ProteoClade: A taxonomic toolkit for multi-species and metaproteomic analysis

We present ProteoClade, a Python toolkit that performs taxa-specific peptide assignment, protein inference, and quantitation for multi-species proteomics experiments. ProteoClade scales to hundreds of millions of protein sequences, requires minimal computational resources, and is open source, multi-platform, and accessible to non-programmers. We demonstrate its utility for processing quantitative proteomic data derived from patient-derived xenografts and its speed and scalability enable a novel de novo proteomic workflow for complex microbiota samples

Altered expression of autoimmune regulator in infant down syndrome thymus, a possible contributor to an autoimmune phenotype.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageDown syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall's corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.Swedish Research Council 80409601 Marianne and Marcus Wallenberg Foundation Region Vastra Gotaland ALFGBG-771712 Arbetsmarknadens Forsakringsaktiebolag 100258 IngaBritt and Arne Lundbergs Research Foundation AnnMari and Per Ahlqvists Foundation Gothenburg Medical Society Wilhelm and Martina Lundgrens Research Foundatio

Site-specific O-glycosylation on the MUC_{2} mucin protein inhibits cleavage by the porphyromonas gingivalis secreted cysteine protease (RgpB)

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation

Neoadjuvant chemoradiotherapy plus surgery versus active surveillance for oesophageal cancer: A stepped-wedge cluster randomised trial

Background: Neoadjuvant chemoradiotherapy (nCRT) plus surgery is a standard treatment for locally advanced oesophageal cancer. With this treatment, 29% of patients have a pathologically complete response in the resection specimen. This provides the rationale for investigating an active surveillance approach. The aim of this study is to assess the (cost-)effectiveness of active surveillance vs. standard oesophagectomy after nCRT for oesophageal cancer. Methods: This is a phase-III multi-centre, stepped-wedge cluster randomised controlled trial. A total of 300 patients with clinically complete response (cCR, i.e. no local or disseminated disease proven by histology) after nCRT will be randomised to show non-inferiority of active surveillance to standard oesophagectomy (non-inferiority margin 15%, intra-correlation coefficient 0.02, power 80%, 2-sided α 0.05, 12% drop-out). Patients will undergo a first clinical response evaluation (CRE-I) 4-6 weeks after nCRT, consisting of endoscopy with bite-on-bite biopsies of the primary tumour site and other suspected lesions. Clinically complete responders will undergo a second CRE (CRE-II), 6-8 weeks after CRE-I. CRE-II will include 18F-FDG-PET-CT, followed by endoscopy with bite-on-bite biopsies and ultra-endosonography plus fine needle aspiration of suspected lymph nodes and/or PET- positive lesions. Patients with cCR at CRE-II will be assigned to oesophagectomy (first phase) or active surveillance (second phase of the study). The duration of the first phase is determined randomly over the 12 centres, i.e., stepped-wedge cluster design. Patients in the active surveillance arm will undergo diagnostic evaluations similar to CRE-II at 6/9/12/16/20/24/30/36/48 and 60 months after nCRT. In this arm, oesophagectomy will be offered only to patients in whom locoregional regrowth is highly suspected or proven, without distant dissemination. The main study parameter is overall survival; secondary endpoints include percentage of patients who do not undergo surgery, quality of life, clinical irresectability (cT4b) rate, radical resection rate, postoperative complications, progression-free survival, distant dissemination rate, and cost-effectiveness. We hypothesise that active surveillance leads to non-inferior survival, improved quality of life and a reduction in costs, compared to standard oesophagectomy. Discussion: If active surveillance and surgery as needed after nCRT leads to non-inferior survival compared to standard oesophagectomy, this organ-sparing approach can be implemented as a standard of care

Study of the colonic mucus layer by mass spectrometry

The mucus covering our internal mucosal surfaces is a part of the innate immune system, and the first line of defense against microbial challenges. The need of an efficient defense system is especially important in the lower parts of the digestive tract where the microbiota reaches its highest density. In the colon, the mucus forms a dense layer that prevents bacteria from accessing the epithelial surface. The gel-forming mucin 2 (MUC2) is the major structural component of the colonic mucus layer, forming large net-like structures by oligomerization in the N- and C- terminal regions. A dysfunctional mucus layer that allows bacteria to pass through and access the underlying epithelium has been associated with inflammatory bowl diseases such as ulcerative colitis. However, detailed understanding of the molecular mechanisms behind the defective mucus layer is lacking. This lack of knowledge can largely be explained by the limited information regarding the composition and processing of the mucus during normal conditions. This thesis aims to broaden the knowledge regarding the protein composition of the human colonic mucus, and the molecular properties of the heavily glycosylated MUC2 mucin. Proteomic and mass spectrometry approaches were used to characterize the composition of the human colonic mucus layer in health an disease, and to determine how alterations in protein abundance and modification of the MUC2 mucin affect the function of the mucus gel. Our results showed that the human colonic mucus is comprised of approximately 50 proteins. The protein composition of the mucus layer was shown to be unaffected in patients with ulcerative colitis, though the relative abundance of 13 mucus proteins including the structural components MUC2 and FCGBP were shown to be decreased during active disease. The mucin protein family is characterized by a heavily O-glycosylated core that is resistant against proteolytic degradation. However, our results showed that the C-terminal part of the protein is also modified by N- and O-glycans, and that site specific O-glycosylation plays an important role in protecting the protein from proteolytic degradation by bacterial proteases. In addition, we could correlate the relative abundance of various glycosyltransferases required for O- glycosylation in the different parts of the colon, to the previously characterized segmental pattern of terminating glycans on the MUC2. Taken together, the results from this thesis show that the human colonic mucus is composed of a relatively small number of proteins that are organized around the heavily O-glycosylated MUC2 mucin, and suggests that decreased amounts of the core mucus proteins in combination with impaired O-glycosylation of the MUC2 renders the mucus layer more permeable to bacteria and susceptible to proteolytic degradation

Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C‑Terminus of the Intestinal MUC2Mucin

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein’s central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites, and 14 <i>O</i>-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins

Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C‑Terminus of the Intestinal MUC2Mucin

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein’s central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites, and 14 <i>O</i>-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins

Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C‑Terminus of the Intestinal MUC2Mucin

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein’s central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites, and 14 <i>O</i>-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins