RESUMO
Cell-free protein synthesis is an attractive method for generating enzyme/protein variants for simplified functional analysis as both in vitro protein expression and analysis may often be performed in a single vial or well. Today, researchers may choose from multiple commercial cell lysate products or reconstituted systems which are compatible with either mRNA, linear DNA or plasmid DNA templates. Here we provide guidance for optimal design of the genetic elements within linear and plasmid DNA templates which are required to reliably practice cell-free protein synthesis. Protocols are presented for generating linear DNA templates, and data are presented to show that linear DNA templates may in many cases provide robust protein yields even when employing an Escherichia coli lysate for protein synthesis. Finally, the use of linear DNA templates makes it possible to bypass all cell cultivation steps and proceed from PCR amplification of synthetic DNA to generation of target protein in a matter of hours.
Assuntos
Escherichia coli , Biossíntese de Proteínas , Sistema Livre de Células/metabolismo , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/genética , RNA Mensageiro/metabolismo , Moldes GenéticosRESUMO
The use of sequential exoglycosidase digestion of oligosaccharides followed by LC-FLD, LC-MS or CE analysis provides detailed carbohydrate structural information. Highly specific exoglycosidases cleave monosaccharides from the nonreducing end of an oligosaccharide and yield information about the linkage, stereochemistry and configuration of the anomeric carbon. Here we use combinations of exoglycosidases to precisely characterize glycans on the Fc domain of therapeutic antibodies and dimeric fusion proteins. The workflow described includes glycan release with Rapid™ PNGase F (NEB #P0710), direct labeling of released glycans with procainamide (PCA) or 2-aminobenzamide (2AB), cleanup of labeled glycans and a 3 h enzymatic digestion with exoglycosidases. This protocol is designed for completion within an 8 h time frame to allow for subsequent LC-FLD, LC-MS, or CE analysis overnight.
Assuntos
Anticorpos Monoclonais/análise , Glicoproteínas/análise , Glicosídeo Hidrolases/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Configuração de Carboidratos , Cromatografia Líquida de Alta Pressão , Corantes Fluorescentes/química , Fluorometria , Glicosilação , Hidrólise , Espectrometria de Massas , Procainamida/química , Proteínas Recombinantes de Fusão/análise , Projetos de Pesquisa , Especificidade por Substrato , Fluxo de Trabalho , ortoaminobenzoatos/químicaRESUMO
Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.
Assuntos
Simulação de Dinâmica Molecular , Polissacarídeos/análise , Receptor para Produtos Finais de Glicação Avançada/química , Glicosilação , Humanos , Pichia/genética , Pichia/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Receptor para Produtos Finais de Glicação Avançada/metabolismoRESUMO
Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG - the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.
Assuntos
Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Animais , Cães , Glicoproteínas/sangue , Glicosilação , Humanos , Polissacarídeos/sangueRESUMO
Transmembrane mucins are highly O-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin N-glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with N-acetyllactosamine, a preferred galectin ligand. In N-glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi N-acetylglucosaminyltransferase I gene (MGAT1) induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for N-glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for N-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.
Assuntos
Antígeno Ca-125/metabolismo , Córnea/metabolismo , Células Epiteliais/metabolismo , Galectina 3/metabolismo , Glicocálix/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Sanguíneas , Antígeno Ca-125/genética , Linhagem Celular Transformada , Galectina 3/genética , Galectinas , Glicocálix/genética , Glicosilação , Humanos , Proteínas de Membrana/genética , Estabilidade ProteicaRESUMO
Glycosylation affects the efficacy, safety and pharmacokinetics/pharmacodynamics properties of therapeutic monoclonal antibodies (mAbs), and glycoengineering is now being used to produce mAbs with improved efficacy. In this work, a glycoengineered version of rituximab was produced by chemoenzymatic modification to generate human-like N-glycosylation with α 2,6 linked sialic acid. This modified rituximab was comprehensively characterized by liquid chromatography-mass spectrometry and compared to commercially available rituximab. As anticipated, the majority of N-glycans were converted to α 2,6 linked sialic acid, in contrast to CHO-produced rituximab, which only contains α 2,3 linked sialic acid. Typical posttranslational modifications, such as pyro-glutamic acid formation at the N-terminus, oxidation at methionine, deamidation at asparagine, and disulfide linkages were also characterized in both the commercial and glycoengineered mAbs using multiple enzymatic digestion and mass spectrometric analysis. The comparative study reveals that the glycoengineering approach does not cause any additional posttranslational modifications in the antibody except the specific transformation of the glycoforms, demonstrating the mildness and efficiency of the chemoenzymatic approach for glycoengineering of therapeutic antibodies.
Assuntos
Espectrometria de Massas , Ácido N-Acetilneuramínico/química , Rituximab/química , Animais , Células CHO , Cricetinae , Cricetulus , Glicosilação , HumanosRESUMO
N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.
Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Corantes Fluorescentes/química , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos/análise , Polissacarídeos/química , Animais , Anticorpos Monoclonais/química , Cromatografia Líquida , Glicoproteínas/química , Glicosilação , Humanos , Imunoglobulina G , Indicadores e Reagentes/química , Camundongos , Modelos Moleculares , Conformação Proteica , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Fatores de TempoRESUMO
UNLABELLED: The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., ß-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, ß-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is ß-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) ß-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of ß-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, ß-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls. IMPORTANCE: We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain ß-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, ß-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of ß-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.
Assuntos
Parede Celular/química , Eimeria/química , Oocistos/química , Toxoplasma/química , beta-Glucanas/análise , Antiprotozoários/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Parede Celular/ultraestrutura , Equinocandinas/metabolismo , Eimeria/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Oocistos/ultraestrutura , Toxoplasma/ultraestruturaRESUMO
Enzymatic deglycosylation followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples. Specific glycosidases were used to remove sugars from glycoproteins in a controlled fashion leaving the protein core intact; the resulting change in molecular weight could be detected as shifts in gel mobility. Alternatively, glycan-sensitive reagents were used to visualize the intensity of glycoprotein bands before and after enzyme treatment. The ease of use of these techniques, which require only basic laboratory instrumentation and reagents, makes them the methodology of choice for initial glycobiology studies. These protocols are also well suited to screen for optimal expression conditions, since multiple glycoprotein samples can be processed at once.
Assuntos
Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Acetilglucosamina/metabolismo , Acetilglucosaminidase/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Eletroforese em Gel de Poliacrilamida , Subunidade alfa de Hormônios Glicoproteicos/química , Subunidade alfa de Hormônios Glicoproteicos/genética , Subunidade alfa de Hormônios Glicoproteicos/isolamento & purificação , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Glicosilação , Células HeLa , Humanos , Membranas Artificiais , Camundongos , Polissacarídeos/metabolismo , Polivinil/química , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , alfa-Cristalinas/química , alfa-Cristalinas/genética , alfa-Cristalinas/isolamento & purificação , alfa-Cristalinas/metabolismoRESUMO
Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms. The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGß), which carries two N-glycans and four O-glycans. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples. Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), ß1-4 Galactosidase, and ß-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans. Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and ß1-3 Galactosidase), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans. SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 µg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.
Assuntos
Glicoproteínas/biossíntese , Glicosídeo Hidrolases/metabolismo , Proteínas/metabolismo , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Glicosilação , Humanos , Modelos Moleculares , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.
Assuntos
Asparagina/química , Entamoeba histolytica/metabolismo , Regulação da Expressão Gênica , Oligossacarídeos/química , Polissacarídeos/química , Animais , Concanavalina A/química , Retículo Endoplasmático/metabolismo , Glicosilação , Lectinas/química , Espectrometria de Massas/métodos , Modelos Químicos , Conformação Molecular , Dobramento de Proteína , alfa-Manosidase/químicaRESUMO
Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.
Assuntos
Candida/fisiologia , Parede Celular/fisiologia , Neutrófilos/fisiologia , Fagocitose/fisiologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Humanos , Neutrófilos/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , beta-Glucanas/farmacologiaRESUMO
Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a approximately 60% decrease in CSIII activity, which is correlated with a approximately 30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.
Assuntos
Quitina Sintase/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Prenilação de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quitina Sintase/genética , Quitina Sintase/isolamento & purificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Frações SubcelularesRESUMO
Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an approximately 30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans.
Assuntos
Quitina/metabolismo , Entamoeba/metabolismo , Lectinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Quitinases/química , Quitinases/metabolismo , Sequência Conservada , Cisteína Endopeptidases/metabolismo , Entamoeba/genética , Genoma de Protozoário , Lectinas/química , Lectinas/genética , Espectrometria de Massas , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismoRESUMO
The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, contains chitin, which is a homopolymer of beta-1, 4-linked N-acetyl-glucosamine (GlcNAc). In fungi and in bacteria that make nodulation factors, chitin deacetylases make chitosan, which is a mixture of GlcNAc and glucosamine and so has a positive charge. The activity of an Ei chitin deacetylase was revealed by a 3-4-fold increase in released GlcNAc when deproteinated cyst walls were chemically acetylated prior to treatment with a commerical chitinase. Because this chitinase releases GlcNAc but not GlcN, increases in released GlcNAc after acetylation suggested the presence of chitosan in Ei cyst walls. Five putative Ei and Eh chitin deacetylase genes resembled those of fungi and bacteria. A predicted Eh chitin deacetylase matched closely the three-dimensional structure of a Bacillus subtilis peptiodglycan deacetylase. A recombinant Eh chitin deacetylase, expressed in Saccharomyces cerevisiae, deacetylated chitooligosaccharides in vitro. These results are consistent with the idea that Ei chitin deacetylases modify chitin to produce chitosan in the Ei cyst wall.
Assuntos
Amidoidrolases/metabolismo , Quitosana/metabolismo , Entamoeba/metabolismo , Proteínas de Protozoários/metabolismo , Acetilação , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Animais , Quitosana/análise , Entamoeba/enzimologia , Entamoeba/genética , Genes de Protozoários , Dados de Sequência Molecular , Fenótipo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes , Alinhamento de SequênciaRESUMO
Mutants of Kluyveromyces lactis denominated vga (vanadate glycosylation affected) bear various combinations of glycosylation and cell-wall defects. The vga3 mutation of K. lactis was mapped in the KlOCH1 gene, encoding the functional homologue of the Saccharomyces cerevisiaealpha1,6-mannosyltransferase. Quantitative analysis of cell-wall components indicated a noticeable increase of chitin and beta1,6-glucans and a severe decrease of mannoproteins in the mutant cells as compared with the wild-type counterparts. Fine-structure determination of the beta1,6-glucan polymer indicated that, in the vga3-1 strain, the beta1,6-glucans are shorter and have more branches than in the wild-type strain. This suggests that cell-wall remodelling changes take place in K. lactis in the presence of glycosylation defects. Moreover, the vga3 cells showed a significantly improved capability of secreting heterologous proteins. Such a capability, accompanied by the highly reduced N-glycosylation, may be of biotechnological interest, especially when hyper-glycosylation of recombinant products must be avoided.
Assuntos
Parede Celular/fisiologia , Kluyveromyces/enzimologia , Manosiltransferases/fisiologia , Transporte Proteico , Sequência de Aminoácidos , Parede Celular/química , Parede Celular/metabolismo , Quitina/análise , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Deleção de Genes , Teste de Complementação Genética , Glicosilação , Kluyveromyces/fisiologia , Manosiltransferases/química , Manosiltransferases/genética , Glicoproteínas de Membrana/análise , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , beta-Glucanas/análise , beta-Glucanas/químicaRESUMO
The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.
Assuntos
Asparagina , Dolicóis/metabolismo , Variação Genética , Glicosiltransferases/metabolismo , Polissacarídeos/química , Animais , Bactérias/enzimologia , Bactérias/genética , Biologia Computacional , Evolução Molecular , Glicopeptídeos/biossíntese , Glicosiltransferases/genética , Humanos , Especificidade da EspécieRESUMO
Purified endoglucanases have been used to determine the composition of Schizosaccharomyces pombe cell wall. This structure has been traditionally studied after isolating its components (mannoproteins, alpha1,3-glucan, beta1,3-glucan, and a branched beta-glucan) with hot alkali. Instead, we sequentially removed the polysaccharides by digesting with endo-beta1,3-glucanase and with a novel endo-alpha1,3-glucanase (mutanase). After this gentle isolation we observed that a branched beta1,3-beta1,6-glucan is much more abundant than previously described. By scaling-up the new protocol we prepared large amounts of the highly branched glucan and determined its structural features. We have named this highly branched beta-glucan diglucan, reflecting its two types of beta linkages. We have also identified an insoluble endoglucanase-resistant type of 1,3-linked glucan present in S. pombe cell walls. We redefined the wall composition of S. pombe vegetative cells by this new method. Finally, to demonstrate its application, we determined the cell wall composition of known mutant strains.
Assuntos
Parede Celular/química , Endo-1,3(4)-beta-Glucanase/metabolismo , Glucanos/química , Glucanos/metabolismo , Schizosaccharomyces/citologia , Sequência de Carboidratos , Parede Celular/metabolismo , Diálise , Espectroscopia de Ressonância MagnéticaRESUMO
The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall beta-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of beta-1,6-glucan and more chitin and beta-1,3-glucan and less mannoprotein than the WT. The remaining beta-1,6-glucan, about 20% of WT levels, exhibits a beta-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a beta-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall beta-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.
Assuntos
Candida albicans/genética , Candida albicans/patogenicidade , Parede Celular/metabolismo , Glicoproteínas/genética , Glicoproteínas/fisiologia , Hifas/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Alelos , Animais , Proliferação de Células , Quitina/metabolismo , Células Epiteliais/microbiologia , Deleção de Genes , Teste de Complementação Genética , Glucosiltransferases/metabolismo , Homozigoto , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Mutação , Oligossacarídeos/química , Filogenia , Saccharomyces cerevisiae/metabolismo , Sensibilidade e Especificidade , Temperatura , Fatores de Tempo , VirulênciaRESUMO
Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.