Secretion of Recombinant Human Annexin V in Fusion with the Super Folder GFP for Labelling Phosphatidylserine-Exposing Membranes

Twair, Aya; Kassem, Issam; Murad, Hossam; Abbady, Abdul Qader

doi:10.1007/s00232-021-00169-y

Cited by 5 publications

(2 citation statements)

References 51 publications

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“…The surface phosphatidylserine (PS) of apoptotic vesicles was identified by flow cytometry. PS is highly expressed on the membrane surface of ABs as a key feature of ABs [ 8 , 9 ], and to further characterize the quality of the collected ABs, we stained them with annexin V and analyzed them by flow cytometry [ 10 ], and the results suggested that the results showed that more than 90% of the examined particles were positive for annexin V ( Figure 1(c) ), suggesting that the majority of the particles we collected were from ABs.…”

Section: Resultsmentioning

confidence: 99%

The Formation of Melanocyte Apoptotic Bodies in Vitiligo and the Relocation of Vitiligo Autoantigens under Oxidative Stress

Tian

Wang

Ding

et al. 2021

Oxidative Medicine and Cellular Longevity

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Background. Oxidative stress has a vital role in the early stages of vitiligo. Autoantigens released from apoptotic melanocytes (MC) under oxidative stress are involved in the presentation and recognition of antigens. However, the transport of autoantigens to the cell surface and their release to the extracellular environment are still unclear. Apoptotic bodies (ABs) have always been considered as a key source of immunomodulators and autoantigens. Yet, the role of ABs in the immune mechanism of vitiligo is still unknown. Purpose. To explore whether MC’s autoantigens translocate into ABs during oxidative stress-induced apoptosis and study the molecular mechanisms underlying autoantigen migration and AB formation. Methods. PIG3V (an immortalized human vitiligo melanocyte cell line) were treated with H2O2, and ABs were separated. Transmission electron microscopy, flow cytometry, Western blot, mass spectrometry, and other methods were used to determine the relocation of specific antigens in PIG3V cells to ABs. After pretreatment with specific inhibitors (Rho kinase (Y-27632), myosin light chain kinase (MLCK, ML-9), pan-caspase (zVAD-FMK), and JNK (SP600125)), the pathway of autoantigen translocation into ABs and the formation of apoptotic bodies were determined. Results. When treated with 0.8 mM H2O2, ABs were released from these cells. Autoantigens such as tyrosinase-related protein 1 (TYRP-1) and cleavage nuclear membrane antigen Lamin A/C (Asp230) were concentrated in ABs. The expression of autoantigens and the formation of ABs increased in a time- and dose-dependent manner after treatment with H2O2, while the application of specific inhibitors inhibited the formation of apoptotic bodies, i.e., the expression of antigens. Conclusion. Vitiligo autoantigens translocate into ABs in the process of apoptosis induced by oxidative stress. The cytoskeletal protein activation pathway and the JNK-related apoptosis pathway are involved in the transport of autoantigens and the formation of ABs. ABs may be the key bridge between MC cell apoptosis and cellular immunity.

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Section: Resultsmentioning

confidence: 99%

The Formation of Melanocyte Apoptotic Bodies in Vitiligo and the Relocation of Vitiligo Autoantigens under Oxidative Stress

Tian

Wang

Ding

et al. 2021

Oxidative Medicine and Cellular Longevity

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“…So, when expressing the target protein fusion with sfGFP tag, we can directly, from the fluorescence, judge the presence of secreted sfGFP-fusion protein in cells supernatant or in the medium. The sfGFP expression system can not only achieve the rapid screening of high-level expression strains by detecting fluorescence, but also achieve the secretion of target proteins [ 29 , 30 ]. It has been reported that sfGFP as fusion tag not only facilitates the solubility but also promotes the stability of target protein in Escherichia coli [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production

Zhao¹,

Yang²,

Xie³

et al. 2022

Biomolecules

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Adenosine triphosphate (ATP), as a universal energy currency, takes a central role in many biochemical reactions with potential for the synthesis of numerous high-value products. However, the high cost of ATP limits industrial ATP-dependent enzyme-catalyzed reactions. Here, we investigated the effect of cell-surface display of phosphotransferase on ATP regeneration in recombinant Escherichia coli. By N-terminal fusion of the super-folder green fluorescent protein (sfGFP), we successfully displayed the phosphotransferase of Pseudomonas brassicacearum (PAP-Pb) on the surface of E. coli cells. The catalytic activity of sfGFP-PAP-Pb intact cells was 2.12 and 1.47 times higher than that of PAP-Pb intact cells, when the substrate was AMP and ADP, respectively. The conversion of ATP from AMP or ADP were up to 97.5% and 80.1% respectively when catalyzed by the surface-displayed enzyme at 37 °C for only 20 min. The whole-cell catalyst was very stable, and the enzyme activity of the whole cell was maintained above 40% after 40 rounds of recovery. Under this condition, 49.01 mg/mL (96.66 mM) ATP was accumulated for multi-rounds reaction. This ATP regeneration system has the characteristics of low cost, long lifetime, flexible compatibility, and great robustness.

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Detection of phosphatidylserine by using liquid crystal supported on the gold-deposited waveform surfaces with the annexin V-based signal enhancement

Duong

Jang²

2023

Microchemical Journal

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Secretion of Recombinant Human Annexin V in Fusion with the Super Folder GFP for Labelling Phosphatidylserine-Exposing Membranes

Cited by 5 publications

References 51 publications

The Formation of Melanocyte Apoptotic Bodies in Vitiligo and the Relocation of Vitiligo Autoantigens under Oxidative Stress

The Formation of Melanocyte Apoptotic Bodies in Vitiligo and the Relocation of Vitiligo Autoantigens under Oxidative Stress

Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production

Detection of phosphatidylserine by using liquid crystal supported on the gold-deposited waveform surfaces with the annexin V-based signal enhancement

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