Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Summary

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

Abstract

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Introduction

Mast cells are an integral part of the immune system and play a crucial role in both innate and adaptive immune responses. Mast cells are primarily activated either by the binding of an antigen to the immunoglobulin E (IgE) - FcεRI receptor complex, or by the recently discovered mas related G-protein receptor-X2 (MRGPRX2)¹. MRGPRX2 activation has been linked to several immune and inflammatory diseases, and hence, it is important to understand the binding mechanism of the receptor to its ligands². To do so, a library of small peptide molecules was developed and screened against MRGPRX2 receptors that were overexpress....

Protocol

1. Design and development of peptide library

1. To identify the ligands of the mast cell MRGPRX2 receptor based on a known ligand i.e., PAMP-12¹³, follow the steps below.
   1. Generate N-truncated peptide library by truncating the N-terminal amino acid residues of the ligand, in succession, by solid-phase peptide synthesis (SPPS).
   2. Generate C-truncated peptide library by truncating the C-terminal amino acid residues of the known ligand, in succession, by SPPS.
   3. Based on the results of 1.1.1 and 1.1.2, generate an N+C-truncated peptide library using SPPS by truncating the desired residues from....

Results

Table 1 contains the peptide sequences generated through terminal amino acid truncation and alanine scanning. As shown in Table 1, peptide sequence RKKWNKWALSR lacks N-terminal phenylalanine (F) with respect to its parent PAMP-12 and hence is a representative peptide in N-truncated library. Similarly, in FRKKWNKWALS, PAMP-12 C-terminal serine has been removed, representing a C-truncated peptide library derived from PAMP-12. In N+C-truncated peptide library, amino acid from both N and C-t.......

Discussion

Calcium signaling is central to mast cell degranulation and has been widely used in the study of receptor-ligand interactions, ligand identification, and drug discovery¹⁴. MRGPRX2 is a recently discovered mast cell receptor that has been found to play a key role in many inflammatory diseases like itch, asthma, and atopic dermatitis, among others². Furthermore, several approved drugs have been shown to elicit an inflammatory response through the MRGPRX2 receptor

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin	Sigma Aldrich	5470
Calcium Chloride	Sigma Aldrich	793939
Corning 96 Well	Sigma Aldrich	CLS3603
Black Polystyrene Microplate	Sigma Aldrich	CLS3603
DMEM	Thermo Fischer	11995065	High Glucose
DMSO	Thermo Fischer	D12345	Sterile, biological grade
EGTA	Sigma Aldrich	E3889
Fetal Bovine Serum	Thermo Fischer	12483-020
Flexstation 3	Molecular devices	FV06060
Fura-2 AM	Thermo Fischer	F1221
Glucose	Sigma Aldrich	D8270
HEPES buffer	Thermo Fischer	15630-080
Ionomycin	Sigma Aldrich	I9657
L Glutamine	Thermo Fischer	25030-081
Pen Strep	Thermo Fischer	15140122
Peptides	RS Syntehsis	Custom	≥95% pure; N terminal - acetyl group C terminal - amide group
Potassium Chloride	Sigma Aldrich	12636
Sodium Chloride	Sigma Aldrich	S9888
TritonX-100	DOW Chemical	166704