Recombinant Expression in Pichia pastoris System of Three Potent Kv1.3 Channel Blockers: Vm24, Anuroctoxin, and Ts6

Borrego, Jesús; Naseem, Muhammad Umair; Sehgal, A; Panda, Lipsa Rani; Shakeel, Kashmala; Gáspár, Attila; Nagy, Cynthia; Varga, Zoltán; Panyi, György

doi:10.3390/jof8111215

Cited by 3 publications

(2 citation statements)

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“…SDS‐PAGE and circular dichroism (CD) spectroscopy were used to verify Alt a 1 protein purity, identity and secondary structure. For detailed information see online supplement 14,28,29 …”

Section: Methodsmentioning

confidence: 99%

Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice

Fakhimahmadi,

Roth‐Walter,

Hofstetter

et al. 2024

Allergy

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BackgroundWe investigated the biological function of the mould allergen Alt a 1 as a carrier of micronutrients, such as the vitamin A metabolite retinoic acid (RA) and the influence of RA binding on its allergenicity in vitro and in vivo.MethodsAlt a 1‐RA complex formation was analyzed in silico and in vitro. PBMCs from Alternaria‐allergic donors were stimulated with Alt a 1 complexed with RA (holo‐Alt a 1) or empty apo‐Alt a 1 and analyzed for cytokine production and CD marker expression. Serum IgE‐binding and crosslinking assays to apo‐ and holo‐protein were correlated to B‐cell epitope analysis. Female BALB/c mice already sensitized to Alt a 1 were intranasally treated with apo‐Alt a 1, holo‐Alt a 1 or RA alone before measuring anaphylactic response, serum antibody levels, splenic cytokines and CD marker expression.ResultsIn silico docking calculations and in vitro assays showed that the extent of RA binding depended on the higher quaternary state of Alt a 1. Holo‐Alt a 1 loaded with RA reduced IL‐13 released from PBMCs and CD3+CD4+CRTh2 cells. Complexing Alt a 1 to RA masked its IgE B‐cell epitopes and reduced its IgE‐binding capacity. In a therapeutic mouse model of Alternaria allergy nasal application of holo‐Alt a 1, but not of apo‐Alt a 1, significantly impeded the anaphylactic response, impaired splenic antigen‐presenting cells and induced IL‐10 production.ConclusionHolo‐Alt a 1 binding to RA was able to alleviate Th2 immunity in vitro, modulate an ongoing Th2 response and prevent anaphylactic symptoms in vivo, presenting a novel option for improving allergen‐specific immunotherapy in Alternaria allergy.

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“…SDS‐PAGE and circular dichroism (CD) spectroscopy were used to verify Alt a 1 protein purity, identity and secondary structure. For detailed information see online supplement 14,28,29 …”

Section: Methodsmentioning

confidence: 99%

Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice

Fakhimahmadi,

Roth‐Walter,

Hofstetter

et al. 2024

Allergy

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“…SDS-PAGE and circular dichroism (CD) spectroscopy were used to verify protein purity, identity, and secondary structure. Measurement of endotoxin content was done by EndoZyme recombinant Factor C Endotoxin Detection Assay (Biomerieux, Craponne, France) and total protein content by BCA assay (Pierce BCA Protein Assay Kit, Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions [99,100]. Batches were sterile-filtered (0.22 µM) before use.…”

Section: Alt a 1 Expression In Pichia Pastorismentioning

confidence: 99%

Nutritional Provision of Iron Complexes by the Major Allergen Alt a 1 to Human Immune Cells Decreases Its Presentation

Fakhimahmadi,

Hasanaj,

Hofstetter

et al. 2023

IJMS

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Alternaria alternata is a common fungus strongly related with severe allergic asthma, with 80% of affected individuals being sensitized solely to its major allergen Alt a 1. Here, we assessed the function of Alt a 1 as an innate defense protein binding to micronutrients, such as iron–quercetin complexes (FeQ2), and its impact on antigen presentation in vitro. Binding of Alt a 1 to FeQ2 was determined in docking calculations. Recombinant Alt a 1 was generated, and binding ability, as well as secondary and quaternary structure, assessed by UV-VIS, CD, and DLS spectroscopy. Proteolytic functions were determined by casein and gelatine zymography. Uptake of empty apo– or ligand-filled holoAlt a 1 were assessed in human monocytic THP1 cells under the presence of dynamin and clathrin-inhibitors, activation of the Arylhydrocarbon receptor (AhR) using the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for phenotypic changes in monocytes by flow cytometry. Alt a 1 bound strongly to FeQ2 as a tetramer with calculated Kd values reaching pico-molar levels and surpassing affinities to quercetin alone by a factor of 5000 for the tetramer. apoAlt a 1 but not holoAlta 1 showed low enzymatic activity against casein as a hexamer and gelatin as a trimer. Uptake of apo– and holo–Alt a 1 occurred partly clathrin-dependent, with apoAlt a 1 decreasing labile iron in THP1 cells and holoAlt a 1 facilitating quercetin-dependent AhR activation. In human PBMCs uptake of holoAlt a 1 but not apoAlt a 1 significantly decreased the surface expression of the costimulatory CD86, but also of HLADR, thereby reducing effective antigen presentation. We show here for the first time that the presence of nutritional iron complexes, such as FeQ2, significantly alters the function of Alt a 1 and dampens the human immune response, thereby supporting the notion that Alt a 1 only becomes immunogenic under nutritional deprivation.

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Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform

Sun,

Ko,

Gao

et al. 2023

Front. Microbiol.

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In Phi29-α–hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies.

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Recombinant Expression in Pichia pastoris System of Three Potent Kv1.3 Channel Blockers: Vm24, Anuroctoxin, and Ts6

Cited by 3 publications

References 50 publications

Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice

Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice

Nutritional Provision of Iron Complexes by the Major Allergen Alt a 1 to Human Immune Cells Decreases Its Presentation

Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform

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