ABSTRACT
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. 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 on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.
Subject(s)
Neuraminidase , Sialic Acids , Adult , Humans , Sialic Acids/chemistry , Neuraminidase/metabolism , Lectins , Oxidoreductases Acting on Sulfur Group DonorsABSTRACT
Glycobiology as a field holds enormous potential for understanding human health and disease. However, few glycobiology studies adequately address the issue of sex differences in biology, which severely limits the conclusions that can be drawn. Numerous CAZymes, lectins, and other carbohydrate-associated molecules have the potential to be differentially expressed and regulated with sex, leading to differences in O-GlcNAc, N-glycan branching, fucosylation, sialylation, and proteoglycan structure, among others. Expression of proteins involved in glycosylation is influenced through hormones, miRNA, and gene dosage effects. In this review, we discuss the benefits of incorporating sex-based analysis in glycobiology research and the potential drivers of sex differences. We highlight examples of where incorporation of sex-based analysis has led to insights into glycobiology. Finally, we offer suggestions for how to proceed moving forward, even if the experiments are already complete. Properly incorporating sex based analyses into projects will substantially improve the accuracy and reproducibility of studies as well as accelerate the rate of discovery in the glycosciences.
Subject(s)
Carbohydrates , Polysaccharides , Female , Humans , Male , Reproducibility of Results , Glycosylation , Polysaccharides/chemistry , Lectins/metabolismABSTRACT
OBJECTIVE: Systemic sclerosis (SSc) is a rare but deadly disease characterized by autoimmunity, vasculopathy, and fibrosis. Fibrotic complications associated with SSc correlate with severe morbidity and mortality. Previous studies in SSc have identified fibroblasts as the primary drivers of fibrosis; however, the mechanism(s) promoting this are not well understood. Aberrant glycosylation, particularly polysialylation (polySia), has been described as a prominent feature of aggressive cancers. Inspired by this observation, we aimed to determine if polySia is dysregulated in various forms of SSc. METHODS: All patients with SSc met the 2013 ACR/EULAR. Patients were sub-classified into limited cutaneous (lSSc, N = 5 or 46 patients for polySia quantification in the dermis or serum; respectively), diffuse cutaneous (dSSc, N = 11 or 18 patients for polySia quantification in the dermis or serum; respectively), or patients with dSSc treated with an autologous stem cell transplantation (post-ASCT, N = 4 patients for quantification in the dermis). Dermal polySia levels were measured via immunofluorescence microscopy in 10 µm dermal sections, quantified in each group (healthy volunteers (HC), lSSc, dSSc, and post-ASCT) and correlated with skin fibrosis (via the modified Rodnan skin score (mRSS)). Similarly, serum polySia was quantified in each group, and correlated with the mRSS. RESULTS: Dermal polySia levels were highest in patients with dSSc (compared to HC < 0.001), and correlated with the degree of fibrosis in all of the groups (P = 0.008). Serum polySia was higher in all SSc groups (p < 0.001) and correlated with the severity of mRSS (p < 0.0001). CONCLUSION: Polysia is more abundant in the skin and sera from patients with SSc and correlates with the degree of skin fibrosis. The aberrant expression of polySia highlights its potential use as a biomarker in patients with progressive forms of SSc. Dysregulated polySia levels in SSc further emphasizes the cancer-like phenotype present in SSc, which may promote fibrosis and immune dysregulation.
ABSTRACT
Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo.
Subject(s)
Flow Cytometry/methods , Image Cytometry/methods , Phenylalanine/chemistry , Protein Biosynthesis/physiology , Tellurium/chemistry , Amino Acids/chemistry , Amino Acids/metabolism , Animals , Brain/diagnostic imaging , Brain/metabolism , Cycloheximide/pharmacology , HCT116 Cells , Humans , Jejunum/diagnostic imaging , Jejunum/metabolism , Jurkat Cells , Mice , Neoplasms, Experimental , Phenylalanine/metabolism , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Tellurium/metabolismABSTRACT
Male and female immune systems are strikingly different and yet little is known about sex differences in immune glycans, though glycans play central roles in regulating the immune response. Polysialic acid (polySia) occurs on the majority of leukocytes and is a potent immunomodulatory glycan which enables cell migration and serves as an immune checkpoint. Due to widespread influence of polySia on the immune system, we aimed to characterize its levels in serum, its presence on specific proteins, and differences in the amounts of polySia in male and female serum. However, polySia is difficult to quantify and detect on specific proteins, which makes it challenging to elucidate the molecular details of polySia function. We developed a sandwich ELISA that allows for the quantification of polySia as well as specific polysialylated proteins in complex mixtures without any pretreatment or harsh conditions. The assay is quick, linear, and robust under a wide variety of conditions and gave a limit of detection of approximately 0.2 ng polySia per mL of serum. We then quantified polySia and polysialylated CD56 in human and mouse serum. These studies strongly support our hypothesis of differences in glycosylation between the sexes as significantly less polySia was observed in female samples than in male samples.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Sialic Acids/blood , Animals , Female , Healthy Volunteers , Humans , Male , Mice , Mice, Inbred C57BL , Sex Characteristics , Sialic Acids/immunologyABSTRACT
Mass cytometry is a revolutionary technology that allows for the simultaneous quantification of >40 different biomarkers with cellular resolution. The biomarkers are detected using metal-labeled antibodies as well as small-molecule probes of cell size, viability, and biochemical status. Barcoding is an important component of sample preparation because it reduces processing time, eliminates sample-to-sample variation, discriminates cell doublets, reduces the amount of antibody needed, and conserves sample. We developed a thiol-reactive tellurium-based barcode, TeMal. TeMal is nontoxic at working concentrations, compatible with metal-labeled antibodies, and can readily be applied to live or fixed cells, making it advantageous and complementary compared to existing barcoding reagents. We have demonstrated the utility of TeMal by barcoding microscale samples in situ to facilitate analysis of cells from an automated cell culture system using mass cytometry.
Subject(s)
Flow Cytometry/methods , Single-Cell Analysis/methods , Staining and Labeling/methods , Tellurium/chemistry , Antibodies/chemistry , Biomarkers/chemistry , HumansABSTRACT
Capsular polysaccharides (CPSs) are high-molecular-mass cell-surface polysaccharides, that act as important virulence factors for many pathogenic bacteria. Several clinically important Gram-negative pathogens share similar systems for CPS biosynthesis and export; examples include Escherichia coli, Campylobacter jejuni, Haemophilus influenzae, Neisseria meningitidis, and Pasteurella multocida. Each CPS contains a serotype-specific repeat-unit structure, but the glycans all possess a lipid moiety at their reducing termini. In E. coli and N. meningitidis, the predominant lipid is a lysophosphatidylglycerol moiety that is attached to the repeat-unit domain of the CPS via multiple residues of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), referred to as a poly-Kdo linker. The Kdo residues are ß-linked, suggesting that they are synthesized by retaining glycosyltransferases. To date, the only characterized Kdo transferases are the inverting enzymes that catalyze the α-linkages found in lipopolysaccharide. Here, we identify two conserved proteins from CPS assembly systems, KpsC and KpsS, as the ß-Kdo-transferases and demonstrate in vitro reconstitution of poly-Kdo linker assembly on a fluorescent phosphatidylglycerol acceptor. KpsS adds the first Kdo residue, and this reaction product is then extended by KpsC. Cross-complementation experiments demonstrate that the E. coli and N. meningitidis protein homologs are functionally conserved.
Subject(s)
Bacterial Capsules/metabolism , Carrier Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Neisseria meningitidis/metabolism , Transferases/metabolism , Bacterial Capsules/genetics , Carrier Proteins/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Genetic Complementation Test , Neisseria meningitidis/genetics , Sugar Acids/metabolism , Transferases/geneticsABSTRACT
Bacterial capsules are surface layers made of long-chain polysaccharides. They are anchored to the outer membrane of many Gram-negative bacteria, including pathogens such as Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida. Capsules protect pathogens from host defenses including complement-mediated killing and phagocytosis and therefore represent a major virulence factor. Capsular polysaccharides are synthesized by enzymes located in the inner (cytoplasmic) membrane and are then translocated to the cell surface. Whereas the enzymes that synthesize the polysaccharides have been studied in detail, the structure and biosynthesis of the anchoring elements have not been definitively resolved. Here we determine the structure of the glycolipid attached to the reducing terminus of the polysialic acid capsular polysaccharides from E. coli K1 and N. meningitidis group B and the heparosan-like capsular polysaccharide from E. coli K5. All possess the same unique glycolipid terminus consisting of a lyso-phosphatidylglycerol moiety with a ß-linked poly-(3-deoxy-d-manno-oct-2-ulosonic acid) (poly-Kdo) linker attached to the reducing terminus of the capsular polysaccharide.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Capsules/metabolism , Escherichia coli/metabolism , Glycolipids/metabolism , Neisseria meningitidis/metabolism , Polysaccharides/metabolism , Adenosine Triphosphate/metabolism , Biological Transport , Gram-Negative Bacteria/metabolism , Magnetic Resonance Spectroscopy , Mass Spectrometry , Methylation , Mutation , Virulence Factors/metabolismABSTRACT
Mass cytometry (MC) is a powerful tool for studying heterogeneous cell populations. In previous work, our laboratory has developed an MC probe for hypoxia bearing a methyl telluride mass tag. The methyl telluride was unoptimized, displaying stability and toxicity limitations. Here, we investigate three classes of organotelluriums as MC mass tags: methyl tellurides, trifluoromethyl tellurides and 2-alkyl-tellurophenes. NMR was used to compare the stability of these compounds in aqueous and organic solutions and the compounds were analysed for toxicity in Jurkat cells. The methyl tellurides were moderately stable to aerobic oxidation in organic solution under dry ambient conditions. The trifluoromethyl tellurides were stable to aerobic oxidation in organic solution but decomposed in aqueous solution. The 2-alkyl-tellurophenes proved to be stable in both organic and aqueous solutions under ambient conditions and showed limited toxicity (IC50 > 200 µM) in cell based assays. The synthetic feasibility, chemically stability, and limited toxicity of tellurophenes suggests these groups will be good choices for MC reagent development.
ABSTRACT
The posttranslational modification of therapeutic proteins with terminal sialic acids is one means of improving their circulating half-life, thereby improving their efficiency. We have developed a two-step in vitro enzymatic modification of glycoproteins, which has previously only been achieved by chemical means [Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Int J Pharm 300:125-130). This two-step procedure uses the Campylobacter jejuni Cst-II α2,8-sialyltransferase to provide a primer on N-linked glycans, followed by polysialylation using the Neisseria meningitidis α2,8-polysialyltransferase. Here, we have demonstrated the ability of this system to modify three glycoproteins with varying N-linked glycan compositions: the human therapeutic proteins alpha-1-antitrypsin (A1AT) and factor IX, as well as bovine fetuin. The chain length of the polysialic acid addition was optimized by controlling reaction conditions. After demonstrating the ability of this system to modify a variety of proteins, the effect of polysialylation on the activity and serum half-life of A1AT was examined. The polysialylation of A1AT did not adversely affect its in vitro inhibition activity against human neutrophil elastase. The polysialylation of A1AT resulted in a significantly improved pharmacokinetic profile when the modified proteins were injected into CD-1 mice. Together, these results suggest that polysialylated A1AT may be useful for improved augmentation therapy for patients with a deficiency in this protein and that this modification may be applied to other therapeutic proteins.
Subject(s)
Campylobacter jejuni/enzymology , Drug Design , Glycoproteins/metabolism , Neisseria meningitidis/enzymology , Protein Processing, Post-Translational/physiology , Sialyltransferases/metabolism , Animals , Cattle , Chromatography , Electrophoresis, Polyacrylamide Gel , Factor IX/metabolism , Fluorescence , Glycoproteins/pharmacokinetics , Humans , In Vitro Techniques , Mass Spectrometry , Mice , alpha 1-Antitrypsin/metabolism , alpha 1-Antitrypsin/pharmacokinetics , alpha-Fetoproteins/metabolismABSTRACT
Sialic acids are key mediators of cell function, particularly with regard to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. While multiple strategies are being explored for the control of cell surface sialosides, none that shows high selectivity between sialyltransferases or that targets a specific sialyl glycoform has yet to emerge. Here, we describe a strategy to block the formation of α2,8-linked sialic acid chains (oligo- and polysialic acid) through the use of 8-keto-sialic acid as a chain-terminating metabolic inhibitor that, if incorporated, cannot be elongated. 8-Keto-sialic acid is nontoxic at effective concentrations and serves to block polysialic acid synthesis in cancer cell lines and primary immune cells, with minimal effects on other sialyl glycoforms.
Subject(s)
N-Acetylneuraminic Acid , Sialic Acids , Sialic Acids/chemistry , Sialyltransferases/metabolism , Cell Membrane/metabolismABSTRACT
Neisseria meningitidis serogroup B and Escherichia coli K1 bacteria produce a capsular polysaccharide (CPS) that is composed of α2,8-linked polysialic acid (PSA). Biosynthesis of PSA in these bacteria occurs via an ABC (ATP-binding cassette) transporter-dependent pathway. In N. meningitidis, export of PSA to the surface of the bacterium requires two proteins that form an ABC transporter (CtrC and CtrD) and two additional proteins, CtrA and CtrB, that are proposed to form a cell envelope-spanning export complex. CtrA is a member of the outer membrane polysaccharide export (OPX) family of proteins, which are proposed to form a pore to mediate export of CPSs across the outer membrane. CtrB is an inner membrane protein belonging to the polysaccharide co-polymerase (PCP) family. PCP proteins involved in other bacterial polysaccharide assembly systems form structures that extend into the periplasm from the inner membrane. There is currently no structural information available for PCP or OPX proteins involved in an ABC transporter-dependent CPS biosynthesis pathway to support their proposed roles in polysaccharide export. Here, we report cryo-EM images of purified CtrB reconstituted into lipid bilayers. These images contained molecular top and side views of CtrB and showed that it formed a conical oligomer that extended â¼125 Å from the membrane. This structure is consistent with CtrB functioning as a component of an envelope-spanning complex. Cross-complementation of CtrA and CtrB in E. coli mutants with defects in genes encoding the corresponding PCP and OPX proteins show that PCP-OPX pairs require interactions with their cognate partners to export polysaccharide. These experiments add further support for the model of an ABC transporter-PCP-OPX multiprotein complex that functions to export CPS across the cell envelope.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , Adenosine Triphosphate/chemistry , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Neisseria meningitidis/metabolism , Polymers/chemistry , Polysaccharides/chemistry , Amino Acid Sequence , Biological Transport , Cryoelectron Microscopy/methods , Genetic Complementation Test , Lipid Bilayers/chemistry , Models, Genetic , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Lichen symbioses are thought to be stabilized by the transfer of fixed carbon from a photosynthesizing symbiont to a fungus. In other fungal symbioses, carbohydrate subsidies correlate with reductions in plant cell wall-degrading enzymes, but whether this is true of lichen fungal symbionts (LFSs) is unknown. Here, we predict genes encoding carbohydrate-active enzymes (CAZymes) and sugar transporters in 46 genomes from the Lecanoromycetes, the largest extant clade of LFSs. All LFSs possess a robust CAZyme arsenal including enzymes acting on cellulose and hemicellulose, confirmed by experimental assays. However, the number of genes and predicted functions of CAZymes vary widely, with some fungal symbionts possessing arsenals on par with well-known saprotrophic fungi. These results suggest that stable fungal association with a phototroph does not in itself result in fungal CAZyme loss, and lends support to long-standing hypotheses that some lichens may augment fixed CO2 with carbon from external sources.
Subject(s)
Ascomycota , Lichens , Ascomycota/metabolism , Carbohydrate Metabolism , Carbon , Cellulose/metabolismABSTRACT
The last two years have seen major advances in understanding the structural basis of bacterial cell envelope glycoconjugate biosynthesis, including capsules, lipopolysaccharide, teichoic acid, cellulose, and peptidoglycan. The recent crystal and cryo-electron microscopy structures of proteins involved in the initial glycosyltransferase steps in the cytoplasm, the transport of large and small lipid-linked glycoconjugates across the inner membrane, the polymerization of glycans in the periplasm, and the export of molecules from the cell have shed light on the mechanisms by which cell envelope glycoconjugates are made. We discuss these recent advances and highlight remaining unanswered questions.
Subject(s)
Anti-Bacterial Agents , Gram-Negative Bacteria , Cell Membrane , Cell Wall , Cryoelectron Microscopy , Gram-Positive Bacteria , PeptidoglycanABSTRACT
There is an overwhelming amount of evidence demonstrating that people from marginalized groups, including women, racialized and Indigenous peoples, people with disabilities, immigrants, and LGBTQ+ individuals, continue to face substantial discrimination in STEM, manifested as both overt bias and unconscious bias. These biases result in discrimination against individuals in marginalized groups, and independent biases collectively contribute to a culture that systematically discriminates against people from marginalized groups. Representation from marginalized groups in postsecondary degrees in natural science and engineering has not substantially improved in over a decade. A set of 10 concrete principles are presented that trainees, principle investigators, departments, and faculties can use to enhance the participation and lived experiences of people in marginalized groups in STEM.
ABSTRACT
The large (1767-amino acid) endo-alpha-N-acetylgalactosaminidase from Streptococcus pneumoniae (SpGH101) specifically removes an O-linked disaccharide Gal-beta-1,3-GalNAc-alpha from glycoproteins. While the enzyme from natural sources has been used as a reagent for many years, very few mechanistic studies have been performed. Using the recently determined three-dimensional structure of the recombinant protein as a background, we report here a mechanistic investigation of the SpGH101 retaining alpha-glycoside hydrolase using a combination of synthetic and natural substrates. On the basis of a model of the substrate complex of SpGH101, we propose D764 and E796 as the nucleophile and general acid-base residues, respectively. These roles were confirmed by kinetic and mechanistic analysis of mutants at those positions using synthetic substrates and anion rescue experiments. pK(a) values of 5.3 and 7.2 were assigned to D764 and E796 on the basis of the pK(a) values derived from the bell-shaped dependence of k(cat)/K(m) upon pH. The enzyme contains several putative carbohydrate binding modules whose glycan binding specificities were probed using the printed glycan array of the Consortium for Functional Glycomics using the inactive D764A and D764F mutants that had been labeled with Alexafluor 488. These studies revealed binding to galacto-N-biose, consistent with a role for these domains in localizing the enzyme near its substrates.
Subject(s)
Bacterial Proteins/metabolism , Streptococcus pneumoniae/enzymology , alpha-N-Acetylgalactosaminidase/chemistry , alpha-N-Acetylgalactosaminidase/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Hydrogen-Ion Concentration , Kinetics , Models, Genetic , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding/genetics , Sequence Homology, Amino Acid , alpha-N-Acetylgalactosaminidase/geneticsABSTRACT
Glycoconjugates containing polysialic acid have many biological activities and represent target molecules for therapeutic interventions. Enzymatic synthesis of these glycoconjugates should give access to these important molecules to evaluate their potential. The polysialyltransferases from both Neisseria meningitidis and Escherichia coli were cloned and expressed as recombinant proteins in E. coli. We have used synthetic acceptors to probe the acceptor requirement of these enzymes and to examine the basic enzymology. The minimum number of sialic acid residues (Neu5Ac) on the acceptor for activity in vitro was shown to be 2 for both enzymes, but a large increase in activity was seen if the acceptor had three Neu5Ac residues. The polysialyltransferase from N. meningitidis generated longer reaction products than the enzyme from E. coli on FCHASE acceptors. Examination of the products showed them to be a heterogeneous mixture, but products with >50 Neu5Ac residues could be seen using capillary zone electrophoresis analyses. In addition we made fusion proteins of these polysialyltransferase enzymes with the bifunctional alpha-2,3/alpha-2,8-sialyltransferase from Campylobacter jejuni to create self priming polysialyltransferases. These bifunctional sialyltransferases utilized various synthetic disaccharide acceptors with a terminal galactose, and we demonstrate here that the PST enzyme from N. meningitidis and its fusion protein with the C. jejuni sialyltransferase can be used to create polysialic acid on O-linked glycopeptides.
Subject(s)
Bacterial Proteins/chemistry , Neisseria meningitidis/enzymology , Sialyltransferases/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli Proteins/chemistry , Kinetics , Magnesium/metabolism , Molecular Sequence Data , N-Acetylneuraminic Acid/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Sialyltransferases/genetics , Sialyltransferases/metabolismABSTRACT
Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacteria/metabolism , Bacterial Capsules/metabolism , Polysaccharides, Bacterial/biosynthesis , Polysaccharides, Bacterial/chemistry , Animals , Bacteria/cytology , Bacteria/enzymology , Carbohydrate Sequence , Humans , Molecular Sequence Data , Polysaccharides, Bacterial/metabolismABSTRACT
Bacteriophages specific for Escherichia coli K1 express a tailspike protein that degrades the polysialic acid coat of E. coli K1 that is essential for bacteriophage infection. This enzyme is specific for polysialic acid and is a member of a family of endo-sialidases. This family is unusual because all other previously reported sialidases outside of this family are exo- or trans-sialidases. The recently determined structure of an endo-sialidase derived from bacteriophage K1F (endoNF) revealed an active site that lacks a number of the residues that are conserved in other sialidases, implying a new, endo-sialidase-specific catalytic mechanism. Using synthetic trifluoromethylumbelliferyl oligosialoside substrates, kinetic parameters for hydrolysis at a single cleavage site were determined. Measurement of kcat/Km at a series of pH values revealed a dependence on a single protonated group of pKa 5. Mutation of a putative active site acidic residue, E581A, resulted in complete loss of sialidase activity. Direct 1H NMR analysis of the hydrolysis of trifluoromethylumbelliferyl sialotrioside revealed that endoNF is an inverting sialidase. All other wild type sialidases previously reported are retaining glycosidases, implying a new mechanism of sialidase action specific to this family of endo-sialidases.
Subject(s)
Bacteriophages/enzymology , Glycoside Hydrolases/metabolism , Glycosides/metabolism , Mutation/genetics , Neuraminidase/genetics , Neuraminidase/metabolism , Animals , Bacteriophages/genetics , Binding Sites , Campylobacter jejuni/enzymology , Chromatography, Thin Layer , Glycoproteins , Glycoside Hydrolases/genetics , Hydrolysis , Magnetic Resonance Spectroscopy , Mutagenesis, Site-Directed , Sialyltransferases/metabolism , Substrate Specificity , Trypanosoma cruzi/enzymologyABSTRACT
Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase is a cell surface-anchored glycoside hydrolase from family GH101 involved in the breakdown of mucin type O-linked glycans. The 189-kDa mature enzyme specifically hydrolyzes the T-antigen disaccharide from extracellular host glycoproteins and is representative of a broadly important class of virulence factors that have remained structurally uncharacterized due to their large size and highly modular nature. Here we report a 2.9 angstroms resolution crystal structure that remarkably captures the multidomain architecture and characterizes a catalytic center unexpectedly resembling that of alpha-amylases. Our analysis presents a complete model of glycoprotein recognition and provides a basis for the structure-based design of novel Streptococcus vaccines and therapeutics.