Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells.
The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Koichiro
K
Saka
Masahiro
M
Kawahara
Jinying
J
Teng
Makoto
M
Otsu
Hiromitsu
H
Nakauchi
Teruyuki
T
Nagamune
D020816:Amino Acid Motifs / Q000235:genetics; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D053628:Receptors, Thrombopoietin / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D013926:Thrombopoietin / Q000276:immunology / Q000378:metabolism
D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; C082218:MPL protein, human; D013926:Thrombopoietin
Chimeric protein; Cytokine receptor; Hematopoietic stem cell; Signal transduction; Tyrosine motif