We thank Fabrice Agou and Alix Boucharlat from the Chemogenomic and Biological Screening Platform, Center for Technological Resources and Research (C2RT) at Institut Pasteur Paris for helpful suggestions on the protocol, all members of the BGPB Unit for support and scientific discussions, and Simon Legood for critical reading of the manuscript. Work was funded by Global Care Initiatives of the Institute Carnot Infectious diseases and the Institute Carnot Microbes and Health (15 CARN 0017-01 and 16 CARN 0023-01).

1. Enzyme and substrate preparation
<ol>
	<li>Purification of enzyme
	<ol>
		<li>Produce and purify Lnt enzyme from detergent solubilized membranes as described previously4,8. Briefly, induce expression of the lnt-strep gene, encoding Lnt with a C-terminal Strep tag, at OD600 of 0.6 with anhydrous tetracycline (200 ng/mL) at 37 &#176;C for 16 h.</li>
		<li>Harvest cells by centrifugation at 4,000 x g for 10 min and ...

<ol>
	<li>Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+bacterial+lipoprotein+modification--how,+when+and+why.">The molecular mechanism of bacterial lipoprotein modification--how, when and why.</a> FEMS Microbiology Reviews. 39 (2), 246-261 (2015).</li><li>Kovacs-Simon, A., Titball, R. W., Michell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+bacterial+pathogens.">Lipoproteins of bacterial pathogens.</a> Infections and Immunity. 79 (2), 548-561 (2010).</li><li>Jackowski, S., Rock, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+fatty+acids+from+the+1-position+of+phosphatidylethanolamine+to+the+major+outer+membrane+lipoprotein+of+Escherichia+coli.">Transfer of fatty acids from the 1-position of phosphatidylethanolamine to the major outer membrane lipoprotein of Escherichia coli.</a> Journal of Biological Chemistry. 261, 11328-11333 (1986).</li><li>Hillmann, F., Argentini, M., Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+and+phospholipid+specificity+of+apolipoprotein+N-acyltransferase.">Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase.</a> Journal of Biological Chemistry. 286 (32), 27936-27946 (2011).</li><li>Rangan, K. J., Yang, Y. Y., Charron, G., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Visualization+and+Large-Scale+Profiling+of+Bacterial+Lipoproteins+with+Chemical+Reporters.">Rapid Visualization and Large-Scale Profiling of Bacterial Lipoproteins with Chemical Reporters.</a> Journal of American Chemical Society. 132 (31), 10628-10629 (2010).</li><li>Thiele, C., 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=Tracing+fatty+acid+metabolism+by+click-chemistry.">Tracing fatty acid metabolism by click-chemistry.</a> ACS Chemical Biology. 7 (12), 2004-2011 (2012).</li><li>Ganesan, L., Shieh, P., Bertozzi, C. R., Levental, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-Chemistry+Based+High+Throughput+Screening+Platform+for+Modulators+of+Ras+Palmitoylation.">Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation.</a> Science Reports. 7, 41147 (2017).</li><li>Nozeret, K., Boucharlat, A., Agou, F., Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+fluorescence-based+assay+to+monitor+enzymatic+activity+of+the+essential+integral+membrane+protein+Apolipoprotein+N-acyltransferase+(Lnt).">A sensitive fluorescence-based assay to monitor enzymatic activity of the essential integral membrane protein Apolipoprotein N-acyltransferase (Lnt).</a> Science Reports. 9 (1), 15978 (2019).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecules Screening. 4 (2), 67-73 (1999).</li><li>Mao, G., 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=Crystal+structure+of+E.+coli+lipoprotein+diacylglyceryl+transferase.">Crystal structure of E. coli lipoprotein diacylglyceryl transferase.</a> Nature Communication. 7, 10198 (2016).</li><li>Vogeley, L., 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=Structural+basis+of+lipoprotein+signal+peptidase+II+action+and+inhibition+by+the+antibiotic+globomycin.">Structural basis of lipoprotein signal peptidase II action and inhibition by the antibiotic globomycin.</a> Science. 351 (6275), 876-880 (2016).</li><li>Wiktor, M., 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=Structural+insights+into+the+mechanism+of+the+membrane+integral+N-acyltransferase+step+in+bacterial+lipoprotein+synthesis.">Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.</a> Nature Communication. 8, 15952 (2017).</li><li>Noland, C. L., 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=Structural+insights+into+lipoprotein+N-acylation+by+Escherichia+coli+apolipoprotein+N-acyltransferase.">Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase.</a> Proceedings of the National Academy of Sciences USA. 114 (30), 6044-6053 (2017).</li><li>Lu, G., 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=Crystal+structure+of+E.+coli+apolipoprotein+N-acyl+transferase.">Crystal structure of E. coli apolipoprotein N-acyl transferase.</a> Nature Communication. 8, 15948 (2017).</li></ol>

The authors have nothing to disclose.

The protocol for the Lnt assay described here, based on fluorescence detection of the triacylated product, is sensitive and reproducible. The specific and efficient binding of biotin to streptavidin is a key element in the assay. Alkyne-POPE substrate left after completion of the Lnt reaction is also fluorescently labeled with FAM but is efficiently removed after binding onto the streptavidin plates by multiple wash steps. Furthermore, addition of DMSO does not affect Lnt activity and has no impact on the assay. Both sub...

Bacterial lipoproteins are characterized by covalently bound fatty acids at their amino-termini through which they are anchored into membranes1,2. The mature part of the protein is highly diverse in structure and function, thereby explaining the role of lipoproteins in various biological processes in the bacterial cell envelope.
Lipoproteins are modified by phospholipid-derived fatty acids after insertion into the cytoplasmic membrane. The prolipoproteins contain a signature motif, the lipobox, which contains an invariant cysteine residue that becomes acylated and the first amino acid in the mature protein. The first step of this pathway is catalyzed by prolipoprotein phosphatidylglycerol::diacylglyceryl transferase (Lgt), which transfers the diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein via a thioether link between diacylglyceryl and cysteine. Signal peptidase II (Lsp) cleaves the signal peptide from diacylglyceryl prolipoprotein, resulting in an apolipoprotein that is anchored into the membrane through its diacylglyceryl moiety. The third and last step is catalyzed by apolipoprotein N-acyltransferase (Lnt), which adds a fatty acid from the sn-1 position of phospholipid onto apolipoprotein, resulting in triacylated mature lipoprotein (Figure 1)3. The Lnt reaction is a two-step ping-pong reaction where a stable thioester acyl enzyme intermediate is formed. The lysophospholipid byproduct is released prior to the acylation of the apolipoprotein substrate in the second step of the reaction.
The phospholipid substrate specificity is determined in a Lnt assay based on the mobility shift of N-acyl diacylglyceryl peptide on a high percentage Tris-Tricine Urea SDS-PAGE4. Phospholipids with small polar headgroups, saturated [sn-1] and nonsaturated [sn-2], were preferred substrates4. The gel shift assay is not appropriate for extensive kinetic studies of apolipoprotein N-acyltransferase nor for HTS to identify inhibitory molecules. Click-chemistry using alkyne fatty acids has been successfully used to study lipoprotein modification in bacteria5 and fatty acid metabolism in eukaryotes6. Recently, an in vitro assay of Ras palmitoylation was reported to identify inhibitors7.
In the method described here, purified active Lnt in detergent is incubated with substrates in mixed micelles to form alkyne-triacylated peptide that is subsequently detected by fluorescence spectrometry.

<table>
	<tbody>
		<tr>
			<td>&#196;kta Purifier FPLC system</td>
			<td>GE Healthcare</td>
			<td>NA</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>alkyne-POPE</td>
			<td>Avanti Polar Lipids</td>
			<td>900414P</td>
			<td>Purity: &#62;99% 
			Lnt substrate</td>
		</tr>
		<tr>
			<td>Azido-FAM</td>
			<td>Lumiprobe</td>
			<td>A4130</td>
			<td>Purity: 100% (pure) 
			Click reagent</td>
		</tr>
		<tr>
			<td>BioPhotometer Plus</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td>Purity: NA 
			OD 600 nm</td>
		</tr>
		<tr>
			<td>BioTek ELX 405 Select plate washer</td>
			<td>BioTek</td>
			<td>N&#176; serie 115800, n&#176; materiel 405 Select</td>
			<td>Purity: NA 
			Wash steps</td>
		</tr>
		<tr>
			<td>biotin-fluorescein</td>
			<td>Sigma</td>
			<td>53608</td>
			<td>Purity: &#8805;90% 
			Fluorescence control</td>
		</tr>
		<tr>
			<td>Copper(II) sulfate pentahydrate</td>
			<td>Sigma</td>
			<td>C3036</td>
			<td>Purity: &#8805;98% 
			Click reagent</td>
		</tr>
		<tr>
			<td>DDM</td>
			<td>Anatrace</td>
			<td>D310A</td>
			<td>Purity: &#8805; 99% &#946;+&#945;; &#60; 15% &#945; 
			Detergent for Lnt purification</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Invitrogen</td>
			<td>D12435</td>
			<td>Purity: anhydrous 
			Solbilization Click reagent</td>
		</tr>
		<tr>
			<td>Electronic pipet Voyager 8 channels 0.5-12.5 uL</td>
			<td>INTEGRA</td>
			<td>4721</td>
			<td>Purity: NA 
			Handling reagents</td>
		</tr>
		<tr>
			<td>French Pressure Cell</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purity: NA 
			Cell disruption</td>
		</tr>
		<tr>
			<td>FSL-1-biotin</td>
			<td>EMC microcollections</td>
			<td>L7030</td>
			<td>Purity: NA 
			Lnt substrate</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 384-well standard CELLSTAR polystyrene microplate</td>
			<td>Greiner</td>
			<td>781091</td>
			<td>Purity: NA 
			Black with transparent bottom (up or bottom reading)</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 96-well sterile polystyrene plate, high binding</td>
			<td>Greiner</td>
			<td>655097</td>
			<td>Purity: NA 
			Black with transparent bottom (up or bottom reading)</td>
		</tr>
		<tr>
			<td>Microplate reader Infinite M1000 pro</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate)</td>
			<td>Anatrace</td>
			<td>S110MT</td>
			<td>Purity: ~100% 
			Thiol specific inhibitor</td>
		</tr>
		<tr>
			<td>Optically Clear Adhesive Seal Sheets</td>
			<td>Thermo Scientific</td>
			<td>AB-1170</td>
			<td>Purity: NA 
			Foil to seal multi-well plate</td>
		</tr>
		<tr>
			<td>Sephacryl S400 HR 16/60 gel filtration column</td>
			<td>GE Healthcare</td>
			<td>GE28-9356-04</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>StrepTactin Sepharose 50 %</td>
			<td>IBA Biotechnology</td>
			<td>2-1201-010</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>streptavidin</td>
			<td>Sigma</td>
			<td>S4762-1MG</td>
			<td>Purity: &#8805;13 units/mg protein 
			Biotin binding</td>
		</tr>
		<tr>
			<td>TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine</td>
			<td>Sigma</td>
			<td>678937</td>
			<td>Purity: 97% 
			Click reagent</td>
		</tr>
		<tr>
			<td>TCEP (tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Sigma</td>
			<td>75259</td>
			<td>Purity: &#8805;98% 
			Click reagent</td>
		</tr>
		<tr>
			<td>TECAN Infinite F500</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>TECAN Infinite M1000 pro</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>Thermomixer C</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td>Purity: NA 
			Heated lid</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>93443</td>
			<td>Purity: 10% in H2O 
			Lnt reaction buffer</td>
		</tr>
		<tr>
			<td>Ultra centrifuge</td>
			<td>Beckman LC</td>
			<td>N/A</td>
			<td>Purity: NA 
			Cell fractionation</td>
		</tr>
	</tbody>
</table>

click-chemistry based fluorometric assay for apolipoprotein n-acyltransferase from enzyme characterization to high-throughput screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

In the Lnt reaction the sn-1 fatty acid from phospholipids is transferred onto a diacylglyceryl peptide, resulting in mature triacylated peptide8. The in vitro Lnt assay described here is designed to use phospholipids containing an alkyne fatty acid (alkyne-POPE) and FSL-1-biotin as substrates, resulting in the formation of alkyne-FSL-1-biotin. Upon a click-chemistry reaction with azido-FAM, this product should become fluorescently labeled and detected by fluorescence spectrometry (<stron...

Watch this Scientific Journal Video about Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening at JoVE.com

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

This protocol addresses two important aspects, characterization of an essential integral membrane, apolipoprotein acyltransferase of bacteria, and development of the assay in HDS format to screen for inhibitors of the enzyme. The method is very sensitive and suitable for screening purposes. It can contribute to the development of potential novel antibiotics.This video will help highlight critical steps of this protocol, such as mixing, vortexing, sonicating, while working with nonhomogeneous lipid and detergent containing suspensions. Demonstrating the procedure will be Karine Nozeret, Aurelia Pernin, both are fellows in my group. Begin by preparing the reagent mixture for the apolipoprotein and acyltransferase or LNT assay.Mix alkyne POPE and FSL-1-biotin with LNT reaction buffer in 1.5 milliliter tubes at a final volume of 18 microliters. Prepare triplicate reagent mixtures for all tested conditions. Sonicate the reagent mixtures for three minutes in an ultrasonic water bath, and incubate them at 37 degrees Celsius for five minutes.Then add active or inactive LNT to a final concentration of one nanogram per microliter, and pipette up and down to mix. Incubate the reaction at 37 degrees Celsius for 16 hours in a thermal mixer with a heated lid. Meanwhile, prepare a stock solution of two milligram per milliliter streptavidin.And dilute it to 10 micrograms per milliliter to create a working solution. Add 100 microliters of the solution to each well of a 96 well plate. Then air dry the wells overnight by incubating the plate at 37 degrees Celsius without a lid.On the next day, prepare stock solutions of five millimolar Azido-FAM, 50 millimolar TCEP, two millimolar TBTA, and 50 millimolar copper sulfate pentahydrate. Perform click chemistry by adding 0.2 microliters of stock Azido-FAM, 0.4 microliters of stock TCEP, and 0.2 microliters of stock TBTA to the 1.5 milliliter LNT reaction tubes. Vortex the solution for five seconds.Then add 0.4 microliters of stock copper sulfate. Vortex for another five seconds and incubate the reaction for one hour at room temperature in the dark. While the click chemistry mixture is incubating, wash the wells of the streptavidin coated plates three times with 200 microliters of PBST1.Transfer 18 microliters of the click chemistry mixture to a streptavidin coated well, and add 100 microliters of PBST1 to bind the N-acyl-FSL-1-biotin FAM product to the plate. Add biotin fluorescein to the plate as a positive control for streptavidin binding and fluorescence readout. Then incubate the plate at room temperature for one hour in the dark, while shaking at 300 RPM.After the incubation use a multi-channel electronic pipette to manually wash the plate six times with 200 microliters of PBST2. Flush it three times with the pipette. Wash it three times with 200 microliters of PBS and flush it three more times.Add 200 microliters of PBS to each well, and detect the fluorescence at 520 nanometers in a microplate reader. Save all results in a spreadsheet, then calculate the standard deviation for each reaction as well as the P-values for negative controls and positive samples with an unpaired T test. The in vitro LNT assay described here uses phospholipids containing an alkyne fatty acid and FSL-1-biotin as substrates to form alkyne FSL-1-biotin.Upon a click chemistry reaction with Azido-FAM, this product becomes fluorescently labeled and detectable by fluorescence spectrometry. The reaction conditions were optimized for maximum fluorescence readout at 520 nanometers. Negative controls included reactions without enzyme, with an inactive variant of the enzyme, or with thiol specific inhibitor, MTSES.Biotin fluorescein was used as an internal control for maximum fluorescence signal. In order to develop a high throughput screening assay, the reactions were performed directly in 384 well plates. Wash steps were automated and quantity of reagents was reduced.The reaction was sensitive and reproducible with a significant difference in fluorescent signal between the negative control and active enzyme. For high throughput screening the Z prime factor determines whether a response in an assay is large enough for screening purposes. The average Z prime factor was greater than 0.6 for the LNT assay performed in a 384 well format, suggesting that it was outstanding.The tube assay in this video has been developed in a multi plate format that is compatible with HDS. It can be used to study other acyltransferases from a variety of organisms.

click-chemistry-based-fluorometric-assay-for-apolipoprotein-n

Research

JoVE Journal

Biochemistry

1.8K ビュー.  Institut Pasteur, Unité Biologie et génétique de la paroi bactérienne, Paris, France ; CNRS, UMR 2001 « Microbiologie intégrative et Moléculaire », Paris, France ; INSERM, Équipe Avenir, Paris, France. This protocol addresses two important aspects, characterization of an essential integral membrane, apolipoprotein acyltransferase of bacteria, and development of the assay in HDS format to screen for inhibitors of the enzyme. The method is very sensitive and suitable for screening purposes. It can contribute to the development of potential novel antibiotics.This video will help highlight critical steps of this protocol, such as mixing, vortexing, sonicating, while working with nonhomog...