Name: screening peptides that activate mrgprx2 using engineered hek cells
Uploaded: 2021-11-06T13:00:00
Description: Watch this Scientific Journal Video about Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells at JoVE.com

SR and LDU would like to acknowledge Alberta Innovates Strategic Research Project, NRC, and NSERC-Discovery grant for this project.

1. Design and development of peptide library
<ol>
	<li>To identify the ligands of the mast cell MRGPRX2 receptor based on a known ligand i.e., PAMP-1213, follow the steps below.
	<ol>
		<li>Generate N-truncated peptide library by truncating the N-terminal amino acid residues of the ligand, in succession, by solid-phase peptide synthesis (SPPS).</li>
		<li>Generate C-truncated peptide library by truncating the C-terminal amino acid residues of the known ligand, in succession, by ...

<ol>
	<li>McNeil, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mast-cell-specific+receptor+crucial+for+pseudo-allergic+drug+reactions.">Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.</a> Nature. 519 (7542), 237-241 (2015).</li><li>Subramanian, H., Gupta, K., Ali, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Mas-related+G+protein-coupled+receptor+X2+on+mast+cell-mediated+host+defense,+pseudoallergic+drug+reactions,+and+chronic+inflammatory+diseases.">Roles of Mas-related G protein-coupled receptor X2 on mast cell-mediated host defense, pseudoallergic drug reactions, and chronic inflammatory diseases.</a> Journal of Allergy and Clinical Immunology. 138 (3), 700-710 (2016).</li><li>R&#229;dinger, M., Jensen, B. M., Kuehn, H. S., Kirshenbaum, A., Gilfillan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+isolation,+and+maintenance+of+human+mast+cells+and+mast+cell+lines+derived+from+peripheral+blood+or+cord+blood.">Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood.</a> Current protocols in immunology. , (2010).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> Journal of Pharmacological and Toxicological Methods. 51 (3), 187-200 (2005).</li><li>Subedi, G. P., Johnson, R. W., Moniz, H. A., Moremen, K. W., Barb, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+yield+expression+of+recombinant+human+proteins+with+the+transient+transfection+of+HEK293+cells+in+suspension.">High yield expression of recombinant human proteins with the transient transfection of HEK293 cells in suspension.</a> Journal of Visualized Experiments. (106), e53568 (2015).</li><li>Occhiuto, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+calcium+entry+via+STIM1+contributes+to+MRGPRX2+induced+mast+cell+functions.">Store-operated calcium entry via STIM1 contributes to MRGPRX2 induced mast cell functions.</a> Frontiers in Immunology. 10, 3143 (2020).</li><li>Baba, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+function+for+the+calcium+sensor+STIM1+in+mast+cell+activation+and+anaphylactic+responses.">Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses.</a> Nature Immunology. 9 (1), 81-88 (2008).</li><li>Hoth, M., Penner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+intracellular+calcium+stores+activates+a+calcium+current+in+mast+cells.">Depletion of intracellular calcium stores activates a calcium current in mast cells.</a> Nature. 355 (6358), 353-356 (1992).</li><li>Assinger, A., Volf, I., Schmid, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+rapid+method+to+quantify+intraplatelet+calcium+dynamics+by+ratiometric+flow+cytometry.">A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.</a> PLoS One. 10 (4), 0122527 (2015).</li><li>Roe, M. W., Lemasters, J. J., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Fura-2+for+measurements+of+cytosolic+free+calcium.">Assessment of Fura-2 for measurements of cytosolic free calcium.</a> Cell Calcium. 11 (2-3), 63-73 (1990).</li><li>Malgaroli, A., Milani, D., Meldolesi, J., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fura-2+measurement+of+cytosolic+free+Ca2++in+monolayers+and+suspensions+of+various+types+of+animal+cells.">Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells.</a> The Journal of Cell Biology. 105 (5), 2145-2155 (1987).</li><li>Grynkiewicz, G., Poenie, M., Tsienb, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Generation+of+Ca2+++Indicators+with+Greatly+Improved+Fluorescence+Properties.">A New Generation of Ca2 + Indicators with Greatly Improved Fluorescence Properties.</a> Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).</li><li>Lu, L., Parmar, M. B., Kulka, M., Kwan, P., Unsworth, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide+nanoscaffold+that+activates+human+mast+cells.">Self-assembling peptide nanoscaffold that activates human mast cells.</a> ACS Applied Materials and Interfaces. 10 (7), 6107-6117 (2018).</li><li>Lansu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+design+of+novel+probes+for+the+atypical+opioid+receptor+MRGPRX2.">In silico design of novel probes for the atypical opioid receptor MRGPRX2.</a> Nature Chemical Biology. 13 (5), 529-536 (2017).</li><li>Navin&#233;s-Ferrer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRGPRX2-mediated+mast+cell+response+to+drugs+used+in+perioperative+procedures+and+anaesthesia.">MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia.</a> Scientific Reports. 8 (1), 11628 (2018).</li><li>Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+store-operated+calcium+(Ca+2+)+entry+(SOCE)+in+HEK293+cells+using+Fura-2.">Calcium imaging of store-operated calcium (Ca 2+) entry (SOCE) in HEK293 cells using Fura-2.</a> Calcium Signalling. , 163-172 (2019).</li><li>Tinning, P. W., Franssen, A. J. P. M., Hridi, S. U., Bushell, T. J., McConnell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+340/380+nm+light-emitting+diode+illuminator+for+Fura-2+AM+ratiometric+Ca2++imaging+of+live+cells+with+better+than+5+nM+precision.">A 340/380 nm light-emitting diode illuminator for Fura-2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision.</a> Journal of Microscopy. 269 (3), 212-220 (2018).</li></ol>

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<sup class='x...

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) - Fc&#949;RI 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 overexpressed in HEK cells. In the study, the peptide library was constructed using the simple and versatile techniques of alanine scanning and amino acid truncation. Alanine scanning involves replacing specific amino acids with an alanine residue. Alanine being small and neutral, strips the peptide of the specific properties conferred by the replaced residue and consecutively highlights the significance of the respective physiochemical properties of the amino acid in receptor interactions. On the contrary, in amino acid truncation, peptide sequences are designed such that it lacks one or more amino acid residues from the N-terminal, C terminal, or both. This set of peptides was used to identify the amino acid sequences crucial to MRGPRX2 binding.
Experience with human mast cells lines (LAD-2) has shown that these cells are difficult to culture and maintain in vitro: a doubling time of two weeks, expensive medium supplements, and direct attention required during passaging3. These attributes make the cells unsuitable for large scale screening of potential ligands. Herein, stably transfected HEK cells expressing MRGPRX2 receptor (HEK-X2) were used to screen the peptide library1. HEK-293 cells are widely used and studied for the heterologous expression of surface receptors due to their high transfection efficiency, faster doubling rate, and the need for non-expensive medium supplements to be cultured in laboratory4. The protocol to transfect HEK-293 cell line has been demonstrated and is well established5. HEK-293 cells stably expressing MRGPRX2 receptor (passage 13-19) were activated with the peptides generated through N-truncation, C-truncation, N+C-truncation, and alanine scanning1. Wild type HEK cells (HEK-WT) (passage 16-21) were used as control. Intracellular calcium release upon activation was monitored to study the MRGPRX2 based activation.
Cell activation by MRGPRX2 is followed by a cytosolic calcium mobilization. This regulated intracellular calcium release in mast cells is regulated by the store operated calcium entry (SOCE), coordinated by the stromal interaction molecule 1 (STIM1); and is central to the immune response cascade6,7. Various methods have been used to detect intracellular calcium concentration, including patch-clamps and fluorescent dyes8. Of all the techniques available, fluorometric calcium dyes in conjugation with various detection techniques are being widely used9. Two types of fluorometric dyes that have gained interests are namely, single wavelength dyes like Fluo-4, and dual wavelength ratiometric dyes like Indo -1 and Fura-2. The advantage that dual wavelength ratiometric dyes bring over single wavelength dyes is that the ratiometric dyes correct for experimental errors like dye loading, photo bleaching, and focusing10,11.
Fura-2 acetoxymethyl ester (Fura-2 AM) is a cell permeating, green-fluorescent dye whose excitation shifts to a lower wavelength upon calcium-binding. Experimentally, Fura-2 is excited at 340 and 380 nm, while the emission is recorded at 510 nm. Upon calcium binding, the fluorescent intensity at 340 nm increases while that of 380 nm decreases, as shown in Figure 1. Data is represented as a ratio of fluorescence intensity after excitation at 340 nm (F340) to that of intensity after excitation at 380 nm (F380) i.e., F340/F380. The F340/F380 ratio is proportional to intracellular calcium, the value of which can be calculated by the Grynkiewicz equation12. Since the fluorescence signal is obtained from the excitation of the dye at two wavelengths (340 nm and 380 nm), the ratio of the fluorescence signals corrects for experimental factors like dye loading, dye leakage, photobleaching, and cell densities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>793939</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 Well</td>
			<td>Sigma Aldrich</td>
			<td>CLS3603</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Polystyrene Microplate</td>
			<td>Sigma Aldrich</td>
			<td>CLS3603</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fischer</td>
			<td>11995065</td>
			<td>High Glucose</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Thermo Fischer</td>
			<td>D12345</td>
			<td>Sterile, biological grade</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fischer</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexstation 3</td>
			<td>Molecular devices</td>
			<td>FV06060</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 AM</td>
			<td>Thermo Fischer</td>
			<td>F1221</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>D8270</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Thermo Fischer</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma Aldrich</td>
			<td>I9657</td>
			<td></td>
		</tr>
		<tr>
			<td>L Glutamine</td>
			<td>Thermo Fischer</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Thermo Fischer</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptides</td>
			<td>RS Syntehsis</td>
			<td>Custom</td>
			<td>&#8805;95% pure; 
			N terminal - acetyl group 
			C terminal - amide group</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>12636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>DOW Chemical</td>
			<td>166704</td>
			<td></td>
		</tr>
	</tbody>
</table>

screening peptides that activate mrgprx2 using engineered hek cells

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.

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...

Watch this Scientific Journal Video about Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells at JoVE.com

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

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.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

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