Growth inhibition of granulocyte-macrophage colony-forming cells by human cytidine deaminase requires the catalytic function of the protein.
Previous studies have indicated that cytidine deaminase (CDD) is a potent growth inhibitor of granulocyte-macrophage colony-forming cells (GM-CFC). In this study, we have undertaken molecular cloning and purification of recombinant human CDD to elucidate the growth regulatory potential and mechanism behind the growth suppressive effect. The purified protein had a specific activity of 1.35 x 10(5) U/mg and a Km value of 30 micromol/L. In the GM-CFC assay, the recombinant protein was shown to reduce colony formation to 50% at 16 pmol/L concentration. Similarly, as was observed with CDD derived from granulocyte extract, the effect depended on the presence of thymidine (>/= 4 x 10(-5) mol/L). These results imply that CDD is an extremely potent inhibitor of GM-CFC and that no additional factor from the granulocyte extract is required for the growth inhibitory effect. Modification of CDD by truncation from the C-terminal end, or by amino acid substitution of an active site glutamate residue, eliminated both the enzyme activity and the growth regulatory potential of CDD. Furthermore, CDD from Escherichia coli was found to be even more effective than human CDD in growth suppression of GM-CFC, with 10-fold higher inhibitory activity corresponding to a 10-fold higher enzymatic activity. Taken together, these results show that the catalytic nucleoside deaminating function of the protein is essential for the growth suppressive effect of CDD. Most probably, CDD exerts growth inhibition by depleting the cytidine and deoxycytidine pool required for DNA synthesis, as addition of deoxycytidine monophosphate, which is not a substrate for CDD, neutralizes the inhibiting effect.
Gran
C
C
Institute of Medical Microbiology, Department of Molecular Biology, University of Oslo, The National Hospital, Oslo, Norway.
Bøyum
A
A
Johansen
R F
RF
Løvhaug
D
D
Seeberg
E C
EC
D000328:Adult; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D006098:Granulocytes / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D019891:Leukopoiesis; D008264:Macrophages / Q000166:cytology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D003564:Cytidine Deaminase