RESUMEN
The emergence of coronavirus disease 2019 (COVID-19) posed a major challenge to healthcare systems worldwide, especially as mutations in the culprit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) complicated the development of vaccines and antiviral drugs. Therefore, the search for natural products with broad anti-SARS-CoV-2 capabilities is an important option for the prevention and treatment of similar infectious diseases. Lectins, which are widely recognized as antiviral agents, could contribute to the development of anti-SARS-CoV-2 drugs. This study evaluated the binding affinity of six lectins (including the cyanobacterial lectin from Microcystis viridis NIES-102 (MVL), and Jacalin, a lectin from the breadfruit, Artocarpus altilis) to the receptor binding domain (RBD) of the spike protein on the original (wild) SARS-CoV-2 and three of its mutants: Alpha, Delta, and Omicron. MVL and Jacalin showed distinct binding affinity to the RBDs of the four SARS-CoV-2 strains. The remaining four lectins (DB1, ConA, PHA-M and CSL3) showed no such binding affinity. Although the glycan specificities of MVL and Jacalin were different, they showed the same affinity for the spike protein RBDs of the four SARS-CoV-2 strains, in the order of effectiveness Alpha > Delta > original > Omicron. The verification of glycan-specific inhibition revealed that both lectins bind to RBDs by glycan-specific recognition, but, in addition, MVL binds to RBDs through protein-protein interactions.
Asunto(s)
Lectinas , Microcystis , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Lectinas/metabolismo , Lectinas/química , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Microcystis/metabolismo , Humanos , COVID-19/virología , COVID-19/metabolismo , Antivirales/farmacología , Antivirales/metabolismo , Dominios y Motivos de Interacción de Proteínas , Cianobacterias/metabolismo , Lectinas de Plantas/metabolismo , Lectinas de Plantas/química , Sitios de Unión , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , MutaciónRESUMEN
A disintegrin and metalloproteinase (ADAM) family proteins are a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell surface protein ectodomains, including amyloid precursor protein (APP). Human ADAM 9, 10, and 17 proteolyze APPs and produce non-amyloid-genic p3 peptides, instead of neurotoxic amyloid-ß peptides (Aßs; Aß40 and Aß42), which form fibrils and accumulate in the brain of patients with Alzheimer's disease (AD). The ADAM family is closely related to snake venom metalloproteinases (SVMPs), which are derived from ancestral ADAMs but act as soluble proteinases. To test the therapeutic potential of SVMPs, we purified SVMPs from Protobothrops flavoviridis venom using metal ion affinity and pooled into a cocktail. Thus, 9 out of 11 SVMPs in the P. flavoviridis genome were identified in the cocktail. SVMPs inhibited Aß secretion when added to human cell culture medium without affecting APP proteolysis. SVMPs degraded synthetic Aß40 and Aß42 peptides at the same cleavage site (α-site of APP) as ADAM9, 10, and 17. SVMPs did not degrade Aß fibrils but interfered with their formation, assessed using thioflavin-T. Thus, SVMPs have therapeutic potential for AD as an Aß-degrading protease, and the finding adds to the discovery of bioactive peptides from venoms as novel therapeutics.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Humanos , Ponzoñas , Proteolisis , Encéfalo , Proteínas de la Membrana , Proteínas ADAMRESUMEN
Amyloid-ß peptides (Aßs) are produced via cleavage of the transmembrane region of the amyloid precursor protein (APP) by γ-secretase and are responsible for Alzheimer's disease. Familial Alzheimer's disease (FAD) is associated with APP mutations that disrupt the cleavage reaction and increase the production of neurotoxic Aßs, i.e., Aß42 and Aß43. Study of the mutations that activate and restore the cleavage of FAD mutants is necessary to understand the mechanism of Aß production. In this study, using a yeast reconstruction system, we revealed that one of the APP FAD mutations, T714I, severely reduced the cleavage, and identified secondary APP mutations that restored the cleavage of APP T714I. Some mutants were able to modulate Aß production by changing the proportions of Aß species when introduced into mammalian cells. Secondary mutations include proline and aspartate residues; proline mutations are thought to act through helical structural destabilization, while aspartate mutations are thought to promote interactions in the substrate binding pocket. Our results elucidate the APP cleavage mechanism and could facilitate drug discovery.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide , Animales , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico/genética , Mutación , Prolina/genéticaRESUMEN
N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.
Asunto(s)
Poríferos , Receptores de Trombopoyetina , Animales , Lectinas , Poríferos/metabolismo , Receptores de Trombopoyetina/metabolismo , Azúcares , TrombopoyetinaRESUMEN
Genes encoding snake venom toxins have been studied extensively. However, genes involved in the modification and functioning of venom proteins are little known. Protobothrops is a genus of pit vipers, which are venomous and inhabit the Nansei (Southwest) islands of Japan, Taiwan China, Vietnam, Thailand, Myanmar, Nepal, Bhutan, and India. Our previous study decoded the genome of Protobothrops flavoviridis, a species endemic to the Nansei Islands, Japan, and revealed unique evolutionary processes of some venom genes. In this study, we analyzed genes that are highly expressed in venom glands to survey genes for candidate enzymes or chaperone proteins involved in toxin folding and modification. We found that, in addition to genes that encode venom proteins and ribosomal proteins, genes that encode protein disulfide isomerase (PDI) family members (orthologs of human P4HB and PDIA3), Selenoprotein M (SELENOM), and Calreticulin (CALR) are highly expressed in venom glands. Since these enzymes or chaperones are involved in protein modification and potentially possess protein folding functions, we propose that P4HB, SELENOM, CALR, and PDIA3 encode candidate enzymes or chaperones to confer toxic functions upon the venom transcriptome.
Asunto(s)
Trimeresurus , Animales , China , Genoma , Humanos , Japón , Procesamiento Proteico-Postraduccional , Trimeresurus/genéticaRESUMEN
Amyloid beta peptides (Aßs) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by γ-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic Aß42/43 species is enhanced by familial Alzheimer's disease (FAD) mutations within the catalytic subunit of γ-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying Aß production. Aph1 is a regulatory subunit of γ-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long Aßs than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of γ-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in γ-secretase activity.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Endopeptidasas/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Mutación , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Dominio Catalítico , Endopeptidasas/metabolismo , Fibroblastos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Presenilina-1/metabolismo , Proteolisis , Saccharomyces cerevisiaeRESUMEN
Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane ß-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component α-hemolysin (α-HL) and two-component γ-hemolysin (γ-HL). The α-HL forms a homo-heptamer, whereas γ-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a ß-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the ß-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT ß-barrels for use in further promising applications.
Asunto(s)
Toxinas Bacterianas , Proteínas Hemolisinas , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólisis , Leucocidinas/química , Leucocidinas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
Snakebites are one of the major causes of death and long-term disability in the developing countries due to the presence of various bioactive peptides and proteins in snake venom. In Japan, the venom of the habu snake (Protobothrops flavoviridis) causes severe permanent damage due to its myonecrotic toxins. Antivenom immunoglobulins are an effective therapy for snakebites, and antivenom was recently developed with effective suppressive activity against myonecrosis induced by snake venom. To compare the properties of an antivenom having anti-myonecrotic activity with those of conventional antivenom with no anti-myonecrotic activity, this study applied focused proteomics analysis of habu venom proteins using 2D gel electrophoresis. As a target protein for antivenom immunoglobulins with anti-myonecrotic activity, we identified a thrombin-like serine protease, TLSP2 (TLf2), which was an inactive proteolytic isoform due to the replacement of the active site, His43 with Arg. Additionally, we identified the unique properties and a novel synergistic function of pseudoenzyme TLf2 as a myonecrosis-enhancing factor. To our knowledge, this is the first report of a function of a catalytically inactive snake serine protease.
RESUMEN
Multicopper oxidases have a wide range of substrate specificity to be involved in various physiological reactions. Pseudomonas syringae, a plant pathogenic bacterium, has a multicopper oxidase, CumA. Multicopper oxidases have ability to degrade plant cell wall component, lignin. Once P. syringae enter apoplast and colonize, they start to disrupt plant immunity. Therefore, deeper understanding of multicopper oxidases from plant pathogens helps to invent measures to prevent invasion into plant cell, which brings agricultural benefits. Several biochemical studies have reported lower activity of CumA compared with other multicopper oxidase called CotA. However, the mechanisms underlying the difference in activity have not yet been revealed. In order to acquire insight into them, we conducted a biophysical characterization of PsCumA. Our results show that PsCumA has weak type I copper EPR signal, which is essential for oxidation activity. We propose that difference in the coordination of copper ions may decrease reaction frequency.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Oxidorreductasas/metabolismo , Plantas/microbiología , Pseudomonas syringae/enzimología , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Oxidorreductasas/clasificación , FilogeniaRESUMEN
BACKGROUND: Clinical evidence indicates that there are various risk factors of tooth loss. However, the degree of this risk among other risk factors remains unclear. In this retrospective cohort study, the authors evaluated the hazard ratios of several risk factors for tooth loss. METHODS: Included patients had all been treated for dental disorders, were in the supportive phase of periodontal therapy by dental hygienists, and visited a Japanese dental office continually during a 10-year period. Periodontal parameters, tooth condition, and general status of all teeth (excluding third molars) at the initial visit and at least 10 years later were evaluated by using multiple classification analysis. RESULTS: The authors evaluated a total of 7584 teeth in 297 patients (average age: 45.3, mean follow-up time: 13.9 years) Non-vital pulp was the most significant predictor of tooth loss according to Cox hazards regression analysis (hazard ratio: 3.31). The 10-year survival rate was approximately 90% for teeth with non-vital pulp and 99% for teeth with vital pulp. Fracture was the most common reason for tooth loss. CONCLUSIONS: Non-vital pulp had the most significant association with tooth loss among the parameters. Therefore, it is very important to minimize dental pulp extirpation.
Asunto(s)
Pérdida de Diente , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Pérdida de Diente/epidemiología , Pérdida de Diente/etiologíaRESUMEN
Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family, jacalin-related ß-prism fold lectins (JRLs). While PPL3s and PPL4 shared only 35%-50% homology to PPL2A, respectively, they exhibited unique carbohydrate binding properties based on the multiple glycan-binding profiling data sets from frontal affinity chromatography analysis. In this paper, we investigated biomineralization properties of these lectins and compared their biomineral functions. It was found that these lectins showed different effects on CaCO3 crystalization, respectively, although PPL3 and PPL2A showed similar carbohydrate binding specificities. PPL3 suppressed the crystal growth of CaCO3 calcite, while PPL2A increased the number of contact polycrystalline calcite composed of more than one crystal with various orientations. Furthermore, PPL4 alone showed no effect on CaCO3 crystalization; however, PPL4 regulated the size of crystals collaborated with N-acetyl-D-glucosamine and chitin oligomer, which are specific in recognizing carbohydrates for PPL4. These observations highlight the unique functions and molecular evolution of this lectin family involved in the mollusk shell formation.
Asunto(s)
Exoesqueleto/química , Biomineralización , Bivalvos/fisiología , Carbonato de Calcio/química , Lectinas/química , Lectinas de Plantas/química , Aminoácidos/química , Animales , Carbohidratos/química , Quitina/química , Cristalización , Fenotipo , Isoformas de ProteínasRESUMEN
OBJECTIVES: The absence of bleeding on probing (BOP) is a good predictor of disease stability. This study investigated whether detection of hemoglobin (Hb) in gingival crevicular fluid (GCF) indicates minute signs of periodontal disease, even in BOP (-) cases. MATERIALS AND METHODS: GCF was collected from gingival sulci of 152 sound maxillary and mandibular teeth from 76 patients who had entered supportive periodontal therapy (SPT) using the split-mouth design. As clinical parameters, plaque index, GCF amount, gingival index, probing depth (PD), clinical attachment level, BOP, and alveolar bone resorption ratio were then recorded. As biochemical parameters, Hb amount, alkaline phosphatase (ALP) activity, and protein amount in GCF were measured. Periodontal conditions of diseased sites (PD ≥ 4 mm, BOP (+)) and healthy sites (PD ≤ 4 mm, BOP (-)) were further classified into two groups using the Hb cutoff value determined by PD and BOP and analyzed. RESULTS: Despite being healthy, ALP activity and protein amount in sulci of the group with Hb level greater than the cutoff value were significantly higher than those in the group with Hb level less than the cutoff value (P < 0.01). CONCLUSIONS: This study indicates that Hb examination is a promising candidate marker of pre-symptomatic periodontal disease because Hb presence in GCF suggests slight tissue damage, even in healthy sites defined as BOP (-). CLINICAL RELEVANCE: Hb examination of GCF is a powerful diagnostic tool for pre-symptomatic diagnosis of periodontal disease.
Asunto(s)
Enfermedades Periodontales , Periodontitis , Líquido del Surco Gingival , Hemoglobinas , Humanos , Pérdida de la Inserción Periodontal , Enfermedades Periodontales/diagnóstico , Enfermedades Periodontales/terapia , Bolsa PeriodontalRESUMEN
The objective of this study was to evaluate the clinical effects of repeated subgingival debridement by air polishing during supportive periodontal therapy. A double-blind, randomized controlled trial of 6 months in duration was conducted on 19 recall patients who were previously treated for chronic periodontitis. Three sites with probing pocket depths (PPD) of 4-9 mm in each of the patients were randomly assigned to the following treatments: Glycine powder/air polishing every 30 days (group 1), glycine powder/air polishing at baseline and on day 90 (group 2), or water irrigation every 30 days (group 3). Clinical parameters were recorded and microbiological sampling was performed at 0, 90, and 180 days post-treatment. Subgingival samples were analyzed using real-time PCR methods for Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Between baseline and 90 days, group 1 showed significantly more PPD reduction compared to group 3 and no significant differences with group 2. Between baseline and 180 days, group 1 displayed a significant increase in clinical attachment level compared with group 3. No differences were observed among the groups in numbers of total bacteria or percentage of investigated bacteria at any time point. This study revealed that routine subgingival air polishing at 30-day intervals had significant clinical effects in moderately deep pockets in patients who underwent supportive periodontal therapy.
Asunto(s)
Periodontitis Crónica , Raspado Dental , Pulido Dental , Humanos , Bolsa Periodontal , Porphyromonas gingivalisRESUMEN
Snake venoms are complex mixtures of toxic proteins encoded by various gene families that function synergistically to incapacitate prey. A huge repertoire of snake venom genes and proteins have been reported, and alternative splicing is suggested to be involved in the production of divergent gene transcripts. However, a genome-wide survey of the transcript repertoire and the extent of alternative splicing still remains to be determined. In this study, the comprehensive analysis of transcriptomes in the venom gland was achieved by using PacBio sequencing. Extensive alternative splicing was observed in three venom protein gene families, metalloproteinase (MP), serine protease (SP), and vascular endothelial growth factors (VEGF). Eleven MP and SP genes and a VEGF gene are expressed as a total of 81, 61, and 8 transcript variants, respectively. In the MP gene family, individual genes are transcribed into different classes of MPs by alternative splicing. We also observed trans-splicing among the clustered SP genes. No other venom genes as well as non-venom counterpart genes exhibited alternative splicing. Our results thus indicate a potential contribution of mRNA alternative and trans-splicing in the production of highly variable transcripts of venom genes in the habu snake.
Asunto(s)
Venenos de Crotálidos/genética , Metaloproteasas/genética , ARN Mensajero/genética , Proteínas de Reptiles/genética , Serina Proteasas/genética , Trimeresurus , Factores de Crecimiento Endotelial Vascular/genética , Empalme Alternativo , Animales , Femenino , Perfilación de la Expresión GénicaRESUMEN
We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + ß, ß + ß, respectively) and PPL4, which is heterodimer consisting of α + ß subunits, isolated from mantle secretory fluid of Pteria penguin (Mabe) pearl shell. Their carbohydrate-binding properties were analyzed, in addition to that of PPL2A, which was previously reported as a matrix protein. PPL3s and PPL4 shared only 35-50% homology to PPL2A, respectively; they exhibited significantly different carbohydrate-binding specificities based on the multiple glycan binding profiling data sets from frontal affinity chromatography analysis. The carbohydrate-binding specificity of PPL3s was similar to that of PPL2A, except only for Man3Fuc1Xyl1GlcNAc2 oligosaccharide, while PPL4 showed different carbohydrate-binding specificity compared with PPL2A and PPL3s. PPL2A and PPL3s mainly recognize agalactosylated- and galactosylated-type glycans. On the other hand, PPL4 binds to high-mannose-and hybrid-type N-linked glycans but not agalactosylated- and galactosylated-type glycans.
Asunto(s)
Lectinas/metabolismo , Pinctada/metabolismo , Lectinas de Plantas/metabolismo , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Lectinas/química , Modelos Moleculares , Pinctada/química , Lectinas de Plantas/química , Polisacáridos/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Alineación de SecuenciaRESUMEN
BACKGROUND: Cation transport regulator 1 (CHAC1), a newly discovered enzyme that degrades glutathione, is induced in Helicobacter pylori (H. pylori)-infected gastric epithelial cells in culture. The CHAC1-induced decrease in glutathione leads to an accumulation of reactive oxygen species and somatic mutations in TP53. We evaluated the possible correlation between H. pylori infection and CHAC1 expression in human gastric mucosa. MATERIALS AND METHODS: Both fresh-frozen and formalin-fixed paraffin-embedded tissue samples of gastric mucosa with or without H. pylori infection were obtained from 41 esophageal cancer patients that underwent esophago-gastrectomy. Fresh samples were used for real-time polymerase chain reaction for H. pylori DNA and CHAC1 mRNA, and formalin-fixed samples were used for immunohistochemistry with anti-CHAC1 and anti-H. pylori monoclonal antibodies. Double-enzyme or fluorescence immunohistochemistry and immuno-electron microscopy were used for further analysis. RESULTS: Significant CHAC1 overexpression was detected in H. pylori-infected parietal cells that expressed the human proton pump/H,K-ATPase α subunit, whereas a constitutively low level of CHAC1 mRNA expression was observed in the other samples regardless of the H. pylori infection status, reflecting the weak CHAC1 expression detected by immunohistochemistry in the fundic-gland areas. Immuno-electron microscopy revealed intact H. pylori cells in the secretory canaliculi of infected parietal cells. Some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic-gland mucosa with H. pylori infection. CONCLUSION: Cation transport regulator 1 overexpression in H. pylori-infected parietal cells may cause the H. pylori-induced somatic mutations that contribute to the development of gastric cancer.
Asunto(s)
Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/genética , Helicobacter pylori/fisiología , gamma-Glutamilciclotransferasa/genética , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Humanos , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/microbiología , Células Parietales Gástricas/patología , gamma-Glutamilciclotransferasa/metabolismoRESUMEN
Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110â¯Å in diameter and 160â¯Å in height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.
Asunto(s)
Decapodiformes/química , Hemocianinas/química , Proteínas Inmovilizadas/química , Animales , Biotina/química , Quelantes/química , Cristalización , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Níquel/química , Ácido Nitrilotriacético/química , Multimerización de Proteína , Estreptavidina/químicaRESUMEN
Phospholipase A2 (PLA2) is one of the representative toxic components of snake venom. PLA2s are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA2 assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA2. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA2 remains unknown. In this study, we analyzed a Lys49-PLA2 homologue from Protobothrops flavoviridis (PflLys49-PLA2 BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.
Asunto(s)
Venenos de Crotálidos/enzimología , Lisina/química , Fosfolipasas A2/química , Multimerización de Proteína , Animales , Bothrops , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Relación Estructura-ActividadRESUMEN
Encapsulation of guest molecules into the hollow spaces of crystals has been applied for a variety of purposes such as structure determination, separation, and catalysis of the guest. Although host-guest studies have been developed mainly in crystals of small molecules, those of biomacromolecules have recently been applied. In those reports, a huge hollow space in the protein crystal is commonly used for encapsulation of the guest. Our previous study revealed that cylindrical hemocyanins stack inside the crystal as a linear hollow structure. The diameter of the linear hollow is approximately 110â¯Å, which is large enough for most proteins to pass through. In the present study, we evaluated the potential of hemocyanin crystals as a host to encapsulate biomacromolecules. Confocal microscopy revealed that hemocyanin crystals encapsulate proteins of molecular mass up to 250â¯kDa, i.e., 27â¯kDa green fluorescence protein, 105â¯kDa allophycocyanin, 220â¯kDa C-phycocyanin, and 250â¯kDa phycoerythrin, and DNAs up to 200-bp long, whereas 440â¯kDa ferritin not. Further analysis revealed that hemocyanin crystals prefer a negatively charged guest rather than a positive charge to encapsulate. Moreover, a photobleaching experiment showed that the guest does not move once entrapped. This knowledge of the host-guest study using the hollow hemocyanin crystal should be of significance for further application of hollow proteinaceous crystals as a host.
Asunto(s)
Cristalización/métodos , Decapodiformes/química , Hemocianinas/química , Animales , Proteínas Fluorescentes Verdes/química , Modelos Moleculares , Ficocianina/química , Ficoeritrina/química , PorosidadRESUMEN
We have developed a cast microfluidic chip for concentration gradient generation that contains a thin (~5 µm² cross-sectional area) microchannel. The diffusion of diffused 185 nm ultraviolet (UV) light from an inexpensive low-pressure mercury lamp exposed a layer of the SU-8 photoresist from the backside and successfully patterned durable 2 µm-high microchannel mold features with smooth bell-shaped sidewalls. The thin channel had appropriate flow resistance and simultaneously satisfied both the rapid introduction of test substance and long-term maintenance of gradients. The average height and width at the half height of the channel, defined by a 2 µm-wide line mask pattern, were 2.00 ± 0.19 µm, and 2.14 ± 0.89 µm, respectively. We were able to maintain the concentration gradient of Alexa Fluor 488 fluorescent dye inside or at the exit of the thin microchannel in an H-shaped microfluidic configuration for at least 48 h. We also demonstrated the cultivation of chick embryo dorsal root ganglion neuronal cells for 96 h, and the directional elongation of axons under a nerve growth factor concentration gradient.