Effect of the His-Tag Location on Decapping Scavenger Enzymes and Their Hydrolytic Activity toward Cap Analogs

Aleksandra Ferenc-Mrozek; Elzbieta Bojarska; Janusz Stepinski; Edward Darzynkiewicz; Maciej Lukaszewicz

doi:10.1021/acsomega.0c00304

Effect of the His-Tag Location on Decapping Scavenger Enzymes and Their Hydrolytic Activity toward Cap Analogs

ACS Omega. 2020 May 6;5(19):10759-10766. doi: 10.1021/acsomega.0c00304. eCollection 2020 May 19.

Authors

Aleksandra Ferenc-Mrozek^{1

2}, Elzbieta Bojarska², Janusz Stepinski¹, Edward Darzynkiewicz^{1

2}, Maciej Lukaszewicz¹

Affiliations

¹ Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-093 Warsaw, Poland.
² Centre of New Technologies, University of Warsaw, 02-093 Warsaw, Poland.

Abstract

Decapping scavenger enzymes (DcpSs) are important players in mRNA degradation machinery and conserved in eukaryotes. Importantly, human DcpS is the recognized target for spinal muscular atrophy (SMA) and acute myeloid leukemia (AML) therapy, and has recently been connected to development of intellectual disability. Most recombinant DcpSs used in biochemical and biophysical studies are prepared as tagged proteins, with polyhistidine (His-tag) at the N-terminus or C-terminus. Our work is the first report on the parallel characterization of three versions of DcpSs (native and N- or C-terminally tagged) of three species (humans, Caenorhabditis elegans , and Ascaris suum). The native forms of all three enzymes were prepared by N-(His)₁₀ tag cleavage. Protein thermal stability, measured by differential scanning fluorimetry (DSF), was unaffected in the case of native and tagged versions of human and A. suum DcpS; however, the melting temperature (T _m) of C. elagans DcpS of was significantly influenced by the presence of the additional N- or C-tag. To investigate the impact of the tag positioning on the catalytic properties of DcpS, we tested the hydrolytic activity of native DcpS and their His-tagged counterparts toward cap dinucleotides (m⁷GpppG and m₃ ^2,2,7GpppG) and m⁷GDP. The kinetic data indicate that dinucleotide substrates are hydrolyzed with comparable efficiency by native human and A. suum DcpS and their His-tagged forms. In contrast, both His-tagged C. elegans DcpSs exhibited higher activity toward m⁷GpppG than the native enzyme. m⁷GDP is resistant to enzymatic cleavage by all three forms of human and nematode DcpS.