Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Gnaccarini, Claudio; Ben-Tahar, Wajih; Lubell, William D.; Pelletier, Joelle N.; Keillor, Jeffrey W.

doi:10.1016/j.bmc.2009.07.031

Cited by 12 publications

(8 citation statements)

References 35 publications

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“…23) N-Hydroxysuccinimide (90.0 mg, 0.780 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiiimide (205 mg, 1.07 mmol) were added to a solution of (+)-biotin (173 mg, 0.710 mmol) in anhydrous DMF (3.00 mL), and the mixture was stirred for 2 h at room temperature under an Ar atmosphere. N-Boc-1,8-diamino-3,6-dioxaoctane (166 mg, 0.710 mmol) in DMF (2.00 mL) and N,N-diisopropylethylamine (367 µL, 2.13 mmol) were then added, and the resulting mixture was stirred for 24 h at room temperature.…”

Section: N-tert-butoxycarbonyl-2-(2-(2-(biotinylamino) Ethoxy)-ethoxymentioning

confidence: 99%

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

Chiba

Hashimoto

Yamaguchi

2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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Chemical probes comprising a ligand moiety, a reactive group (e.g. epoxide, haloacetyl or photoreactive group) and a tag unit (e.g. fluorophore or radioisotope) are widely used in affinity labeling to identify the target proteins of bioactive molecules. However, design and synthesis of highly functionalized chemical probes are often time-consuming. In this paper, we propose a simple design strategy for chemical probes bearing a small 2,3-dichloromaleimide (diCMI) unit, which serves as a combined reactive group and tag unit by reacting with a nucleophilic lysine residue near the ligand-binding site of the target protein to generate the 2-amino-3-chloromaleimide fluorophore. Model ligand-protein experiments confirmed that the diCMI unit has suitable reactivity and fluorogenic capability for efficient affinity labeling.Key words affinity labeling; dichloromaleimide; protein modification Affinity labeling is a powerful method to label and visualize target proteins of bioactive molecules.1,2) This method generally utilizes a chemical probe composed of a ligand moiety, a reactive group and a tag unit (Fig. 1a). The reactive group serves to form a covalent bond with the target protein. For efficient and specific target labeling, the reactive group should be stable in aqueous media, have low reactivity towards non-target molecules, and react appropriately with the target protein after ligand-target binding. The tag unit serves to visualize the target protein. Fluorophores are often used as a tag unit because of the ease of detection. However, a major obstacle to affinity labeling is often synthesis of the highly functionalized chemical probe. More importantly, the ligand must retain its binding affinity after introduction of these two functional groups (reactive group and tag unit). For these reasons, a small alkyne tag is often used in affinity labeling, since it can be subsequently conjugated with a detection unit (e.g. azide-fluorophore, azide-biotin), despite the inconvenience of the additional conjugation step(s).3) We recently reported a small alkoxy nitrobenzoxadiazole (O-NBD, 180 Da) unit as a fluorogenic reactive group for affinity labeling.4) Here, we show that 2,3-dichloromaleimide (diCMI, 164 Da) can serve as an even smaller fluorogenic reactive group (Fig. 1b). Results and DiscussionMolecular Design, Model Reaction and Spectrometric Analysis The maleimide motif is a highly reactive Michael acceptor and has been widely used for chemical modification of thiols of biomolecules.5-7) For instance, fluorophore-conjugated maleimides have been employed for the modification of cysteine residues. 8,9) Recently, 2-bromomaleimide and 2,3-dibromomaleimide were developed as novel cysteine-labeling reagents, which react with thiol in an addition-elimination sequence (nucleophilic substitution) to afford thiomaleimide. 10-18)In an organic solvent such as tetrahydrofuran (THF), 2,3-dibromomaleimide also reacts with amine, affording 2-amino-3-bromomaleimide as an amine-conjugation product. Interestingly, 2-aminomaleimides we...

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Section: N-tert-butoxycarbonyl-2-(2-(2-(biotinylamino) Ethoxy)-ethoxymentioning

confidence: 99%

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

Chiba

Hashimoto

Yamaguchi

2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…An alternative approach has been to label a protein substrate of interest in a reaction mediated by TG2 with a biotinylated fluorophore and subsequently isolate the newly biotinylated protein with streptavidin beads, allowing for immobilization and separation of the product [67]. The sensitivity of this assay allows for detection of 0.6 mU purified TG2, and can also be applied to crude lysates, making it possible to screen for low transpeptidase activities.…”

Section: Assaysmentioning

confidence: 99%

Biotechnological Applications of Transglutaminases

Rachel

Pelletier

2013

Biomolecules

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In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. This specific function has long been exploited in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool, and leather. In recent years, biotechnological applications of transglutaminases have come to light in areas ranging from material sciences to medicine. There has also been a substantial effort to further investigate the fundamentals of transglutaminases, as many of their characteristics that remain poorly understood. Those studies also work towards the goal of developing transglutaminases as more efficient catalysts. Progress in this area includes structural information and novel chemical and biological assays. Here, we review recent achievements in this area in order to illustrate the versatility of transglutaminases.

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“…A TGase-mediated ligation assay was recently reported based on the use of a fluorescent acyl-donor substrate and a biotinylated acyl-acceptor substrate. 26 Seeking a ligation assay that was more amenable to high-throughput screening, direct detection of the ligation reaction has now been achieved without the need for pull down products (Scheme 1). Fluorescein-tagged peptide 1 was employed as acyl-donor substrate.…”

Section: Assay Developmentmentioning

confidence: 99%

Site-specific protein propargylation using tissue transglutaminase

et al. 2012

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Transglutaminases (TGases) catalyse the transamidation of glutamine residues with primary amines. Herein we report the first FRET-based activity assay for the direct detection of the ligation (transamidation) reaction mediated by tissue TGase (TG2). This novel assay was then used in a microtiter plate-based screen of a library of 18 potential amine substrates. From this screen it was discovered that propargyl amine serves as an excellent substrate for TG2. Subsequently, propargyl amine and 2-azidoethyl amine were validated independently as TG2 substrates with K(M) values of 44 ± 4 μM, and 0.99 ± 0.06 mM, respectively. In a proof-of-principle protein labelling experiment, the protein casein was selectively functionalized with propargyl amine using TG2 and subsequently fluorescently labelled through a dipolar cycloaddition reaction with an azido-fluorescein conjugate. This application demonstrates the strong potential of using TG2 for site-specific protein modification through a combination of enzymatic and bioorthogonal chemistry.

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Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Cited by 12 publications

References 35 publications

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

Biotechnological Applications of Transglutaminases

Site-specific protein propargylation using tissue transglutaminase

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