Elsevier

Biochimie

Volume 107, Part A, December 2014, Pages 124-134
Biochimie

Research paper
Supported inhibitor for fishing lipases in complex biological media and mass spectrometry identification

https://doi.org/10.1016/j.biochi.2014.07.015Get rights and content

Highlights

  • Synthesis of grafted phosphonate inhibitor.

  • A tool for fishing lipolytic enzymes coupled to mass spectrometry analysis.

  • Increased sensitivity to proteases for proteins trapped in complex mixtures.

Abstract

A synthetic phosphonate inhibitor designed for lipase inhibition but displaying a broader range of activity was covalently immobilized on a solid support to generate a function-directed tool targeting serine hydrolases. To achieve this goal, straightforward and reliable analytical techniques were developed, allowing the monitoring of the solid support's chemical functionalization, enzyme capture processes and physisorption artifacts. This grafted inhibitor was tested on pure lipases and serine proteases from various origins, and assayed for the selective capture of lipases from several complex biological extracts. The direct identification of captured enzymes by mass spectrometry brought the proof of concept on the efficiency of this supported covalent inhibitor. The features and limitations of this “enzyme-fishing” proteomic tool provide new insight on solid–liquid inhibition process.

Introduction

Protein class-directed chemical probes deserve considerable attention as innovative technologies in chemoproteomics [1], [2]. In this context, the optimized design of immobilized inhibitor provides access to activity-based probes (ABP) that can serve as specific capture agents for enzymes. The main advantage of such ABPs is that they can be used on complex biological material to selectively sequester target proteins with minimal sample preparation and processing. Such a technology can thereby be used for preparative purposes in affinity chromatography or for the identification of so-far unknown biomacromolecular targets [3], [4], [5]. In addition, this method can inform on the degree of selectivity of the grafted molecule towards various enzymes within the medium investigated [1], [2]. In various biological contexts, grafted inhibitors serve to considerably improve comparative proteomic analyses, i.e. to monitor phenotypic changes upon exposure to chemical or biological stimuli [6]. Two alternative approaches can be envisaged for the function-directed capture of enzymes: the first one consists in the incubation of soluble inhibitors followed by the capture of the enzyme–inhibitor complex by a functionalized solid matrix. The second involves the preliminary immobilization of the inhibitor on the support and a subsequent solid/liquid inhibition-capture process [7].

Within the hydrolytic enzyme family, serine and cysteine hydrolases, including proteases, lipases and carboxylesterases, are the most abundant classes of enzymes in the living world [8]. In particular, lipases represent a specific class of carboxylesterases hydrolyzing insoluble triacylglycerol substrates and play key roles in fat metabolism, energy mobilization, and bacterial growth. Reactive para-nitrophenyl phosphonate (pNPP)-based inhibitors revealed as relevant probes, targeting the active site of such hydrolases by forming an irreversible covalent bond with the catalytic residue [9], [10], [11], [12]. More generally, phosphonate inhibitors are amongst the most often used probes to efficiently capture lipolytic enzymes or proteases in solution, followed by solid capture of the resulting complex [13], [14], [15]. The alternative strategy of direct solid–liquid capture was envisaged during pioneering studies conducted in the 1970s using immobilized pNPP in order to purify acetylcholinesterase (AChE) from biological extracts [16]. Such systems were subsequently synthetically revisited and applied to the selective removal of chymotrypsin-like proteases from biological samples using diphenyl α-aminoalkylphosphonate immobilized on sepharose gel [17]. We have chosen to evaluate the potency and limitations of this second option, as we were particularly interested in the physical and chemical features of interfacial, heterogeneous and covalent inhibition process of lipases and carboxylesterases using pNPP-based probes.

In the perspective of exploring the strength and limitations of covalent solid–liquid inhibition, we tried to minimize the synthetic and analytical investment required to build and use the grafted inhibitor. One of the main difficulties of solid–liquid covalent capture relies in the analysis of both the supported inhibitor and immobilized protein. Early reports by Reetz et al. [12] suggested the need for routinely accessible analytical techniques to monitor the chemical construction of this ABP and assess the effective amount of available supported active species. The design of a reliable, yet accessible set of analytical assays is therefore crucial to envisage the use of such a molecular architecture for the capture of proteins from complex biological mixtures. The chemical immobilization of the inhibitor on the solid support is far from being a trivial issue either [18]. An additional challenge while targeting lipolytic enzymes comes from the specific nature of the enzyme activation, which is sometimes related to conformational changes involving the opening of a lid that provides access to the active site and can be triggered by the presence of lipids or amphiphiles [19], [20]. Molecular elements that can induce this conformational transition toward the active form prior to capture must therefore be implemented in the design either of the immobilized grafted inhibitor (by selecting an amphiphilic inhibitor whose profile shall be preserved after grafting) or of the assay (by the use of amphiphilic auxiliaries in the incubation medium).

The objective of this study consisted in developing, characterizing and testing on complex mixtures such as entire cellular media or digestive fluids, a molecular tool directed toward serine hydrolases and more specifically lipases and carboxylesterases.

Section snippets

Solid support

Poly[acryloyl-bis(aminopropyl)polyethylene glycol] (PL-PEGA) resins, reagents and chemicals were purchased from Sigma–Aldrich–Fluka Chimie (St-Quentin-Fallavier, France). The methods describing the synthesis and chemical characterization of the compounds 1 to 5 are available in Supporting information.

Purified enzymes

Fusarium solani (Fs) cutinase, human pancreatic lipase (HPL), dog gastric lipase (DGL) and LipY lipase from Mycobacterium tuberculosis were produced as recombinant enzymes and purified following

Chemical synthesis

Starting from prototypes developed by Reetz et al. [12] pNPP were selected for their well-known wide spectrum of inhibition on serine hydrolases and, in particular, on lipases [33], [34]. Alkenylphosphonates 1 and 3 were obtained by adapting reported procedures (Scheme 1A) [35]. The biochemically inert, robust but reversible olefin linkage was chosen to connect the phosphorus building block and the intermediate spacing unit to be linked to the solid support. Indeed, olefin cross-metathesis is a

Conclusion

As regards the increasingly number of ABP reported in the recent literature, we intended to design a tool directed toward lipases and able to uncover some important feature of solid–liquid capture processes. This simple chemical system associated with robust assays to monitor the captured species could be directly conducted without preliminary and arduous treatments of the biological samples. Performances and limitations of our chemical tool and the validity of our monitoring assays were

Conflict of interest

None.

Acknowledgments

Financial support from the Agence Nationale de la Recherche (ANR PCV 2007–184840 PHELIN) (PKK and SM), from the LISA Carnot Institute (ANR n°07-CARN-009-01), from the Ministère de l’Enseignement Supérieur et de la Recherche (VD and BR), Ecole Centrale Marseille (VD) and CNRS are acknowledged.

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