Title : Human
thrombopoietin structure-function relationships: identification of functionally important
residues
Abstract :
- Thrombopoietin ( TPO ) is a haematopoietic growth factor responsible for megakaryocyte progenitor proliferation and differentiation
- It belongs to the four-helix-bundle cytokine family and exerts its biological effects through binding to a specific receptor , c-Mpl
- With the use of site-directed mutagenesis we have generated 20 TPO mutants
- Each of the TPO mutants was produced in a eukaryotic expression system and the mutants' ability to induce the proliferation of factor-dependent c-Mpl-expressing megakaryoblastic M-O7e cells was compared with that of wild-type TPO
- Among the mutations studied, 10 lead to a significant decrease in TPO bioactivity
- Of these ten residues, three are located in helix A of the protein ( Arg10, Lys14 and Arg17 ) and four in helix D ( His133, Gln132, Lys138 and Phe141 ), indicating that in TPO , as in other cytokines, these two helices are important for functional cytokine/ receptor interactions
- Surprisingly, mutant Arg10--> Ala (R10A) lacked any proliferative activity, despite the fact that this mutation was recently reported to have no effect on TPO / c-Mpl binding in a TPO phage ELISA [Pearce, Potts, Presta, Bald, Fendly and Wells (1997) J. Biol
- Chem
- 272, 20595-20602]
- The lack of M-O7e proliferation is probably due to an inability of R10A mutant to promote receptor dimerization and thus receptor activation
- Moreover we found that the Arg10 and Arg17 residues of TPO seem to be specific determinants for TPO / c-Mpl recognition
- We also demonstrate that the O-glycosylation site located at position 110 of TPO is not necessary for the bioactivity of the cytokine