RESUMEN
Surface expression of the common vertebrate sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) by commensal and pathogenic microbes appears structurally to represent "molecular mimicry" of host sialoglycans, facilitating multiple mechanisms of host immune evasion. In contrast, ketodeoxynonulosonic acid (Kdn) is a more ancestral Sia also present in prokaryotic glycoconjugates that are structurally quite distinct from vertebrate sialoglycans. We detected human antibodies against Kdn-terminated glycans, and sialoglycan microarray studies found these anti-Kdn antibodies to be directed against Kdn-sialoglycans structurally similar to those on human cell surface Neu5Ac-sialoglycans. Anti-Kdn-glycan antibodies appear during infancy in a pattern similar to those generated following incorporation of the nonhuman Sia N-glycolylneuraminic acid (Neu5Gc) onto the surface of nontypeable Haemophilus influenzae (NTHi), a human commensal and opportunistic pathogen. NTHi grown in the presence of free Kdn took up and incorporated the Sia into its lipooligosaccharide (LOS). Surface display of the Kdn within NTHi LOS blunted several virulence attributes of the pathogen, including Neu5Ac-mediated resistance to complement and whole blood killing, complement C3 deposition, IgM binding, and engagement of Siglec-9. Upper airway administration of Kdn reduced NTHi infection in human-like Cmah null (Neu5Gc-deficient) mice that express a Neu5Ac-rich sialome. We propose a mechanism for the induction of anti-Kdn antibodies in humans, suggesting that Kdn could be a natural and/or therapeutic "Trojan horse" that impairs colonization and virulence phenotypes of free Neu5Ac-assimilating human pathogens.IMPORTANCE All cells in vertebrates are coated with a dense array of glycans often capped with sugars called sialic acids. Sialic acids have many functions, including serving as a signal for recognition of "self" cells by the immune system, thereby guiding an appropriate immune response against foreign "nonself" and/or damaged cells. Several pathogenic bacteria have evolved mechanisms to cloak themselves with sialic acids and evade immune responses. Here we explore a type of sialic acid called "Kdn" (ketodeoxynonulosonic acid) that has not received much attention in the past and compare and contrast how it interacts with the immune system. Our results show potential for the use of Kdn as a natural intervention against pathogenic bacteria that take up and coat themselves with external sialic acid from the environment.
Asunto(s)
Antígenos CD/inmunología , Infecciones por Haemophilus/inmunología , Haemophilus influenzae/inmunología , Interacciones Huésped-Patógeno/inmunología , Ácido N-Acetilneuramínico/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/inmunología , Ácidos Siálicos/inmunología , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Antígenos CD/metabolismo , Transporte Biológico , Complemento C3/inmunología , Complemento C3/metabolismo , Femenino , Glicoconjugados/química , Glicoconjugados/inmunología , Infecciones por Haemophilus/genética , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/química , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Ratones , Ratones Endogámicos C57BL , Imitación Molecular/genética , Imitación Molecular/inmunología , Ácido N-Acetilneuramínico/inmunología , Unión Proteica , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Ácidos Siálicos/química , Azúcares Ácidos/química , Azúcares Ácidos/inmunologíaRESUMEN
Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.
Asunto(s)
Aterosclerosis/metabolismo , Fallo Renal Crónico/metabolismo , Ácido N-Acetilneuramínico/biosíntesis , Polisacáridos/biosíntesis , Aterosclerosis/genética , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fallo Renal Crónico/genética , Ácido N-Acetilneuramínico/genética , Polisacáridos/genéticaRESUMEN
Novel therapies to counteract multidrug-resistant gonorrhea are urgently needed. A unique gonococcal immune evasion strategy involves capping of lipooligosaccharide (LOS) with sialic acid by gonococcal sialyltransferase (Lst), utilizing host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation renders gonococci resistant to complement and cationic peptides, and down-regulates the inflammatory response by engaging siglecs. CMP-sialic acid analogs (CMP-nonulosonates [CMP-NulOs]) such as CMP-Leg5,7Ac2 and CMP-Kdn are also utilized by Lst. Incorporation of these NulO analogs into LOS maintains gonococci susceptible to complement. Intravaginal administration of CMP-Kdn or CMP-Leg5,7Ac2 attenuates gonococcal colonization of mouse vaginas. Here, we identify a key mechanism of action for the efficacy of CMP-NulOs. Surprisingly, CMP-NulOs remained effective in complement C1q-/- and C3-/- mice. LOS Neu5Ac, but not Leg5,7Ac2 or Kdn, conferred resistance to the cathelicidins LL-37 (human) and mouse cathelicidin-related antimicrobial peptide in vitro. CMP-NulOs were ineffective in Camp-/- mice, revealing that cathelicidins largely mediate the efficacy of therapeutic CMP-NulOs.
Asunto(s)
Catelicidinas/farmacología , Citidina Monofosfato/análogos & derivados , Citidina Monofosfato/metabolismo , Citidina Monofosfato/farmacología , Gonorrea/tratamiento farmacológico , Ácido N-Acetilneuramínico/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas del Sistema Complemento , Citidina Monofosfato/genética , Femenino , Lipopolisacáridos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/metabolismo , Ácidos Neuramínicos , Ácidos Siálicos , Sialiltransferasas/metabolismoRESUMEN
Neisseria gonorrhoeae deploys a unique immune evasion strategy wherein the lacto-N-neotetraose termini of lipooligosaccharide (LOS) are "capped" by a surface LOS sialyltransferase (Lst), using extracellular host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation enhances complement resistance by recruiting factor H (FH; alternative complement pathway inhibitor) and also by limiting classical pathway activation. Sialylated LOS also engages inhibitory Siglecs on host leukocytes, dampening innate immunity. Previously, we showed that analogues of CMP-sialic acids (CMP-nonulosonates [CMP-NulOs]), such as CMP-Leg5,7Ac2 and CMP-Neu5Ac9N3, are also substrates for Lst. Incorporation of Leg5,7Ac2 and Neu5Ac9N3 into LOS results in N. gonorrhoeae being fully serum sensitive. Importantly, intravaginal administration of CMP-Leg5,7Ac2 attenuated N. gonorrhoeae colonization of mouse vaginas. In this study, we characterize and develop additional candidate therapeutic CMP-NulOs. CMP-ketodeoxynonulosonate (CMP-Kdn) and CMP-Kdn7N3, but not CMP-Neu4,5Ac2, were substrates for Lst, further elucidating gonococcal Lst specificity. Lacto-N-neotetraose LOS capped with Kdn and Kdn7N3 bound FH to levels â¼60% of that seen with Neu5Ac and enabled gonococci to resist low (3.3%) but not higher (10%) concentrations of human complement. CMP-Kdn, CMP-Neu5Ac9N3, and CMP-Leg5,7Ac2 administered intravaginally (10 µg/d) to N. gonorrhoeae-colonized mice were equally efficacious. Of the three CMP-NulOs above, CMP-Leg5,7Ac2 was the most pH and temperature stable. In addition, Leg5,7Ac2-fed human cells did not display this NulO on their surface. Moreover, CMP-Leg5,7Ac2 was efficacious against several multidrug-resistant gonococci in mice with a humanized sialome (Cmah-/- mice) or humanized complement system (FH/C4b-binding protein transgenic mice). CMP-Leg5,7Ac2 and CMP-Kdn remain viable leads as topical preventive/therapeutic agents against the global threat of multidrug-resistant N. gonorrhoeae.
Asunto(s)
Ácido N-Acetilneuramínico Citidina Monofosfato/farmacología , Citidina Monofosfato/análogos & derivados , Citidina Monofosfato/fisiología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Gonorrea/tratamiento farmacológico , Neisseria gonorrhoeae/efectos de los fármacos , Ácidos Neuramínicos/farmacología , Ácidos Siálicos/farmacología , Animales , Línea Celular Tumoral , Factor H de Complemento/metabolismo , Proteínas del Sistema Complemento/farmacología , Citidina Monofosfato/farmacología , Femenino , Gonorrea/metabolismo , Gonorrea/microbiología , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Oligosacáridos/fisiología , Sialiltransferasas/farmacologíaRESUMEN
Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, the dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/enzimología , Proteínas Bacterianas/genética , Campylobacter jejuni/genética , Catálisis , Clonación Molecular , Cristalografía por Rayos X , Cinética , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Especificidad por SustratoRESUMEN
Legionaminic acids are analogs of sialic acid that occur in cell surface glycoconjugates of several bacteria. Because legionaminic acids share the same stereochemistry as sialic acid but differ at C7 and C9, they are interesting analogs to probe the impact of varying exocyclic moieties (C7-C9) on biological activities such as susceptibilities to sialidases, interactions with Siglecs and immunogenicity. There are currently no reports on the bacterial enzymes that transfer legionaminic acids to these cell surface glycoconjugates, but some mammalian and bacterial sialyltransferases display donor promiscuity and can use CMP-Leg5,7Ac2 efficiently enough to transfer Leg5,7Ac2 to their natural acceptor glycans. When the natural activity with CMP-Leg5,7Ac2 is significant but relatively low, an alternate strategy has been to engineer versions with improved activity to transfer Leg5,7Ac2. Importantly, we have found that some bacterial sialyltransferases are very efficient for transferring Leg5,7Ac2 to small synthetic glycans with various aglycones. The two mammalian sialyltransferases that have been tested so far (porcine ST3Gal1 and human ST6Gal1) were found to be more efficient than the bacterial sialyltransferases for the modification of glycoproteins. We provide a review of the sialyltransferases selected to modify different types of glycoconjugates with Leg5,7Ac2, including small synthetic acceptors, glycolipids, and glycoproteins. In the first part, we also propose an optimized biosynthetic pathway for in vitro preparation of the donor CMP-Leg5,7Ac2, based on enzymes selected from two bacteria that naturally produce legionaminic acid.
Asunto(s)
Bacterias/enzimología , Ingeniería Metabólica/métodos , Ácidos Siálicos/biosíntesis , Sialiltransferasas/biosíntesis , Animales , Glicoconjugados/biosíntesis , Glicoconjugados/química , Glicoconjugados/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/química , Polisacáridos/genética , Ácidos Siálicos/química , Ácidos Siálicos/genética , Sialiltransferasas/química , Sialiltransferasas/genética , PorcinosRESUMEN
The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.
Asunto(s)
Aciltransferasas/genética , Campylobacter jejuni/genética , Galactosa/genética , Glicosiltransferasas/genética , Nucleotidiltransferasas/genética , Aciltransferasas/química , Aciltransferasas/metabolismo , Vías Biosintéticas/genética , Campylobacter jejuni/enzimología , Cristalografía por Rayos X , Escherichia coli/genética , Galactosa/química , Galactosa/metabolismo , Glucosa/química , Glucosa/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/genética , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Nucleótidos de Timina/química , Nucleótidos de Timina/metabolismoRESUMEN
Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes' functions. Using capillary electrophoresis (CE), CE-mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs.
Asunto(s)
Vías Biosintéticas/genética , Proteínas de la Membrana/genética , Tannerella forsythia/genética , Genoma Bacteriano/genética , Glicosilación , Interacciones Huésped-Patógeno/genética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Oligosacáridos/genética , Oligosacáridos/metabolismo , Ácidos Siálicos/biosíntesis , Azúcares Ácidos/metabolismo , Tannerella forsythia/enzimología , Tannerella forsythia/patogenicidadRESUMEN
Legionaminic acid, Leg5,7Ac2 , a nonulosonic acid like 5-acetamido neuraminic acid (Neu5Ac, sialic acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac2 has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a deâ novo Leg5,7Ac2 biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over-expression of this deâ novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac2 (≈120â mg L(-1) of culture broth) to be produced. Pure Leg5,7Ac2 could be isolated and converted into CMP-activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac2 enabling study of its role in prokaryotic and eukaryotic glycobiology.
Asunto(s)
Vías Biosintéticas , Campylobacter jejuni/metabolismo , Escherichia coli/metabolismo , Legionella pneumophila/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Saccharomyces cerevisiae/metabolismo , Ácidos Siálicos/metabolismo , Campylobacter jejuni/genética , Escherichia coli/genética , Legionella pneumophila/genética , Ingeniería Metabólica , Saccharomyces cerevisiae/genética , Ácidos Siálicos/genéticaRESUMEN
Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5'-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic acid (Neu5Ac), a sialic acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation ("serum-resistance"). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, legionaminic acid (Leg5Ac7Ac) and pseudaminic acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.
Asunto(s)
Citidina Monofosfato/análogos & derivados , Farmacorresistencia Microbiana/inmunología , Resistencia a Múltiples Medicamentos/inmunología , Gonorrea/inmunología , Ácidos Siálicos/farmacología , Animales , Western Blotting , Proteínas del Sistema Complemento/inmunología , Citidina Monofosfato/farmacología , Ácido N-Acetilneuramínico Citidina Monofosfato/análogos & derivados , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Gonorrea/metabolismo , Humanos , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/metabolismoRESUMEN
Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble ß-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 µM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.
Asunto(s)
Polisacáridos/metabolismo , Ácidos Siálicos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Ingeniería Metabólica , Estructura Molecular , N-Acetil-Lactosamina Sintasa/metabolismo , Polisacáridos/química , Ácidos Siálicos/metabolismoRESUMEN
Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.
Asunto(s)
Glicoconjugados/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferasas/metabolismo , Animales , Conformación de Carbohidratos , Glicoconjugados/química , Modelos Moleculares , Mutación , Pasteurella multocida/enzimología , Photobacterium/enzimología , PorcinosRESUMEN
Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 µM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 µM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 µM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.
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Antibacterianos/farmacología , Flagelos/efectos de los fármacos , Flagelina/antagonistas & inhibidores , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/patogenicidad , Bibliotecas de Moléculas Pequeñas/farmacología , Azúcares Ácidos/metabolismo , Antibacterianos/química , Transporte Biológico , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular , Descubrimiento de Drogas , Flagelos/genética , Flagelos/metabolismo , Flagelina/biosíntesis , Flagelina/genética , Expresión Génica , Glicosilación/efectos de los fármacos , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Ensayos Analíticos de Alto Rendimiento , Simulación del Acoplamiento Molecular , Movimiento/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Interfaz Usuario-Computador , VirulenciaRESUMEN
BACKGROUND: Anandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH). In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide. RESULTS: A putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates. CONCLUSIONS: This study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.
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Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Ácidos Araquidónicos/metabolismo , Dictyostelium/enzimología , Dictyostelium/genética , Endocannabinoides/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Biología Computacional , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Dictyostelium/metabolismo , Escherichia coli/genética , Expresión Génica , Perfilación de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Glycomics which is the study of saccharides and genes responsible for their formation requires the continuous development of rapid and sensitive methods for the identification of glycan structures. It involves glycoanalysis which relies upon the development of methods for determining the structure and interactions of carbohydrates. For the application of functional glycomics to microbial virulence, carbohydrates and their associated metabolic and carbohydrate processing enzymes and respective genes can be identified and exploited as targets for drug discovery, glyco-engineering, vaccine design, and detection and diagnosis of diseases. Glycomics also encompasses the detailed understanding of carbohydrate-protein interactions and this knowledge can be applied to research efforts focused toward the development of vaccines and immunological therapies to alleviate infectious diseases.
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Glicómica/métodos , Espectroscopía de Resonancia Magnética/métodos , Bioquímica/métodos , Carbohidratos/química , Endopeptidasa K/química , Glicopéptidos/química , Glicoproteínas/química , Humanos , Ligandos , Modelos Químicos , Biología Molecular/métodos , Polisacáridos/química , Unión Proteica , Programas InformáticosRESUMEN
Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-beta-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 A resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His(17)-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His(17) functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C-O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.
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Vías Biosintéticas , Campylobacter jejuni/enzimología , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Azúcares Ácidos/metabolismo , Azúcares de Uridina Difosfato/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Biocatálisis , Simulación por Computador , Cristalografía por Rayos X , Helicobacter pylori/enzimología , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , N-Acetilglucosaminiltransferasas/química , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Catch a tiger by the tail: We have demonstrated that by feeding nonmotile mutant C. jejuni bacteria with a neutral azide-labelled pseudaminic acid precursor we can restore their ability to generate functional flagella. The presence of azido-pseudaminic acid on the surface of the flagella provides a bio-orthogonal chemical handle that can be used to modify the flagellar proteins.
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Azidas/química , Campylobacter jejuni/química , Flagelina/química , Azúcares Ácidos/metabolismo , Ingeniería de Proteínas , Coloración y Etiquetado , Azúcares Ácidos/químicaRESUMEN
The sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid, or legion-aminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an alpha2,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.
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Campylobacter jejuni/metabolismo , Citidina Monofosfato/metabolismo , Glucosamina/biosíntesis , Guanosina Difosfato/biosíntesis , Ácidos Siálicos/metabolismo , Guanosina Difosfato/metabolismoRESUMEN
UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5'' stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP+ to oxidize UDP-GlcNAc at C-4'', enabling dehydration. The alpha,beta unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6'' and a proton to C-5''. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5'' position of product during catalysis in D2O. Likewise, PseB catalyzes solvent isotope exchange into the H-5'' position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.