RESUMO
Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.
Assuntos
Escherichia coli/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/biossíntese , Bibliotecas de Moléculas Pequenas , Glicosiltransferases/isolamento & purificação , Glicosiltransferases/metabolismo , Humanos , Lectinas/metabolismo , Polissacarídeos/química , Saccharomyces cerevisiae/metabolismoRESUMO
Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.
Assuntos
Bacteroidetes/metabolismo , Trato Gastrointestinal/microbiologia , Mananas/metabolismo , Modelos Biológicos , Leveduras/química , Animais , Bacteroidetes/citologia , Bacteroidetes/enzimologia , Bacteroidetes/genética , Evolução Biológica , Configuração de Carboidratos , Dieta , Enzimas/genética , Enzimas/metabolismo , Feminino , Loci Gênicos/genética , Vida Livre de Germes , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Masculino , Mananas/química , Manose/metabolismo , Camundongos , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Periplasma/enzimologiaRESUMO
Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.
Assuntos
Antivirais/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Proteínas de Membrana/farmacologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Antivirais/uso terapêutico , Cães , Feminino , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/uso terapêutico , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Oligopeptídeos/química , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Vírion/efeitos dos fármacosRESUMO
Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.
Assuntos
Coronavirus Felino/genética , Peritonite Infecciosa Felina/virologia , Glicoproteína da Espícula de Coronavírus/genética , Motivos de Aminoácidos , Animais , Gatos , Sequência Conservada , Coronavirus Felino/patogenicidade , Fezes/virologia , Mutação , Proteólise , Análise de Sequência de DNA , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
A critical step in the influenza virus replication cycle is the cleavage activation of the HA precursor. Cleavage activation of influenza HA enables fusion with the host endosome, allowing for release of the viral genome into the host cell. To date, studies have determined that HA activation is driven by trypsin-like host cell proteases, as well as yet to be identified bacterial proteases. Although the number of host proteases that can activate HA is growing, there is still uncertainty regarding which secreted proteases are able to support multicycle replication of influenza. In this study, we have determined that the kallikrein-related peptidases 5 and 12 are secreted from the human respiratory tract and have the ability to cleave and activate HA from the H1, H2, and H3 subtypes. Each peptidase appears to have a preference for particular influenza subtypes, with kallikrein 5 cleaving the H1 and H3 subtypes most efficiently and kallikrein 12 cleaving the H1 and H2 subtypes most efficiently. Cleavage analysis using HA cleavage site peptide mimics revealed that the amino acids neighboring the arginine cleavage site affect cleavage efficiency. Additionally, the thrombolytic zymogens plasminogen, urokinase, and plasma kallikrein have all been shown to cleave and activate influenza but are found circulating mainly as inactive precursors. Kallikrein 5 and kallikrein 12 were examined for their ability to activate the thrombolytic zymogens, and both resulted in activation of each zymogen, with kallikrein 12 being a more potent activator. Activation of the thrombolytic zymogens may therefore allow for both direct and indirect activation of the HA of human-adapted influenza viruses by kallikrein 5 and kallikrein 12.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Calicreínas/química , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Cães , Ativação Enzimática , Precursores Enzimáticos , Fibrinolisina/química , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Calicreínas/fisiologia , Cinética , Líquido da Lavagem Nasal , Nariz/enzimologia , Fragmentos de Peptídeos/química , Proteólise , Especificidade por Substrato , Ativador de Plasminogênio Tipo Uroquinase/química , Células VeroRESUMO
A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H7N7/genética , Mutagênese Insercional , Proteínas Virais de Fusão/genética , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Análise Mutacional de DNA , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hidrólise , Vírus da Influenza A Subtipo H7N7/fisiologia , Células Vero , Proteínas Virais de Fusão/metabolismo , Internalização do VírusRESUMO
Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Influenza Humana/virologia , Orthomyxoviridae/fisiologia , Virologia/métodos , Internalização do Vírus , Animais , Membrana Celular/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Orthomyxoviridae/química , Orthomyxoviridae/genéticaRESUMO
Cleavage activation of the hemagglutinin (HA) precursor is an essential step in the influenza virus replication cycle that is driven by host cell proteases. HA cleavage activation is required for virus-endosome membrane fusion and the subsequent release of the influenza virus genome into the cytoplasm. Previous studies have determined that HA cleavage is most likely driven by either membrane-bound or extracellular trypsin-like proteases that reside in the respiratory tract. However, there is still uncertainty regarding which proteases are critical for HA cleavage in vivo. Therefore, further investigation of HA cleavage activation is needed in order to gain insight into the critical proteases involved. Matriptase is a member of the type II transmembrane serine protease family that is highly expressed in a membrane-bound form throughout the respiratory tract. One feature of matriptase is that, once activated, the catalytic domain is secreted into the extracellular space and so serves as a functional extracellular protease. In this study, we have determined that the secreted, catalytic domain of matriptase has the ability to cleave and activate HA from the influenza virus H1 subtype but not the H2 and H3 subtypes. Furthermore, matriptase selectively cleaved the HA of particular strains within the H1 subtype, revealing both subtype and H1 strain specificity. Matriptase was also found to activate thrombolytic zymogens that have been shown to cleave and activate the influenza virus HA. Our data demonstrate that matriptase has the ability to cleave HA directly or indirectly by activating HA-cleaving zymogens.
Assuntos
Orthomyxoviridae/genética , Serina Endopeptidases/metabolismo , Animais , Domínio Catalítico , Bovinos , Chlorocebus aethiops , Precursores Enzimáticos/química , Células HEK293 , Humanos , Influenza Humana/virologia , Orthomyxoviridae/metabolismo , Inibidores de Proteases/química , Sistema Respiratório/virologia , Serina Endopeptidases/química , Especificidade da Espécie , Terapia Trombolítica , Fatores de Tempo , Células VeroRESUMO
Dimerization can be utilized to double the molecular weight of proteins and peptides and potentially increase their avidity of binding to target receptors. These dimerization effects may be utilized to increase in vivo half-lives in a manner similar to PEGylation and may also improve biological activity. In this paper, we report a new strategy for the synthesis of N-terminally linked protein and peptide homodimers utilizing native chemical ligation to conjugate a short dithioester linker to the N-terminal cysteines of protein and peptide monomers to form dimers in a single step. This strategy is general and has been applied to the production of dimers from three recombinantly expressed polypeptides, the IgG binding domain Protein G, an HIV entry inhibitor peptide C37H6, and human interleukin-1 receptor antagonist (IL-1ra). The biological activities of the C37H6 and IL-1ra dimers produced by these methods were retained or even slightly increased when compared to their corresponding monomers.
Assuntos
Ésteres/química , Proteínas do Tecido Nervoso/síntese química , Peptídeos/síntese química , Dimerização , Ésteres/síntese química , Humanos , Imunoglobulina G/química , Estrutura Molecular , Peso Molecular , Proteínas do Tecido Nervoso/química , Peptídeos/químicaRESUMO
Interleukin-1 receptor antagonist is an inhibitor of the pro-inflammatory action of interleukin-1. The gene encoding for interleukin-1 receptor antagonist (IL-1ra) was cloned into a Pichia pastoris expression vector pPICzalphaA (Invitrogen, USA) and transformed into P. pastoris strain SMD1168H. Multi-copy selection of the gene produced a high expressing strain of IL-1ra that produced 17mg/L of total secreted purified protein. The IL-1ra produced in P. pastoris was a mixture of glycosylated and non-glycosylated IL-1ra where 70% of the total protein was glycosylated. SP-Sepharose purification allowed for separation of the two expressed forms of IL-1ra, which permits biochemical investigation of glycosylated and non-glycosylated IL-1ra using one expression system. Mass spectrometric analysis revealed the expression of the full-length protein and that the glycosylated IL-1ra contained high mannose glycoforms that ranged from Man(9)GlcNAc(2) to Man(14)GlcNAc(2).