Degradation of chondroitin sulfate A by a PUL-like operon in Tannerella forsythia.

Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. T. forsythia shares a distant genetic linkage to Bacteroidetes thetaiotaomicron and may therefore produce analogous polysaccharide utilization loci (PUL) which enable complex carbohydrate degradation, import, and use, although this capacity has yet to be demonstrated. Chondroitin sulfate A is a linear, sulfated carbohydrate linked to periodontal disease as the principal species of glycosaminoglycan appended on the surface of cortical bone of teeth and in supporting dental ligaments. Through genomic comparisons with B. thetaiotaomicron, a new PUL-like operon (Bfo2285-Bfo2295, and Bfo3043) was identified in T. forsythia and the crystal structure of two proteins from this PUL-like operon, Bfo2290 and Bfo2294, were reported using X-ray crystallography. Enzyme kinetics for Bfo2290 were reported using a pH-dependent assay and suggested a Km of 0.75 mg/ml ± 0.60 mg/ml, Kcat of 3.74 min-1 ± 0.88 min-1, and Vmax of 7.48 µM/min ± 1.76 µM/min with partially degraded chondroitin sulfate A. Fluorophore-assisted carbohydrate electrophoresis was used to show the processive degradation of chondroitin sulfate A by the proteins encoded in T. forsythia PUL-like operon, and revealed Bfo2291 and Bfo2290 to be an endolytic chondroitin sulfate A lyase and exolytic ΔDi-4S chondroitin sulfate A sulfatase, respectively.

Genome-wide association meta-analysis identifies five modifier loci of lung disease severity in cystic fibrosis.

The identification of small molecules that target specific CFTR variants has ushered in a new era of treatment for cystic fibrosis (CF), yet optimal, individualized treatment of CF will require identification and targeting of disease modifiers. Here we use genome-wide association analysis to identify genetic modifiers of CF lung disease, the primary cause of mortality. Meta-analysis of 6,365 CF patients identifies five loci that display significant association with variation in lung disease. Regions on chr3q29 (MUC4/MUC20; P=3.3 × 10(-11)), chr5p15.3 (SLC9A3; P=6.8 × 10(-12)), chr6p21.3 (HLA Class II; P=1.2 × 10(-8)) and chrXq22-q23 (AGTR2/SLC6A14; P=1.8 × 10(-9)) contain genes of high biological relevance to CF pathophysiology. The fifth locus, on chr11p12-p13 (EHF/APIP; P=1.9 × 10(-10)), was previously shown to be associated with lung disease. These results provide new insights into potential targets for modulating lung disease severity in CF.

Mucin variable number tandem repeat polymorphisms and severity of cystic fibrosis lung disease: significant association with MUC5AC.

Variability in cystic fibrosis (CF) lung disease is partially due to non-CFTR genetic modifiers. Mucin genes are very polymorphic, and mucins play a key role in the pathogenesis of CF lung disease; therefore, mucin genes are strong candidates as genetic modifiers. DNA from CF patients recruited for extremes of lung phenotype was analyzed by Southern blot or PCR to define variable number tandem repeat (VNTR) length polymorphisms for MUC1, MUC2, MUC5AC, and MUC7. VNTR length polymorphisms were tested for association with lung disease severity and for linkage disequilibrium (LD) with flanking single nucleotide polymorphisms (SNPs). No strong associations were found for MUC1, MUC2, or MUC7. A significant association was found between the overall distribution of MUC5AC VNTR length and CF lung disease severity (p = 0.025; n = 468 patients); plus, there was robust association of the specific 6.4 kb HinfI VNTR fragment with severity of lung disease (p = 6.2×10(-4) after Bonferroni correction). There was strong LD between MUC5AC VNTR length modes and flanking SNPs. The severity-associated 6.4 kb VNTR allele of MUC5AC was confirmed to be genetically distinct from the 6.3 kb allele, as it showed significantly stronger association with nearby SNPs. These data provide detailed respiratory mucin gene VNTR allele distributions in CF patients. Our data also show a novel link between the MUC5AC 6.4 kb VNTR allele and severity of CF lung disease. The LD pattern with surrounding SNPs suggests that the 6.4 kb allele contains, or is linked to, important functional genetic variation.

Hydra: software for tailored processing of H/D exchange data from MS or tandem MS analyses.

BACKGROUND: Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) experiments implemented to characterize protein interaction and protein folding generate large quantities of data. Organizing, processing and visualizing data requires an automated solution, particularly when accommodating new tandem mass spectrometry modes for H/DX measurement. We sought to develop software that offers flexibility in defining workflows so as to support exploratory treatments of H/DX-MS data, with a particular focus on the analysis of very large protein systems and the mining of tandem mass spectrometry data. RESULTS: We present a software package ("Hydra") that supports both traditional and exploratory treatments of H/DX-MS data. Hydra's software architecture tolerates flexible data analysis procedures by allowing the addition of new algorithms without significant change to the underlying code base. Convenient user interfaces ease the organization of raw data files and input of peptide data. After executing a user-defined workflow, extracted deuterium incorporation values can be visualized in tabular and graphical formats. Hydra also automates the extraction and visualization of deuterium distribution values. Manual validation and assessment of results is aided by an interface that aligns extracted ion chromatograms and mass spectra, while providing a means of rapidly reprocessing the data following manual adjustment. A unique feature of Hydra is the automated processing of tandem mass spectrometry data, demonstrated on a large test data set in which 40,000 deuterium incorporation values were extracted from replicate analysis of approximately 1000 fragment ions in one hour using a typical PC. CONCLUSION: The customizable workflows and user-friendly interfaces of Hydra removes a significant bottleneck in processing and visualizing H/DX-MS data and helps the researcher spend more time executing new experiments and interpreting results. This increased efficiency will encourage the analysis of larger protein systems. The ability to accommodate the tandem MS dimension supports alternative data collection and analysis strategies, as well as higher resolution localization of deuteration where permitted by the fragmentation mechanism.