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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4127-4135
By
From the Institute of Medical Microbiology, Department of Molecular
Biology, University of Oslo, The National Hospital, Oslo; the Norwegian
Defense Research Establishment, Division for Environmental Toxicology,
Kjeller; and NYCOMED Imaging A/S, Oslo, Norway.
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 × 105 U/mg and a Km value of 30 µmol/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 (
THE DEVELOPMENT of blood cells from
hematopoietic stem cells in the bone marrow is a complex process
dependent on the microenvironment constituted by stromal cells,
cytokines, and the extracellular matrix.1-4 The cytokines
mediate communication between the cells and exert their biological
functions mostly through specific receptors expressed on the surface of
the target cells. These signal substances have the capacity to
stimulate, enhance, or suppress the proliferation of the stem and
progenitor cells. Some cytokines even have pleiotropic effects, with
different activities depending on the target cell or assay system
used.5 The inhibitors constitute a rather heterogeneous group of molecules, ranging from higher molecular weight protein factors like the transforming growth factor- We have previously shown that mature human blood granulocytes produce
an inhibitor of granulopoiesis in diffusion chambers11 and
granulocyte-macrophage colony-forming cells (GM-CFC) in agar assays.12 The inhibitor was identified as cytidine
deaminase (CDD), as judged from different experimental
criteria.13 The inhibitor copurified with CDD activity, the
molecular weight of the inhibitor was found to be in the same size
range of that reported for CDD ( To distinguish between these possibilities and further characterize the
molecular effects of CDD on granulopoiesis, we have undertaken
molecular cloning, purification, and characterization of human CDD. It
is shown that human recombinant CDD (rhCDD) is as effective as
authentic CDD from mature granulocytes in suppressing GM-CFC.
Furthermore, site-directed mutagenesis of active site residues of CDD indicates that the growth inhibitory function of CDD in
granulopoiesis is correlated to the enzymatic activity of the protein
and is the result of depleting the pool of cytidine and dC required for
genome replication.
Materials.
Random priming kit and T4 DNA ligase were obtained from Boehringer
Mannheim (Mannheim, Germany).
Synthesis of DNA probe and screening of cDNA library.
The incomplete CDD nucleotide sequence of Kühn et
al16 was used to synthesize the 5
Enzyme activity analysis by complementation.
Escherichia coli (E coli) strain JF611 (pyrE60 cdd
thi-1 argE3 his-4 proA2 thr-1 leu-6 mtl-1 xyl-5 ara-14 galK2 lacY1 rpsL supE44) lacks CDD activity and pyrimidine de novo synthesis and is
phenotypically unable to use cytidine or dC as sole pyrimidine source.21 Thus, the double mutant requires either uracil or uridine for growth. The strain was used to check plasmid constructs for
functional expression of human CDD by growth tests in the absence of
uracil or uridine. The pBK-CMV transformed bacteria was induced with
0.2 mmol/L isopropyl- Isolation of bacterial cell extracts by plasmolysis.
The procedure was performed on ice.22 A total of 4 mL of
cell culture was centrifuged at 3,500 rpm and the cell pellet washed in
0.04 mol/L Tris-HCl, pH 8. The cells were pelleted, resuspended in 84%
sucrose solution (50 µL 0.04 mol/L Tris-HCl, pH 8), and incubated 10 minutes before the addition of 250 µL 50 mmol/L
4-morpholinepropanesulfonate (pH 7.5)/1 mmol/L EDTA/100 mmol/L KCl/1
mmol/L dithiothreitol (DTT)/125 µg/mL lysozyme. After a 45-minute
incubation, cell debris and DNA was pelleted at 13,000 rpm for 15 minutes and the supernatant frozen on dry ice/ethanol.
CDD assays.
CDD activity in cell extract isolated by plasmolysis was determined by
radiochemical and spectrophotometric assays. The radiochemical assay is
described previously,13 with the modification that the
enzyme was diluted in buffers containing 200 µg/mL bovine serum
albumin (BSA) and 1 mmol/L DTT to stabilize the enzyme. The
spectrophotometric assay is based on the decrease of absorbance when dC
is converted to deoxyuridine (dU) at 235 nm where DNA sequencing.
The insert in pBK-CMV was sequenced on both strands using Sequenase
version 2.0 (United States Biochemical, Cleveland, OH). The primers used were: P1, P2, P3 (complementary to P1), P4
(5 Expression of rhCDD in E coli from pT7-SC.
The CDD cDNA was cloned into the vector pT7-SC (USB) and used
for transformation of E coli BL21 (E coli B
F- dcm ompT hsdS(rB-
mB-) gal). The T7 RNA polymerase
(encoded on a lysogenic lambda bacteriophage), and hence, CDD, were
induced with 1 mmol/L IPTG for 2 hours. Expressed proteins were
analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as
described by Schägger and von Jagow23 using a 16.5%
T separating gel made from the 49.5% T/3% C acrylamide-bisacrylamide mixture and a 4% T stacking gel.
Purification of rhCDD.
All purification steps were performed at 4°C. The cell extract,
from 4 L of bacterial cells prepared as described,22 was applied to an Affi-Gel Blue column (Bio-Rad) preequilibrated with buffer A (20 mmol/L Tris-HCl, pH 7.5; 50 mmol/L KCl; 1 mmol/L EDTA; 1 mmol/L DTT; and 20% glycerol). The column was washed with buffer A and
proteins were eluted with 1 mol/L KCl in buffer A. The eluate was
desalted by PD-10 chromatography (Pharmacia), and applied to a Mono Q
anion exchange column (0.7 × 5.5 cm; Pharmacia Fast Protein
Liquid Chromatography, FPLC). After washing, the column
was developed with a linear gradient to 0.2 mol/L KCl in buffer A at a
flow rate 0.2 mL/min. CDD active fractions were desalted by PD-10 and
applied to a Mono S cation exchange column (0.7 × 5.5 cm;
Pharmacia). The column was eluted with 0.2 mol/L KCl in buffer A. The
final step yielded a single ultraviolet (UV)-absorbing (280 nm) peak of rhCDD. Protein concentration was determined by the
Bio-Rad protein assay (Bio-Rad), with BSA as a standard.
Purification of E coli CDD.
The E coli CDD gene was amplified by polymerase chain reaction
(PCR) from strain AB1157 (wild-type). The primers were selected from
the CDD sequence entered in the EMBL Data Library
(Accession no. X63144). The 5 Enzyme kinetics.
For the determination of Km in a Lineweaver-Burk plot,
initial rates (vi) were measured as a function of substrate
concentrations covering the range at least from 0.5 to 5 × Km. Quartz cuvettes of 1-cm light path were used.
Cells.
Bone marrow cells (BMC) were obtained from the femurs of
female (C57BL/6JxDBA/2) F1 hybrid mice (Bomholdt Gaard,
Ejby, Denmark). MNC from human peripheral blood and buffy
coat samples were separated as previously described.13,24 A
human bladder carcinoma cell line (5637) was kindly provided by Dr
Andrew King (SmithKline Beecham, Philadelphia, PA). The
cell line was cultured in RPMI 1640 medium with 10% FCS and
subcultured once or twice weekly. The medium conditioned by the
carcinoma cell line is a rich source of human granulocyte
colony-stimulating factor (G-CSF) (CM 5637).
GM-CFC assay.
Mouse BMC (5 × 104/mL) were cultured in
0.3% agar (Bacto-Agar; DIFCO, Detroit, MI) or 1%
methylcellulose (Methocel MC 4000; Fluka) in McCoy's 5A medium with
16% FCS, 1 mL per culture dish. A total of 10% CM 5637 was used as
stimulator in the routine agar assay, 5 ng/mL interleukin (IL)-1 Site-directed in vitro mutagenesis of CDD.
cDNAs expressing truncated forms of CDD were constructed by PCR using
primers with translation stop codons introduced at defined positions.
The sequence of the PCR primers were: P8,
5 Isolation of human CDD cDNA clones.
Approximately 5 × 105 plaques from a human
blood cDNA library constructed in the Expression of human CDD in E coli.
CDD cDNA excised from the pBK-CMV phagemid was ligated into
pT7-SC, a T7 RNA polymerase-based expression system useful for high
production of toxic proteins in E coli. After 2 hours of IPTG
induction, a strong band of about 17.5 kD was observed by SDS-PAGE analysis (Fig 2, lane
2), which is in reasonable agreement with the predicted monomeric
molecular mass of CDD of 16.2 kD. Enzyme activity analysis of crude
extract showed 80-fold higher activity than in bacteria transformed
with the CDD cDNA in the original pBK-CMV vector.
Purification of rhCDD.
The recombinant CDD was purified by a three-step procedure
(Table 1). The Affi-Gel Blue column was quite effective,
removing 85% of the protein with only 8% loss of the enzyme activity,
and yielding a fraction producing only one distinct protein band on the
gels. However, minor contaminants were removed by additional Mono Q and
Mono S chromatography steps (Fig 2, lane 3). At the final step, the
enzyme was purified 18-fold with a 31% recovery implying that the
rhCDD constitute nearly 6% of the soluble protein in the extract.
Cross-linking of rhCDD in 0.01% glutaraldehyde overnight at room
temperature followed by SDS-PAGE suggested a homotetramer composition
of the enzyme (Fig 2, lane 4). The final specific activity of the pure
enzyme was about 1.35 × 105 U/mg. In a
Lineweaver-Burk plot the characteristic Km-value for CDD
was measured to 30 µmol/L at pH 7.5 and room temperature.
Recombinant CDD is an inhibitor of GM colony formation.
Previous studies have shown that CDD in granulocyte extract acts as an
inhibitor of GM-CFC13; an effect dependent on the presence
of thymidine in the growth medium.13,14 In those
experiments, partially purified CDD from granulocytes was investigated,
and it could not be excluded that additional factors in the extract
would be required for the CDD effect. However, rhCDD mediates efficient
inhibition of GM colony formation of both mouse BMC and human MNC, thus
excluding that any additional protein factor is needed
(Fig 3). These results imply that CDD produced in
bacteria is fully active and can be used in further studies of the
growth regulatory effect. RhCDD appears to be a very potent inhibitor
of GM-CFC; 1 ng/mL (16 pmol/L) enzyme was found to reduce the GM colony
formation of mouse BMC to approximately 50%, whereas 6 ng/mL (96 pmol/L) produced almost a complete inhibition. Thymidine at greater
than or equal to 4 × 10-5 mol/L concentration is
required for significant effects of recombinant CDD on colony formation
(Fig 4) in agreement with results obtained previously
from similar experiments with granulocyte extract preparations. Thymidine alone at these concentrations has no effect on GM-CFC (data
not shown).
The effect of mutant CDD proteins on GM colony formation.
The mechanism of action for the growth suppressive effect of CDD has
not been studied in any detail. We have investigated the possible
enzyme activity requirement for the growth regulatory function of CDD
by producing different modified forms of the enzyme. Three different
truncated forms of the enzyme were made, deleted from the C-terminal
end, including 115, 123, and 134 amino acids of the 146 amino acid CDD
sequence, respectively. Possible enzymatic activity of the three
truncated forms of CDD was measured in cell extracts from IPTG-induced
BL21, transformed by the expression plasmids, using both the
spectrophotometric and radiochemical assay methods. The truncated
enzymes were all inactive, the measured activity being equivalent to
the background value caused by E coli CDD in the crude extract
(data not shown). The truncated forms were purified through the Affi
Gel step, quantified by immunologic staining (Gran and Seeberg,
unpublished observations, January 1997), and analyzed for
possible growth suppressive effect on GM-CFC. The mutant proteins were
all unable to inhibit colony formation (Fig
5, and data not shown). These results strongly suggest a correlation
between the enzyme activity and the growth regulatory effect of CDD.
However, the possibility remained that the truncated CDD fragments did
not fold properly and therefore were inactive due to an altered
structural conformation. Therefore, we also constructed three point
mutant forms of CDD; E67D (primer P12), E67Q (P13), and E67A (P14). E67
is positioned in a conserved sequence cluster common to all the CDD
sequences known (Fig 1B) and is localized to the active site of the
enzyme interacting with the substrate.25 Replacing the
corresponding residue in the E coli enzyme by alanine has
previously been shown to reduce kcat by 8 orders of
magnitude.26 Spectrophotometric CDD assays showed no enzyme
activity above background in extract from mutant forms expressed in
E coli by IPTG induction. GM-CFC assays with the site-specific
CDD mutant proteins confirmed that the catalytic function of CDD is
essential for the growth suppressive function of CDD (Fig
5).
E coli CDD is a potent inhibitor of GM-CFC.
Another approach to investigate the significance of enzyme activity in
regulating GM-CFC is to compare growth suppressive effects of CDD from
different organisms relative to the specific activities of the enzymes.
The coding region of E coli CDD was PCR-amplified and expressed
with the pT7-SC system. High expression of a 31-kD protein was observed
by SDS-PAGE and the enzyme was purified to homogeneity by a three-step
purification procedure (Table 2). The
specific activity of pure E coli CDD was measured to 1.25 × 106 U/mg, an approximately 10-fold higher activity
than for the human enzyme. The Km value was estimated to
130 µmol/L compared with 30 µmol/L for rhCDD. The potential
inhibiting effect of E coli CDD was tested in a GM-CFC assay
using both human MNC and mouse BMC. The bacterial form of the enzyme
appeared to be a more potent inhibitor of GM-CFC than the human enzyme
(Fig 6). In combination with thymidine
(10-4 mol/L), an enzyme concentration of 50 pg/mL (0.8 pmol/L) yielded a 50% reduction in GM colony formation of human MNC
from peripheral blood, 400 pg/mL (6.5 pmol/L) producing nearly complete
inhibition. This is about 1/15 of the amount of the rhCDD required to
produce the same growth inhibitory effect. When considering the 10-fold higher specific activity of E coli CDD, these results strongly argue that the catalytic function of CDD is essential for the growth
regulatory effect.
The effect of nucleotide precursors on the inhibitory activity of
CDD.
In view of the results showing that the catalytic activity of CDD is
essential for the growth suppressive function, we tested the effect of
different DNA precursors on GM-CFC growth inhibition. Addition of
deoxyadenosine (dA) and deoxyguanosine (dG) up to 10-4
mol/L concentration had no effect or even enhanced growth inhibition (Table 3). Addition of dC produced variable
results depending on the concentration of dC and rhCDD (data not
shown). Because dC is the natural substrate for CDD, any results
obtained with dC will be difficult to interpret, as addition of dC in
any case will compete for the catalytic function of CDD. Therefore, to evaluate if growth inhibition is due to cytidine nucleotide deficiency, experiments with deoxycytidine monophosphate (dCMP) additions were
performed (Fig 7). DeoxyCMP is not a
substrate for CDD,27 but will be taken up by the cells to
an extent sufficient for restoring DNA synthesis if the availability of
cytidine nucleotides is the limiting factor for DNA synthesis and cell
growth. Addition of 10-3 mol/L dCMP together with thymidine
reversed the inhibitory activity of CDD, indicating that the growth
inhibitory effect of CDD can be explained by depletion of the
cytidine and dC pool.
In the present work, we have isolated human CDD cDNA from a
blood cDNA library and expressed and purified rhCDD to homogeneity. The
nucleotide sequence of the open reading frame of the CDD cDNA clone is in agreement with the recently published sequence of Laliberté and Momparler.28 Enzyme activity
measurements showed a functional product with a specific activity of
1.35 × 105 U/mg and a Km value of 30 µmol/L, which agrees with values reported previously.18,28-30 In a GM-CFC assay, we have confirmed
the growth suppressive action of CDD on progenitor cells as previously
described by Bøyum et al13 and excluded the need for other
cofactors, apart from thymidine.14 Approximately 16 pmol/L
of rhCDD in combination with an optimal concentration of thymidine
(10-4 mol/L, Fig 4) reduced GM colony formation of mouse
BMC to approximately 50% (Fig 3). This implies that CDD exerts its
effect at very low concentrations, within the same range or even less
than required for inhibition by transforming growth factor- Submitted August 11, 1997;
accepted January 29, 1998.
We are grateful to Dr Jan Neuhard for the gift of E coli strain
JF611, to Dr Anders Høgset for stimulating discussions and support,
and to Vivi-Ann Tennfjord for technical assistance with some of the
growth experiments.
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Abstract
Introduction
4 × 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.
Abstract
, tumor necrosis factor-
and -
50 kD), and the growth suppressive
effect of granulocyte extract was abolished by
3,4,5,6-Tetrahydrouridine (THU), a well-known inhibitor of
CDD activity. CDD was found to be a species nonspecific, but seemingly
lineage-specific suppressor
- and
3
gt11;
Clontech, Palo Alto, CA). The sequences of these primers
were: P1, 5
70°C. Isolated positive clones were
converted to the pBK-CMV phagemid by in vivo excision as described
(Stratagene).
= 2,250 mol/L-1 for a 1-cm light path and the initial rate is
vi = (
K, and a
silent point mutation at nucleotide 441 from C 
) or absence (
) of 10-4 mol/L thymidine.
The cells were cultured for 7 days in a GM-CFC agar assay with 10% CM
5637 as stimulator. Colonies of more than 50 cells were counted. Mean
values ± standard deviation (SD) are given as percent of
controls.
), Mutant E67D (
), Mutant
E67A (
), rhCDD (
).
), dCMP + rhCDD (