin K Sinnecker Heike H Frey Andreas A

mAbs 20161202 2

In chronic inflammatory airway diseases, mucins display disease-related alterations in quantity, composition and glycosylation. This opens the possibility to diagnose and monitor inflammatory airway disorders and their exacerbation based on mucin properties. For such an approach to be reasonably versatile and diagnostically meaningful, the mucin of interest must be captured in a reliable, patient-independent way. To identify appropriate mucin-specific reagents, we tested anti-mucin antibodies on ...[more]

PMID: 27911216

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Project description:Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitic and emphysematous components. In one biophysical model, the concentration of mucin on the airway surfaces is hypothesized to be a key variable that controls mucus transport in healthy persons versus cessation of transport in persons with muco-obstructive lung diseases. Under this model, it is postulated that a high mucin concentration produces the sputum and disease progression that are characteristic of chronic bronchitis.We characterized the COPD status of 917 participants from the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) using questionnaires administered to participants, chest tomography, spirometry, and examination of induced sputum. Total mucin concentrations in sputum were measured with the use of size-exclusion chromatography and refractometry. In 148 of these participants, the respiratory secreted mucins MUC5AC and MUC5B were quantitated by means of mass spectrometry. Data from chronic-bronchitis questionnaires and data on total mucin concentrations in sputum were also analyzed in an independent 94-participant cohort.Mean (±SE) total mucin concentrations were higher in current or former smokers with severe COPD than in controls who had never smoked (3166±402 vs. 1515±152 ?g per milliliter) and were higher in participants with two or more respiratory exacerbations per year than in those with zero exacerbations (4194±878 vs. 2458±113 ?g per milliliter). The absolute concentrations of MUC5B and MUC5AC in current or former smokers with severe COPD were approximately 3 times as high and 10 times as high, respectively, as in controls who had never smoked. Receiver-operating-characteristic curve analysis of the association between total mucin concentration and a diagnosis of chronic bronchitis yielded areas under the curve of 0.72 (95% confidence interval [CI], 0.65 to 0.79) for the SPIROMICS cohort and 0.82 (95% CI, 0.73 to 0.92) for the independent cohort.Airway mucin concentrations may quantitate a key component of the chronic bronchitis pathophysiologic cascade that produces sputum and mediates disease severity. Studies designed to explore total mucin concentrations in sputum as a diagnostic biomarker and therapeutic target for chronic bronchitis appear to be warranted. (Funded by the National Heart, Lung, and Blood Institute and others.).

Project description:Background: Posttranscriptional gene regulation (PTGR) contributes to inflammation through alterations in messenger RNA (mRNA) turnover and translation rates. RNA-binding proteins (RBPs) coordinate these processes but their role in lung inflammatory diseases is ill-defined. We evaluated the expression of a curated list of mRNA-binding RBPs (mRBPs) in selected Gene Expression Omnibus (GEO) transcriptomic databases of airway epithelium isolated from chronic obstructive pulmonary disease (COPD), severe asthma (SA) and matched control subjects, hypothesizing that global changes in mRBPs expression could be used to infer their pathogenetic roles and identify novel disease-related regulatory networks. Methods: A published list of 692 mRBPs [Nat Rev Genet 2014] was searched in GEO datasets originated from bronchial brushings of stable COPD patients (C), smokers (S), non-smokers (NS) controls with normal lung function (n = 6/12/12) (GEO ID: GSE5058) and of (SA) and healthy control (HC) (n = 6/12) (GSE63142). Fluorescence intensity data were extracted and normalized on the medians for fold change (FC) comparisons. FCs were set at ≥ |1.5| with a false discovery rate (FDR) of ≤ 0.05. Pearson correlation maps and heatmaps were generated using tMEV tools v4_9_0.45. DNA sequence motifs were searched using PScan-ChIP. Gene Ontology (GO) was performed with Ingenuity Pathway Analysis (IPA) tool. Results: Significant mRBP expression changes were detected for S/NS, COPD/NS and COPD/S (n = 41, 391, 382, respectively). Of those, 32% of genes changed by FC ≥ |1.5| in S/NS but more than 60% in COPD/NS and COPD/S (n = 13, 267, 257, respectively). Genes were predominantly downregulated in COPD/NS (n = 194, 73%) and COPD/S (n = 202, 79%), less so in S/NS (n = 4, 31%). Unsupervised cluster analysis identified in 4 out of 12 S the same mRBP pattern seen in C, postulating subclinical COPD. Significant DNA motifs enrichment for transcriptional regulation was found for downregulated RBPs. Correlation analysis identified five clusters of co-expressed mRBPs. GO analysis revealed significant enrichments in canonical pathways both specific and shared among comparisons. Unexpectedly, no significant mRBPs modulation was found in SA compared to controls. Conclusions: Airway epithelial mRBPs profiling reveals a COPD-specific global downregulation of RBPs shared by a subset of control smokers, the potential of functional cooperation by coexpressed RBPs and significant impact on relevant pathogenetic pathways in COPD. Elucidation of PTGR in COPD could identify disease biomarkers or pathways for therapeutic targeting.

Dataset Information

Validation of antibody reagents for mucin analysis in chronic inflammatory airway diseases.

Publications

Validation of antibody reagents for mucin analysis in chronic inflammatory airway diseases.

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