Project description:Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown, in part due to the lack of tools with which to study the glycan. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN and EndoNDM on streptavidin-coated magnetic or agarose beads. We showed that immobilizing EndoN improves enzyme usefulness by allowing for effective removal of the enzyme from samples, while retaining hydrolase activity. Additionally, immobilization of EndoNDM allowed for the enrichment of polysialylated proteins from complex mixtures for their identification via mass spectrometry. We identified QSOX2 as a novel polysialylated protein secreted from MCF-7 cells. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.
Project description:Melioidosis is a potentially fatal infection caused by Burkholderia pseudomallei, a bacterium that is intrinsically resistant to many commonly used antibiotics. Therefore, the identification of new drug targets is essential for the development of new and effective therapies. This study demonstrates that the Trigger Factor protein, encoded by BPSL1402, is important for the establishment of B. pseudomallei infection and is involved in multiple facets of virulence including motility, cell cytotoxicity and resistance to stress. With reports of B. pseudomallei isolated from new regions and resistance to current treatment options, the significance of this research is the identification of a novel B. pseudomallei virulence factor that can be potentially exploited for the development of new therapeutics to treat this deadly infection.
Project description:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.
Project description:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.
Project description:Legumain is cysteine protease primarily localised to the endo-lysosomal system. Upregulated legumain activity is associated with inflammation, neurodegeneration, and tumorigenesis. Whilst inhibiting legumain in mouse models has demonstrated therapeutic benefit, the proteolytic mechanisms underpinning its various functions are not well known and thus, further characterisation of its physiological substrates is required. Here, we developed FAIMS-enabled N-terminomics for sensitive and streamlined identification of both protein abundance changes and N-termini in complex samples. Comparison of wildtype and legumain-deficient murine spleens identified 6,366 proteins and 2,528 N-termini, of which 235 were enriched in wildtype spleens. These included 119 with asparaginyl cleavages corresponding to 110 proteins, indicating legumain-specific activity. Surprisingly, many of these localised to the nucleus and cytoplasm, hinting at novel extra-lysosomal roles of legumain. We further confirmed the direct processing of selected substrates by legumain in vitro; together, validating FAIMS-enabled N-terminomics for protease substrate detection in an unbiased and systematic manner.
Project description:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.
Project description:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.
Project description:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.