RESUMO
Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.
Assuntos
Cannabis , Doenças Cardiovasculares , Alucinógenos , Analgésicos , Animais , Agonistas de Receptores de Canabinoides/farmacologia , Dronabinol/farmacologia , Células Endoteliais , Genisteína/farmacologia , Genisteína/uso terapêutico , Inflamação/tratamento farmacológico , Camundongos , Receptor CB1 de Canabinoide , Receptores de CanabinoidesRESUMO
Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non-catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C-terminus region, enabling strong interactions with thrombin exosite-I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic-rich C-terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc-SPPS, hirudin dodecapeptides homologous to the C-terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin-thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.
Assuntos
Hirudinas , Trombina , Tirosina/análogos & derivados , Hirudinas/farmacologia , Hirudinas/química , Hirudinas/metabolismo , Aminoácidos , Peptídeos/farmacologia , Sítios de LigaçãoRESUMO
The outbreak of COVID-19 caused by SARS-CoV-2 has resulted in more than 50 million confirmed cases and over 1 million deaths worldwide as of November 2020. Currently, there are no effective antivirals approved by the Food and Drug Administration to contain this pandemic except the antiviral agent remdesivir. In addition, the trimeric spike protein on the viral surface is highly glycosylated and almost 200,000 variants with mutations at more than 1,000 positions in its 1,273 amino acid sequence were reported, posing a major challenge in the development of antibodies and vaccines. It is therefore urgently needed to have alternative and timely treatments for the disease. In this study, we used a cell-based infection assay to screen more than 3,000 agents used in humans and animals, including 2,855 small molecules and 190 traditional herbal medicines, and identified 15 active small molecules in concentrations ranging from 0.1 nM to 50 µM. Two enzymatic assays, along with molecular modeling, were then developed to confirm those targeting the virus 3CL protease and the RNA-dependent RNA polymerase. Several water extracts of herbal medicines were active in the cell-based assay and could be further developed as plant-derived anti-SARS-CoV-2 agents. Some of the active compounds identified in the screen were further tested in vivo, and it was found that mefloquine, nelfinavir, and extracts of Ganoderma lucidum (RF3), Perilla frutescens, and Mentha haplocalyx were effective in a challenge study using hamsters as disease model.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Adulto , Animais , Antivirais/química , Antivirais/uso terapêutico , COVID-19/epidemiologia , COVID-19/virologia , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Reposicionamento de Medicamentos/métodos , Feminino , Humanos , Masculino , Pandemias , Extratos Vegetais/farmacologia , SARS-CoV-2/genética , Células VeroRESUMO
Multicolor labeling for monitoring the intracellular localization of the same target type in the native environment using chemical fluorescent dyes is a challenging task. This approach requires both bioorthogonal and biocompatible ligations with an excellent fluorescence signal-to-noise ratio. Here, we present a metabolic glycan labeling technique that uses homemade fluorogenic dyes to investigate glycosylation patterns in live cells. These dyes allowed us to demonstrate rapid and efficient simultaneous multilabeling of glycoconjugates with minimum fluorescence noise. Our results demonstrate that this approach is capable of not only probing sialylation and GlcNAcylation in cells but also specifically labeling the cell-surface and intracellular sialylated glycoconjugates in live cells. In particular, we performed site-specific dual-channel fluorescence imaging of extra and intracellular sialylated glycans in HeLa and PC9 cancer cells as well as identified fluorescently labeled sialylated glycoproteins and glycans by a direct enrichment approach combined with an MS-based proteomic analysis in the same experiment. In conclusion, this study provides multilabeling tools in cellular systems for simultaneous site-specific glycan imaging and glycoproteomic analysis to study potential cancer- and disease-associated glycoconjugates.
Assuntos
Glicoproteínas , Proteômica , Humanos , Corantes Fluorescentes/metabolismo , Glicoconjugados/metabolismo , Polissacarídeos/metabolismoRESUMO
Fungal predatory behavior on nematodes has evolved independently in all major fungal lineages. The basidiomycete oyster mushroom Pleurotus ostreatus is a carnivorous fungus that preys on nematodes to supplement its nitrogen intake under nutrient-limiting conditions. Its hyphae can paralyze nematodes within a few minutes of contact, but the mechanism had remained unclear. We demonstrate that the predator-prey relationship is highly conserved between multiple Pleurotus species and a diversity of nematodes. To further investigate the cellular and molecular mechanisms underlying rapid nematode paralysis, we conducted genetic screens in Caenorhabditis elegans and isolated mutants that became resistant to P. ostreatus We found that paralysis-resistant mutants all harbored loss-of-function mutations in genes required for ciliogenesis, demonstrating that the fungus induced paralysis via the cilia of nematode sensory neurons. Furthermore, we observed that P. ostreatus caused excess calcium influx and hypercontraction of the head and pharyngeal muscle cells, ultimately resulting in rapid necrosis of the entire nervous system and muscle cells throughout the entire organism. This cilia-dependent predatory mechanism is evolutionarily conserved in Pristionchus pacificus, a nematode species estimated to have diverged from C. elegans 280 to 430 million y ago. Thus, P. ostreatus exploits a nematode-killing mechanism that is distinct from widely used anthelmintic drugs such as ivermectin, levamisole, and aldicarb, representing a potential route for targeting parasitic nematodes in plants, animals, and humans.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Micotoxinas/toxicidade , Pleurotus/fisiologia , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Cálcio/metabolismo , Cílios/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Necrose/induzido quimicamenteRESUMO
BACKGROUND: Colorectal cancer (CRC) is a worldwide cancer with rising annual incidence. New medications for patients with CRC are still needed. Recently, fluorescent chemical probes have been developed for cancer imaging and therapy. Signal transducer and activator of transcription 1 (STAT1) has complex functions in tumorigenesis and its role in CRC still needs further investigation. METHODS: RNA sequencing datasets in the NCBI GEO repository were analyzed to investigate the expression of STAT1 in patients with CRC. Xenograft mouse models, tail vein injection mouse models, and azoxymethane/dextran sodium sulfate (AOM/DSS) mouse models were generated to study the roles of STAT1 in CRC. A ligand-based high-throughput virtual screening approach combined with SWEETLEAD chemical database analysis was used to discover new STAT1 inhibitors. A newly designed and synthesized fluorescently labeled 4',5,7-trihydroxyisoflavone (THIF) probe (BODIPY-THIF) elucidated the mechanistic actions of STAT1 and THIF in vitro and in vivo. Colonosphere formation assay and chick chorioallantoic membrane assay were used to evaluate stemness and angiogenesis, respectively. RESULTS: Upregulation of STAT1 was observed in patients with CRC and in mouse models of AOM/DSS-induced CRC and metastatic CRC. Knockout of STAT1 in CRC cells reduced tumor growth in vivo. We then combined a high-throughput virtual screening approach and analysis of the SWEETLEAD chemical database and found that THIF, a flavonoid abundant in soybeans, was a novel STAT1 inhibitor. THIF inhibited STAT1 phosphorylation and might bind to the STAT1 SH2 domain, leading to blockade of STAT1-STAT1 dimerization. The results of in vitro and in vivo binding studies of THIF and STAT1 were validated. The pharmacological treatment with BODIPY-THIF or ablation of STAT1 via a CRISPR/Cas9-based strategy abolished stemness and angiogenesis in CRC. Oral administration of BODIPY-THIF attenuated colitis symptoms and tumor growth in the mouse model of AOM/DSS-induced CRC. CONCLUSIONS: This study demonstrates that STAT1 plays an oncogenic role in CRC. BODIPY-THIF is a new chemical probe inhibitor of STAT1 that reduces stemness and angiogenesis in CRC. BODIPY-THIF can be a potential tool for CRC therapy as well as cancer cell imaging.
Assuntos
Neoplasias Colorretais , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Oncogenes , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismoRESUMO
A highly diastereoselective indium-mediated allylation of 2-N-acetyl glycosyl sulfinylimines in brine under mild reaction conditions is reported. The method allows the achievement of a highly remarkable dichotomous selectivity for substrates, providing a single diastereoisomer of the product in 80-98% yield. With chiral (S)-homoallylic sulfinamide (RS)-5 and (RS)-8 formed as key intermediates, two potent anti-influenza agents, zanamivir and zanaphosphor, were synthesized in 50% and 41% overall yields, respectively.
Assuntos
Índio , Sais , Antivirais/farmacologiaRESUMO
N-Glycans are major constituents of several cellular glycoproteins. One-pot strategies for the synthesis of N-glycans are crucial for the rapid generation of pure samples to determine their biological functions. Herein, we describe a double one-pot strategy for the synthesis of N-glycans assisted by an IM-MS analysis approach for rapid screening of optimized glycosylation reaction conditions. This research includes triflate-mediated direct ß-mannosylation and tandem glycosylation in a one-pot strategy for the synthesis of the challenging N-linked trisaccharide core ß-5. Furthermore, a one-pot sequential glycosylation of the N-linked trisaccharide core 7 furnishes diverse high-mannose type N-glycans with excellent stereo- and regioselectivities. In particular, ion mobility-mass spectrometry-based quantitative analysis is applied to identify the stereo- and regioselective outcomes of the crude reaction mixtures to develop a highly efficient one-pot protocol.
Assuntos
Oligossacarídeos , Polissacarídeos , Glicosilação , Espectrometria de Massas , Oligossacarídeos/química , Polissacarídeos/química , TrissacarídeosRESUMO
Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.
Assuntos
Fator de Crescimento Epidérmico , Simulação de Dinâmica Molecular , Sistemas CRISPR-Cas/genética , Análise por Conglomerados , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Células HeLa , Humanos , Ligantes , Neuraminidase/genética , Neuraminidase/metabolismo , FosforilaçãoRESUMO
Secondary metabolites are structurally diverse natural products (NPs) and have been widely used for medical applications. Developing new tools to enrich NPs can be a promising solution to isolate novel NPs from the native and complex samples. Here, we developed native and deuterated chemoselective labeling probes to target phenol-containing glycopeptides by the ene-type labeling used in proteomic research. The clickable azido-linker was included for further biotin functionalization to facilitate the enrichment of labeled substrates. Afterward, our chemoselective method, in conjunction with LC-MS and MSn analysis, was demonstrated in bacterial cultures. A vancomycin-related phenol-containing glycopeptide was labeled and characterized by our labeling strategy, showing its potential in glycopeptide discovery in complex environments.
Assuntos
FenolRESUMO
Protein O-glycosylation by attachment of ß-N-acetylglucosamine (GlcNAc) to the Ser or Thr residue is a major posttranslational glycosylation event and is often associated with protein folding, stability, and activity. The methylation of histone H3 at Lys-27 catalyzed by the methyltransferase EZH2 was known to suppress gene expression and cancer development, and we previously reported that the O-GlcNAcylation of EZH2 at S76 stabilized EZH2 and facilitated the formation of H3K27me3 to inhibit tumor suppression. In this study, we employed a fluorescence-based method of sugar labeling combined with mass spectrometry to investigate EZH2 glycosylation and identified five O-GlcNAcylation sites. We also find that mutation of one or more of the O-GlcNAcylation sites S73A, S76A, S84A, and T313A in the N-terminal region decreases the stability of EZH2, but does not affect its association with the PRC2 components SUZ12 and EED. Mutation of the C-terminal O-GlcNAcylation site (S729A) in the catalytic domain of EZH2 abolishes the di- and trimethylation activities, but not the monomethylation of H3K27, nor the integrity of the PRC2/EZH2 core complex. Our results show the effect of individual O-GlcNAcylation sites on the function of EZH2 and suggest an alternative approach to tumor suppression through selective inhibition of EZH2 O-GlcNAcylation.
Assuntos
Acetilglucosamina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Mutação de Sentido Incorreto , Acetilglucosamina/química , Acetilglucosamina/genética , Substituição de Aminoácidos , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste/química , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Estabilidade Enzimática , Glicosilação , Humanos , Domínios ProteicosRESUMO
For decades, high-resolution 1H NMR spectroscopy has been routinely utilized to analyze both naturally occurring steroid hormones and synthetic steroids, which play important roles in regulating physiological functions in humans. Because the 1H signals are inevitably superimposed and entangled with various JH-H splitting patterns, such that the individual 1H chemical shift and associated JH-H coupling identities are hardly resolved. Given this, applications of thess information for elucidating steroidal molecular structures and steroid/ligand interactions at the atomic level were largely restricted. To overcome, we devoted to unraveling the entangled JH-H splitting patterns of two similar steroidal compounds having fully unsaturated protons, i.e., androstanolone and epiandrosterone (denoted as 1 and 2, respectively), in which only hydroxyl and ketone substituents attached to C3 and C17 were interchanged. Here we demonstrated that the JH-H values deduced from 1 and 2 are universal and applicable to other steroids, such as testosterone, 3ß, 21-dihydroxygregna-5-en-20-one, prednisolone, and estradiol. On the other hand, the 1H chemical shifts may deviate substantially from sample to sample. In this communication, we propose a simple but novel scheme for resolving the complicate JH-H splitting patterns and 1H chemical shifts, aiming for steroidal structure determinations.
Assuntos
Espectroscopia de Ressonância Magnética , Acoplamento Oxidativo , Esteroides/química , Modelos Biológicos , Conformação Molecular , Estrutura MolecularRESUMO
A series ofN-acyl glucosamine-bearingtriterpenoidsaponins has been synthesized with cytotoxic activities evaluated against HL-60, PC-3, HCT-116, and CT-26 tumor cells. Saponins incorporated anoleanolic acid (OA) triterpenoidal core exhibited the highest cytotoxic activity. To study the influence of the lengths of acyl-carbon chain onN-position of glucosamine, cells were treated with28-propargylamides and then reacted with an azido-fluorogenic probe under CuAACclickreactions to visualize the intact distributions of these compounds by confocal microscopy and flow cytometry; it was found that cytotoxic-active compounds (30-32) located in the cytosol and inactivecompounds bearing longer carbon chains (33-35) were impenetrable across cell membranes.Our study demonstrated the defined lipophilic acyl-carbon chain length can precisely regulate thecytotoxic activityof saponins, which is useful for the future development of cytotoxic agents.Furthermore, using quantitative proteomics and immunolabeling,the mechanism ofcytotoxicity induced by the synthetic saponin after membrane penetration could be a result of activation of death receptor pathway and inhibition of PI3K/Akt/mTOR pathway.
Assuntos
Antineoplásicos/farmacologia , Glucosamina/farmacologia , Ácido Oleanólico/farmacologia , Saponinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glucosamina/química , Humanos , Estrutura Molecular , Ácido Oleanólico/química , Saponinas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Influenza is a long-standing health problem. For treatment of seasonal flu and possible pandemic infections, there is a need to develop new anti-influenza drugs that have good bioavailability against a broad spectrum of influenza viruses, including the resistant strains. Relenza™ (zanamivir), Tamiflu™ (the phosphate salt of oseltamivir), Inavir™ (laninamivir octanoate) and Rapivab™ (peramivir) are four anti-influenza drugs targeting the viral neuraminidases (NAs). However, some problems of these drugs should be resolved, such as oral availability, drug resistance and the induced cytokine storm. Two possible strategies have been applied to tackle these problems by devising congeners and conjugates. In this review, congeners are the related compounds having comparable chemical structures and biological functions, whereas conjugate refers to a compound having two bioactive entities joined by a covalent bond. The rational design of NA inhibitors is based on the mechanism of the enzymatic hydrolysis of the sialic acid (Neu5Ac)-terminated glycoprotein. To improve binding affinity and lipophilicity of the existing NA inhibitors, several methods are utilized, including conversion of carboxylic acid to ester prodrug, conversion of guanidine to acylguanidine, substitution of carboxylic acid with bioisostere, and modification of glycerol side chain. Alternatively, conjugating NA inhibitors with other therapeutic entity provides a synergistic anti-influenza activity; for example, to kill the existing viruses and suppress the cytokines caused by cross-species infection.
Assuntos
Antivirais/química , Desenvolvimento de Medicamentos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Neuraminidase/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Desenho de Fármacos , HumanosRESUMO
We have designed a low fluorescent azido-BODIPY-based probe AzBOCEt (Az10) that undergoes copper(I)-catalyzed 1,3-dipolar cycloadditions with alkynes to yield strongly fluorescent triazole derivatives. The fluorescent quantum yield of a triazole product T10 is enhanced by 52-fold as compared to AzBOCEt upon excitation at a wavelength above 500 nm. Quantum mechanical calculations indicate that the increase in fluorescence upon triazole formation is due to the lowering of the HOMO energy level of the aryl moiety to reduce the process of acceptor photoinduced electron transfer. AzBOCEt is shown to label alkyne-functionalized proteins in vitro and glycoproteins in cells with excellent selectivity, and enables cell imaging and visualization of glycoconjugates in alkynyl-saccharide-treated cells at extremely low concentration (0.1 µM). Furthermore, the alkyne-tagged glycoproteins from cell lysates can be directly detected with AzBOCEt in gel electrophoresis.
Assuntos
Azidas/química , Compostos de Boro/química , Corantes Fluorescentes/química , Triazóis/síntese química , Linhagem Celular Tumoral , Cobre/química , Ciclização , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Neoplasias Pulmonares/patologiaRESUMO
This study demonstrates that compounds 1-4 from an extract of Plectranthus amboinicus inhibit the binding of AP-1 to its consensus DNA sequence. Thymoquinone (5) was further identified as a nonpolar ingredient from the hexane extract of P. amboinicus to suppress the expression of lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-α). We then synthesized 2-alkylidenyl-4-cyclopentene-1,3-diones as the designed biomimetics of thymoquinone, and found that compounds 8a, 8b and 8d were more potent TNF-α inhibitors.
Assuntos
Anti-Inflamatórios/farmacologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Óleos de Plantas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Humanos , Extratos Vegetais/química , Óleos de Plantas/química , Óleos de Plantas/isolamento & purificaçãoRESUMO
The assembly and disassembly of biomolecular condensates are crucial for the subcellular compartmentalization of biomolecules in the control of cellular reactions. Recently, a correlation has been discovered between the phase transition of condensates and their maturation (aggregation) process in diseases. Therefore, modulating the phase of condensates to unravel the roles of condensation has become a matter of interest. Here, we create a peptide-based phase modulator, JSF1, which forms droplets in the dark and transforms into amyloid-like fibrils upon photoinitiation, as evidenced by their distinctive nanomechanical and dynamic properties. JSF1 is found to effectively enhance the condensation of purified fused in sarcoma (FUS) protein and, upon light exposure, induce its fibrilization. We also use JSF1 to modulate the biophysical states of FUS condensates in live cells and elucidate the relationship between FUS phase transition and FUS proteinopathy, thereby shedding light on the effect of protein phase transition on cellular function and malfunction.
Assuntos
Peptídeos , Transição de Fase , Proteína FUS de Ligação a RNA , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Humanos , Peptídeos/química , Peptídeos/metabolismo , Amiloide/metabolismo , Amiloide/química , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , LuzRESUMO
Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its Km and kcat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.
Assuntos
alfa-L-Fucosidase , alfa-L-Fucosidase/metabolismo , alfa-L-Fucosidase/química , Humanos , Glicosilação , Engenharia de Proteínas , Prevotella/enzimologia , CinéticaRESUMO
The exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis have recently been shown to include (1,3;1,4)-ß-D-glucans. In the present study, we examined another Clostridia bacterium Clostridium ventriculi that has long been considered to contain abundant amounts of cellulose in its exopolysaccharides. We treated alcohol insoluble residues of C. ventriculi that include the exopolysaccharides with the enzyme lichenase that specifically hydrolyses (1,3;1,4)-ß-D-glucans, and examined the oligosaccharides released. This showed the presence of (1,3;1,4)-ß-D-glucans, which may have previously been mistaken for cellulose. Through genomic analysis, we identified the two family 2 glycosyltransferase genes CvGT2-1 and CvGT2-2 as possible genes encoding (1,3;1,4)-ß-D-glucan synthases. Gain-of-function experiments in the yeast Saccharomyces cerevisiae demonstrated that both of these genes do indeed encode (1,3;1,4)-ß-D-glucan synthases.