Universität zu Köln

Hauser, Sandra ORCID: 0000-0001-8206-6000, Sommerfeld, Paul ORCID: 0000-0002-8182-0347, Wodtke, Johanna, Hauser, Christoph ORCID: 0000-0003-4103-5843, Schlitterlau, Paul, Pietzsch, Jens ORCID: 0000-0002-1610-1493, Loeser, Reik, Pietsch, Markus ORCID: 0000-0002-8110-2265 and Wodtke, Robert ORCID: 0000-0001-7462-7111 (2022). Application of a Fluorescence Anisotropy-Based Assay to Quantify Transglutaminase 2 Activity in Cell Lysates. Int. J. Mol. Sci., 23 (9). BASEL: MDPI. ISSN 1422-0067

Full text not available from this repository.

Abstract

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/mu g protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Hauser, Sandra UNSPECIFIED orcid.org/0000-0001-8206-6000 UNSPECIFIED Sommerfeld, Paul UNSPECIFIED orcid.org/0000-0002-8182-0347 UNSPECIFIED Wodtke, Johanna UNSPECIFIED UNSPECIFIED UNSPECIFIED Hauser, Christoph UNSPECIFIED orcid.org/0000-0003-4103-5843 UNSPECIFIED Schlitterlau, Paul UNSPECIFIED UNSPECIFIED UNSPECIFIED Pietzsch, Jens UNSPECIFIED orcid.org/0000-0002-1610-1493 UNSPECIFIED Loeser, Reik UNSPECIFIED UNSPECIFIED UNSPECIFIED Pietsch, Markus UNSPECIFIED orcid.org/0000-0002-8110-2265 UNSPECIFIED Wodtke, Robert UNSPECIFIED orcid.org/0000-0001-7462-7111 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-675554
DOI:	10.3390/ijms23094475
Journal or Publication Title:	Int. J. Mol. Sci.
Volume:	23
Number:	9
Date:	2022
Publisher:	MDPI
Place of Publication:	BASEL
ISSN:	1422-0067
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language TISSUE TRANSGLUTAMINASE; COVALENT INHIBITORS; SUBSTRATE PEPTIDE; CLICK CHEMISTRY; FACTOR-XIII; PROTEIN; IDENTIFICATION; EXPRESSION; GTP; TRANSAMIDATION Multiple languages Biochemistry & Molecular Biology; Chemistry, Multidisciplinary Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/67555