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Amino Acids

, Volume 49, Issue 3, pp 567–583 | Cite as

A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase

  • Christoph Hauser
  • Robert Wodtke
  • Reik Löser
  • Markus Pietsch
Original Article

Abstract

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2’s transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein‐ and rhodamine B‐conjugated cadaverines 13 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 13 were obtained by solid‐phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published l‐lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z′ factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Keywords

Active-site titration Cadaverine Enzyme inhibition Fluorescent labeling Transglutaminases Xanthene dyes 

Abbreviations

ANOVA

Analysis of variance

BHNA

α-Bromo-4-hydroxy-3-nitroacetophenone

BFP

Blue fluorescent protein

2-ClTrtCl

2-Chlorotrityl chloride

DIPEA

N,N-Diisopropylethylamine

DMC

N,N-Dimethylated casein

DMF

N,N-Dimethylformamide

DMSO

Dimethyl sulfoxide

DTT

1,4-Dithio-d-threitol

EDTA

Ethylenediaminetetraacetic acid

ESI–MS

Electrospray ionization mass spectrometry

Etot

Theoretical total enzyme concentration

FA

Fluorescence anisotropy

FITC

Fluorescein-5-isothiocyanate

Fmoc

9-Fluorenylmethyloxycarbonyl

FRET

Förster resonance energy transfer

GFP

Green fluorescent protein

GMP-PNP

Guanosine 5′-[β,γ-imido]triphosphate

gp

Guinea pig

GTP

Guanosine triphosphate

HATU

1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

HEPES

(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HTS

High-throughput screening

Inp

Isonipecotyl

KXD

(S)-tert-butyl 6-amino-1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido) ethylamino)-1-oxohexan-2-ylcarbamate (Boc-Lys-en-dansyl)

l-PACK

N-(2-Hydroxy-5-nitrophenylacetyl)-l-2-amino-4-oxo-5-chloropentanoate

MOPS

3-(N-Morpholino)propanesulfonic acid

NMR

Nuclear magnetic resonance

PP

Polypropylene

PyBOP

Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

RFU

Relative fluorescence units

RP-HPLC

Reversed-phase high-pressure liquid chromatography

Sar

Sarcosyl

SD

Standard deviation

SEM

Standard error of the mean

SNAP-25

25 kDa synaptosome-associated protein

TEA

Triethylamine

TES

2-[[1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid

TFA

Trifluoroacetic acid

TGase

Transglutaminase

TMS

Tetramethylsilane

UV

Ultraviolet

Notes

Acknowledgments

The authors thank Martin Lohse (Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research) for assisting in the synthesis of compound 3. C. H. and M. P. are grateful for support to the Graduate Program in Pharmacology and Experimental Therapeutics of the University of Cologne and the Bayer Health Care AG (Project No. O23). C. H. acknowledges financial support by the Friedrich-Naumann-Stiftung für die Freiheit (ST 6479/P 622). Partial financial support by the Helmholtz Portfolio Topic “Technologie und Medizin—Multimodale Bildgebung zur Aufklärung des in vivo-Verhaltens von polymeren Biomaterialien” (R. W. and R. L.) and by the Fonds der Chemischen Industrie (R. L.) is gratefully acknowledged.

Compliance with ethical standards

Conflict of interest

Funding by the Bayer Health Care AG to C. H. and M. P. has been received via the University of Cologne without any economic obligation. R. W. and R. L. declare that they have no conflict of interest.

Research involving human participants and/or animals and informed consent

This article does not contain any studies with human participants or animals performed by any of the authors. Obtaining informed consent was, therefore, not necessary.

Supplementary material

726_2016_2192_MOESM1_ESM.pdf (1.1 mb)
Supplementary material 1 (PDF 1135 kb)

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Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  1. 1.Center of Pharmacology, Medical FacultyUniversity of CologneCologneGermany
  2. 2.Helmholtz-Zentrum Dresden-RossendorfInstitute of Radiopharmaceutical Cancer ResearchDresdenGermany
  3. 3.Department of Chemistry and Food ChemistryTechnical University DresdenDresdenGermany

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