MrgX2-SNAP-tag/cell membrane chromatography model coupled with liquid chromatography-mass spectrometry for anti-pseudo-allergic compound screening in Arnebiae Radix

Abstract

Pseudo-allergic reactions (PARs) are IgE-independent hypersensitivity reactions. Mas-related G protein-coupled receptor-X2 (MrgX2) was proved the key receptor of PAR. The anti-pseudo-allergic compound discovery based on MrgX2 was of great value. Cell membrane chromatography (CMC) based on MrgX2 provides a convenient and effective tool in anti-pseudoallergic compound screening and discovery, and further improvements of this method are still needed. In this work, SNAPtag was introduced at C-terminal of Mas-related G protein-coupled receptor (MrgX2-SNAP-tag), and an MrgX2-SNAP-tag/ CMC model was then conducted using CMC technique. Comparative experiments showed that the new model not only satisfied the good selectivity and specificity of screening but also exhibited more stable and longer life span than traditional MrgX2/CMC model. By coupling with HPLC-MS, two compounds were screened out from Arnebiae Radix and identified as shikonin and acetylshikonin. Nonlinear chromatography was performed to study the interactions between two screened compounds and MrgX2, and binding constant (K A ) of shikonin and acetylshikonin with MrgX2 were 2075.67 ± 0.34 M −1 and 32201.36 ± 0.35 M −1 , respectively. Furthermore, β-hexosaminidase and histamine release assay in vitro demonstrated that shikonin (1-5 μM) and acetylshikonin (2.5-10 μM) could both antagonize C48/80-induced allergic reaction. In conclusion, the MrgX2-SNAP-tag/CMC could be a reliable model for screening pseudo-allergy-related components from complex systems.

Quality evaluation methods of chinese medicine based on scientific supervision: recent research progress and prospects

“…However, as the membrane receptors attached to the silica gel can easily fall off or lose their activity, the life of the CMC column is short and repeatability is poor. Due to the uncertainty of the number of membrane receptors on the CMC stationary phase, this can affect the accuracy of the analysis results, so cell membrane chromatography is mostly used in conjunction with multi-dimensional chromatographic techniques, such as LC–MS [ 75 ], GC–MS, HPLC–MS, and UPLC-MS/MS [ 76 ] as a common method for screening, analyzing and identifying target components of specific receptor constituent groups. Lin and colleagues successfully isolated the anti-allergic components in Houpo ( Magnolia officinalis Rehd.et Wils. )…”

Modulation of the Mas-Related G Protein-Coupled Receptor X2 (MRGPRX2) by Xenobiotic Compounds and Its Relevance to Human Diseases

“…Exogenous ligands of MRGPRX2 include the cationic polymer compound 48/80 (C48/80), which is commonly used in receptor functional assays, and a variety of drugs approved by the Food and Drug Administration (FDA), such as fluoroquinolones (e.g., ciprofloxacin), neuromuscular blocking agents (e.g., rocuronium, atracurium), opioids (e.g., morphine), and many others [ 4 , 9 , 28 ]. MRGPRX2 can also be activated or inhibited by other exogenous agents, such as bacterial quorum sensing proteins, insect venoms [ 3 , 29 , 30 ], or many different plant xenobiotics ( Figure 1 ) [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ]; representatives of which are described in the following part of this review.…”