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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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

Abstract

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 γ-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.

Introduction

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 ac....

Protocol

1. Enzyme and substrate preparation

  1. Purification of enzyme
    1. 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 °C for 16 h.
    2. Harvest cells by centrifugation at 4,000 x g for 10 min and .......

Representative Results

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 (

Discussion

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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
Äkta Purifier FPLC systemGE HealthcareNAPurity: NA
Lnt purification
alkyne-POPEAvanti Polar Lipids900414PPurity: >99%
Lnt substrate
Azido-FAMLumiprobeA4130Purity: 100% (pure)
Click reagent
BioPhotometer PlusEppendorfN/APurity: NA
OD 600 nm
BioTek ELX 405 Select plate washerBioTekN° serie 115800, n° materiel 405 SelectPurity: NA
Wash steps
biotin-fluoresceinSigma53608Purity: ≥90%
Fluorescence control
Copper(II) sulfate pentahydrateSigmaC3036Purity: ≥98%
Click reagent
DDMAnatraceD310APurity: ≥ 99% β+α; < 15% α
Detergent for Lnt purification
Dimethylsulfoxide (DMSO)InvitrogenD12435Purity: anhydrous
Solbilization Click reagent
Electronic pipet Voyager 8 channels 0.5-12.5 uLINTEGRA4721Purity: NA
Handling reagents
French Pressure CellN/AN/APurity: NA
Cell disruption
FSL-1-biotinEMC microcollectionsL7030Purity: NA
Lnt substrate
Greiner Bio-One 384-well standard CELLSTAR polystyrene microplateGreiner781091Purity: NA
Black with transparent bottom (up or bottom reading)
Greiner Bio-One 96-well sterile polystyrene plate, high bindingGreiner655097Purity: NA
Black with transparent bottom (up or bottom reading)
Microplate reader Infinite M1000 proTecanN/APurity: NA
Fluorescence detection
MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate)AnatraceS110MTPurity: ~100%
Thiol specific inhibitor
Optically Clear Adhesive Seal SheetsThermo ScientificAB-1170Purity: NA
Foil to seal multi-well plate
Sephacryl S400 HR 16/60 gel filtration columnGE HealthcareGE28-9356-04Purity: NA
Lnt purification
StrepTactin Sepharose 50 %IBA Biotechnology2-1201-010Purity: NA
Lnt purification
streptavidinSigmaS4762-1MGPurity: ≥13 units/mg protein
Biotin binding
TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amineSigma678937Purity: 97%
Click reagent
TCEP (tris(2-carboxyethyl)phosphine hydrochlorideSigma75259Purity: ≥98%
Click reagent
TECAN Infinite F500TecanN/APurity: NA
Fluorescence detection
TECAN Infinite M1000 proTecanN/APurity: NA
Fluorescence detection
Thermomixer CEppendorf5382000015Purity: NA
Heated lid
Triton X-100Sigma93443Purity: 10% in H2O
Lnt reaction buffer
Ultra centrifugeBeckman LCN/APurity: NA
Cell fractionation

References

  1. Buddelmeijer, N. The molecular mechanism of bacterial lipoprotein modification--how, when and why. FEMS Microbiology Reviews. 39 (2), 246-261 (2015).
  2. Kovacs-Simon, A., Titball, R. W., Michell, S. L. Lipoproteins of bacterial pathogens.<....

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