(Received for publication, March 21, 1995)
From the
Epidermal growth factor (EGF) is a 53-amino-acid mitogenic
polypeptide present in a variety of tissues and fluids including
kidney, urine, and amniotic fluid. An EGF isoform,
des-Arg
Epidermal growth factor (EGF)
The significance of the conversion of EGF to
des-Arg
The goals of the present
study were 3-fold: 1) to determine whether EGF is a substrate for CPM;
2) to investigate whether the enzyme on cells and in nonserous
biological fluids that converts EGF to des-Arg
Figure 1:
Hydrolysis of EGF by purified
carboxypeptidase M. EGF (7.5 µM) was incubated with CPM
(62.6 ng/ml) at 37 °C for varying times, and the reaction products
were analyzed by HPLC as described under ``Experimental
Procedures.'' A, control, EGF incubated alone in buffer
for 60 min; B, EGF + CPM, 20 min; C, EGF +
CPM, 40 min; D, EGF + CPM, 60 min; E, EGF +
CPM, 60 min in the presence of 10 µM MGTA.
Figure 2:
Metabolism of EGF by MDCK cells. EGF (5
µM) was incubated with confluent monolayers of MDCK cells
at 37 °C for 2 or 4 h, the medium was collected, and the products
were analyzed by HPLC as described under ``Experimental
Procedures.'' A, EGF + MDCK cells, 2 h; B,
EGF + MDCK cells, 4 h; C, EGF + MDCK cells in the
presence of 10 µM MGTA, 4 h.
Figure 3:
Metabolism of EGF by human urine. EGF (7.5
µM) was incubated with 22 µl of urine (5-fold
concentrated) in a final volume of 50 µl for 1 h at 37 °C, and
products were analyzed by HPLC as described under ``Experimental
Procedures.'' A, control, EGF incubated alone in buffer; B, control, urine incubated alone in buffer; C, EGF
+ urine; D, EGF + urine after immunoprecipitation
with anti-CPM antiserum; E, EGF + urine in the presence
of 10 µM MGTA.
Figure 4:
Metabolism of EGF by human amniotic fluid.
EGF (7.5 µM) was incubated with 17 µl of amniotic
fluid for 1 h (A-C) or 22 µl of amniotic fluid for 2
h (D-F) at 37 °C in a final volume of 50 µl. A, EGF + amniotic fluid in the presence of 10 µM MGTA; B, EGF + amniotic fluid; C, amniotic
fluid alone, no EGF added; D, EGF + amniotic fluid
pretreated by immunoprecipitation with specific anti-CPM antiserum; E, EGF + amniotic fluid; F, amniotic fluid
alone, no EGF added.
Figure 5:
Mitogenic effects of EGF and
des-Arg
Figure 6:
Effect
of a carboxypeptidase inhibitor on the mitogenic effects of EGF and
des-Arg
The enzymes responsible for the proteolytic processing of the
1207-amino-acid EGF precursor have not been identified, but recent data
suggest that kallikrein-like enzymes may be involved in some tissues (21, 22) . EGF concentrations in the kidney are
relatively high compared to most other tissues, but 2000-fold lower
than submandibular gland from which the peptide was first
isolated(23) . Nevertheless, the prepro-EGF mRNA level in
kidney is about 50% of that found in submandibular gland and 100- to
1000-fold higher than in other tissues, indicating EGF is synthesized
at a high rate, but is secreted rather than stored
there(23, 24) . This is consistent with the finding
that urine contains high levels of EGF and that urinary EGF is derived
primarily from renal tubules, most likely secreted from cells of the
thick ascending limb of Henle or distal convoluted tubule(23) .
The function of renal and urinary EGF is unclear, but it inhibits
sodium absorption in the cortical collecting
duct(25, 26) , and it may exert a mitogenic effect on
tubular cells and maintain the epithelial surface (23, 27) .
In one of the first purifications of EGF
from human urine, des-Arg
Another biological
fluid that contains EGF is amniotic fluid(1) . In the present
study, exogenously added EGF was metabolized to only
des-Arg
A
membrane-bound carboxypeptidase is involved in the initial
processing/proteolysis of EGF during or after receptor binding and
internalization(3, 4, 5, 6) .
Studies on early processing of mature EGF in fibroblasts (3, 4, 5) and isolated perfused rat livers (6) revealed that EGF is first cleaved by a basic
carboxypeptidase to remove the COOH-terminal Arg, producing
des-Arg
Whether or not removal of the COOH-terminal Arg could
alter other activities or intracellular transport of EGF is not known.
For example, in MDCK cells, 5-30% of the EGF bound to basolateral
receptors is transcytosed to the apical side without the
receptor(30, 31) , during which time (90-120
min) it would be expected that one or more of the initial processing
steps would have taken
place(3, 4, 5, 6) . Although it was
claimed that transcytosed EGF is intact, the methods used to assess
degradation, SDS-polyacrylamide gel electrophoresis (31) or gel
filtration(30) , would not have been able to differentiate EGF
from a metabolite lacking one or a few amino acids. Thus, the initial
processing of EGF may serve as a signal or, alternatively, remove a
signal for targeting of EGF to different extra- or intracellular
locations where it could either exhibit additional activities or be
further degraded. The COOH-terminal Arg does have one known important
function in both EGF and nerve growth factor (NGF). In both cases, the
COOH-terminal Arg is required for association with their respective
binding proteins, as removal of this residue completely abolishes the
ability of EGF and NGF to form high molecular weight
complexes(32, 33) . The functional importance of the
growth factor complexes is not clear, but both EGF and NGF are
protected from hydrolysis by carboxypeptidase B in this
form(34) .
We gratefully acknowledge the cooperation of Dr. Bruno
Michel in purifying human placental carboxypeptidase M and Dr. M.
Fejgin of the Dept. of Obstetrics and Gynecology in supplying the
amniotic fluid samples.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-EGF, has been identified in urine and is the
earliest metabolite generated in target cells upon EGF binding. In this
study, purified carboxypeptidase M efficiently released the
COOH-terminal arginine residue from EGF with a K
= 56 µM, k
= 388 min
, and k
/K
= 6.9
µM
min
. When EGF was
incubated with urine or amniotic fluid, des-Arg
-EGF was
the only metabolite detected. This conversion was blocked by
immunoprecipitation with specific antiserum to carboxypeptidase M or by
10 µMDL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (a
carboxypeptidase M inhibitor), indicating that the major EGF
metabolizing enzyme in these fluids is carboxypeptidase M. When
incubated on a confluent monolayer of Madin-Darby canine kidney (MDCK)
cells, EGF was readily converted to a single metabolite,
des-Arg
-EGF, by carboxypeptidase M. To investigate one
possible functional consequence of this conversion, mitogenic
activities of EGF and des-Arg
-EGF were tested. Both
peptides were equipotent in stimulating
[
H]thymidine incorporation in MDCK cells at all
doses tested. In addition, inhibition of the conversion of EGF to
des-Arg
-EGF by the carboxypeptidase M inhibitor did not
affect the mitogenic potency of EGF. These data indicate that
carboxypeptidase M, present in a variety of cells and biological
fluids, can convert EGF to des-Arg
-EGF. However, in
contrast to many other peptide hormones whose activity depends on a
final carboxypeptidase processing step, removal of Arg
of
EGF is not required for its mitogenic activity.
(
)is a
53-amino-acid polypeptide originally isolated from the mouse
submandibular gland(1) . EGF was subsequently shown to be
synthesized in high amounts in the kidney, whereas most other tissues
generally contain low amounts(1) . Biological fluids also
contain EGF. One of the highest concentrations is in urine from which
two forms were originally isolated; native EGF
and
EGF
(or des-Arg
-EGF), lacking the
COOH-terminal arginine residue (2) . Studies of EGF metabolism
have shown that COOH-terminal processing of EGF occurs rapidly in
target cells (3, 4, 5, 6) with the
earliest metabolite being identified as
des-Arg
-EGF(4) . The carboxypeptidase(s) involved
in the initial metabolism of EGF has not been identified, but it is
likely to be a B-type carboxypeptidase that specifically releases
COOH-terminal Arg or Lys residues from peptides and
proteins(7) . We have previously identified and characterized a
membrane-bound member of this group of peptidases, carboxypeptidase M
(CPM)(7, 8) . CPM was originally purified, cloned, and
sequenced from human placenta (8, 9) and was
subsequently found to be widely distributed. Relatively rich sites
include: organs such as lung, kidney, intestine, brain, and peripheral
nerves; biological fluids such as seminal plasma and amniotic fluid;
cultured cells including Madin-Darby canine kidney (MDCK) cells,
fibroblasts, endothelial cells, and amnion-derived cell
lines(7, 10, 11, 12, 13, 14, 15) .
Naturally occurring substrates for CPM include biologically active
peptides such as kinins, dynorphin A, and enkephalin
hexapeptides(8) . The physiological role of CPM in tissues and
nonserous fluids has not been clearly defined, but may be comparable to
that of carboxypeptidase N in blood(7) . For example, CPM in
kidney and urine can inactivate kinins which affect sodium and water
excretion. CPM in placenta and amniotic fluid could either activate,
alter the properties of, or inactivate a peptide substrate which is
important in fetal development, fetal-maternal exchange, or
parturition. One peptide that has been implicated in all of these
processes is EGF.
-EGF in biological fluids or on target cells is not
clear. In cases where receptor binding and biological activity have
been investigated, des-Arg
-EGF is as potent or more potent
than the parent EGF(5, 16, 17) . However, in
no studies was this conversion blocked to determine whether it is a
necessary processing step for biological activity, as has been found in
other peptide hormone systems. For example, most neuropeptides are
synthesized as a larger precursor protein which must be enzymatically
processed in intracellular secretory granules; first, by
endoproteolytic cleavage at basic residues by a prohormone convertase
(KEX2/subtilisin-like serine protease) and, next, by a B-type
carboxypeptidase (carboxypeptidase E or H) to remove the remaining
COOH-terminal Arg or Lys residue(7, 18) . In
comparison, EGF is synthesized as a 1207-amino-acid, membrane-bound
extracellular protein that must be processed, by still undefined steps,
to release the 53-amino-acid EGF(1) . Because of the relatively
ubiquitous distribution of CPM, the extracellular conversion of EGF to
des-Arg
-EGF could potentially be a normal second
processing step in many or most tissues.
-EGF is CPM;
3) to determine whether the conversion is required for the mitogenic
activity of EGF. MDCK cells were used as a model system to investigate
these questions for two reasons. First, we have previously shown that
CPM is highly expressed on the membrane of these cells as the only
neutral pH-optimum B-type carboxypeptidase(13) . Second, MDCK
cells, being derived from distal tubular epithelial cells, would be
representative of cells exposed to EGF synthesized in the kidney and
secreted into urine in vivo.
Materials
EGF was from Boehringer Mannheim
Biochemica. des-Arg-EGF was from Upstate Biotechnology
Inc. or, alternatively, was generated by hydrolysis of EGF (0.15 mg/ml)
with carboxypeptidase B (1.5 µg/ml) in Tris buffer, pH 8, for
30-60 min at 37 °C(4) . Fetal bovine serum was from
Atlanta Biologicals, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA)
was from Calbiochem, and [
H]thymidine was from
Amersham. Dulbecco's modified Eagle's medium (DMEM),
nutrient mixture F-12 Ham, Hanks' balanced salt solution (HBSS),
carboxypeptidase B, Triton X-100, and protein A-Sepharose 4B were from
Sigma. Trifluoroacetic acid was from Pierce. All other chemicals were
from Fisher Scientific.
Purification and Assay of CPM
CPM was solubilized
from placental microvilli with phosphatidylinositol-specific
phospholipase C and purified as described(8, 19) .
Routine assays of CPM activity were carried out using dansyl-Ala-Arg
substrate as described(8, 19) .
Metabolism of EGF by Purified CPM
In initial
studies, EGF (7.5 µM) was incubated for 0-60 min
with 62.6 ng/ml pure CPM in a 50 mM Hepes buffer, pH 7.5
containing 1 mg/ml bovine serum albumin. Bovine serum albumin was
included to prevent the nonspecific loss of EGF, and the potential for
introducing contaminating protease activity was eliminated by heat
inactivation for 30 min at 60 °C. The products were analyzed by
HPLC using a Waters µBondapak C18 reverse phase column (0.39
30 cm) and an increasing linear gradient of acetonitrile/0.05%
trifluoroacetic acid in water/0.05% trifluoroacetic
acid(12, 15) . Peptides were detected at 214 nm, and
the peaks were quantitated by integration of the peak area and
comparison to the area obtained with a known quantity of authentic
standard treated identically, except lacking CPM. For kinetic studies,
triplicate reactions were run at each of 5 substrate concentrations
ranging from 5 to 100 µM with 31.3 ng/ml pure CPM in the
same buffer as above. The initial rates of product formation (v) (between 5% and 13% of substrate hydrolysis) were measured
at each substrate concentration ([S]), and kinetic constants
were determined by plotting [S] versus [S]/v(15) .
Metabolism of EGF by MDCK Cells
MDCK cells were
grown to confluency in 96-well plates in DMEM supplemented with 2.2
g/liter sodium bicarbonate, 25 mM Hepes, 100 units/liter
penicillin, 0.1 mg/ml streptomycin, and 10% fetal bovine serum. The
monolayers were incubated for 24 h in serum-free DMEM:nutrient mixture
F-12 Ham (1:1), followed by a 1-h preincubation in HBSS supplemented
with 2.2 g/liter sodium bicarbonate, 100 units/liter penicillin, 0.1
mg/ml streptomycin, and 25 mM Hepes with or without 10
µM MGTA. EGF was added to the cells (final concentration
= 5 µM) and incubated at 37 °C for 2 or 4 h, at
which time the medium was collected and the products were analyzed by
HPLC as above.
Metabolism of EGF in Biological Fluids
Amniotic
fluid samples (clinical discards drawn for other purposes) were
obtained from Dr. M. Fejgin of the Dept. of Obstetrics and Gynecology
at the University of Illinois. Human urine was obtained from normal
volunteers and was concentrated 15-fold with Immersible CX-10
ultrafiltration units (10,000-dalton cutoff, Millipore). EGF (7.5
µM final) was incubated with 17-22 µl of either
amniotic fluid or concentrated urine in 50 mM Hepes buffer, pH
7.4 (final volume = 50 µl) with or without 10 µM MGTA for 1-2 h at 37 °C. The products were then analyzed
by HPLC as described above.
Immunoprecipitation
Polyclonal antiserum to
purified human placental CPM was raised in rabbits(8) .
Amniotic fluid or urine (150 µl) was mixed with 70 µl of 50
mM Tris, pH 7.4, containing 0.75 mM NaCl and 5%
Triton X-100, and 120 µl HO, and then preadsorbed with
50 µl of a protein A-Sepharose suspension (10%) for 30 min at 4
°C. The protein A-Sepharose was removed by centrifugation for 4 min
in a microcentrifuge, and the supernatant was incubated with 10 µl
of antiserum against CPM (diluted 1:2) overnight at 4 °C. Protein
A-Sepharose (50 µl of a 10% suspension) was added, and the
incubation was continued for 30 min at 4 °C followed by
centrifugation for 4 min to pellet the immune complexes. Supernatants
were removed and assayed for remaining CPM activity. Control
experiments were conducted in the same way, except either normal rabbit
serum or H
O was used instead of the CPM antiserum.
Mitogenic Effect of EGF and des-Arg
MDCK cells were grown to confluency in 24-well
plates, incubated in serum-free DMEM:F-12 Ham (1:1) for 24 h, and then
pre-incubated for 1 h in HBSS with or without 10 µM MGTA
as above. EGF or des-Arg-EGF on
MDCK Cells
-EGF (1-100 ng/ml) was added
for 20-24 h followed by 2 µCi/ml
[
H]thymidine for an additional 4 h. The buffer
was then removed, and the cells were washed sequentially with
phosphate-buffered saline (0.5 ml/well), 10% trichloroacetic acid (0.25
ml/well twice), methanol (0.25 ml/well) and then solubilized in 0.5 M NaOH (0.25 ml/well) for 20 min followed by neutralization
with 0.5 M HCl (0.25 ml/well). The radioactivity in each well
was counted in a Beckman LS 2800 scintillation counter.
Metabolism of EGF by CPM
Incubation of EGF with
CPM resulted in a time-dependent decrease in EGF and a corresponding
increase in a single product that coeluted with authentic
des-Arg-EGF. The metabolism was blocked in the presence of
10 µM MGTA, a CPM inhibitor (Fig. 1). The
production of only des-Arg
-EGF is consistent with the
known specificity of CPM, which cleaves only COOH-terminal Arg or Lys
from peptide substrates(8) . An identical product was observed
when EGF was incubated with pancreatic carboxypeptidase B (not shown)
which others have shown to yield only
des-Arg
-EGF(4) . These initial studies indicated
that EGF was a relatively good substrate for CPM, compared with other
biologically active peptides. This was confirmed by determination of
the kinetic constants for EGF hydrolysis (Table 1). Thus, the
specificity constant (k
/K
of 6.9 µM
min
) of EGF is similar to that of bradykinin
and about one-third lower than the best substrate,
[Arg
-Met
]enkephalin (Table 1).
Interestingly, whereas EGF has a K
similar to that of
[Arg
-Leu
]enkephalin (which both have
a COOH-terminal -Leu-Arg sequence), the turnover number is about 3-fold
higher for EGF (Table 1).
Metabolism of EGF by MDCK Cells
To investigate
whether des-Arg-EGF can be generated by renal cells or if
other metabolic pathways predominate, EGF was incubated with confluent
monolayers of MDCK cells. A single metabolite,
des-Arg
-EGF, appeared in the medium, with 59.8 ±
1.0% (± S.E., n = 4) converted after 2 h and
84.6 ± 0.3% (n = 4) after 4 h. The metabolism
was completely blocked by 10 µM MGTA, the CPM inhibitor (Fig. 2). Because we previously determined that CPM is the only
B-type carboxypeptidase on the plasma membrane of MDCK cells active at
physiological pH(13) , these data show that CPM is the primary
EGF metabolizing enzyme on these cells.
Metabolism of EGF in Biological Fluids
When
measured with dansyl-Ala-Arg substrate, the CPM-like activity in urine
was 16.6 ± 2.0 nmol/h/ml (n = 7). Specific
antiserum to human CPM precipitated 95.7 ± 0.9% (n = 3) of the activity, showing that the B-type
carboxypeptidase activity in urine is indeed a soluble form of CPM.
When EGF was incubated with three different samples of urine, a single
product, des-Arg-EGF, was formed in all cases, an example
of which is shown in Fig. 3. The rate of conversion of EGF
ranged from 0.41-0.48 nmol/h/ml and was reduced by 85% ±
8% (n = 3) after immunoprecipitation with anti-CPM
antiserum and by 67 ± 9% (n = 3) with 10
µM MGTA (Fig. 3). These data indicate that
des-Arg
-EGF can be generated in urine and that CPM is the
primary EGF metabolizing enzyme here, although another enzyme or
enzymes might play a minor role.
Amniotic fluid hydrolyzed EGF at a
much higher rate, 4.19-8.82 nmol/h/ml (n = 4),
but again, only a single product, des-Arg-EGF, was
produced (Fig. 4). The higher rate of EGF conversion is
consistent with the (on average) 13-fold higher activity of CPM found
in amniotic fluid compared with urine when measured with dansyl-Ala-Arg
substrate(14) . Proof that CPM in the amniotic fluid was
responsible for the hydrolysis of EGF was obtained by
immunoprecipitation with specific antiserum to CPM which reduced the
EGF hydrolysis by 98 ± 1% (n = 3) and by the use
of 10 µM MGTA which completely (100%; n =
3) inhibited hydrolysis (Fig. 4). Thus, in amniotic fluid under
these conditions, CPM is essentially the only EGF metabolizing enzyme.
Effect of Conversion of EGF to des-Arg
Incubation of EGF with MDCK cells resulted
in a dose-dependent increase in [-EGF
on Mitogenic Potency
H]thymidine
incorporation, to a maximum of 160 ± 11% (n =
11) at 100 ng/ml (Fig. 5). The response to EGF is similar to
that reported previously for MDCK cells(20) .
des-Arg
-EGF also stimulated
[
H]thymidine incorporation in MDCK cells, with a
dose-response relationship that was indistinguishable from that of EGF (Fig. 5). These results do not rule out the possibility that the
metabolism of EGF is required for mitogenic activity (i.e. that des-Arg
-EGF is the active form). In order to
investigate this, [
H]thymidine incorporation was
measured in the presence of MGTA (Fig. 6), at a concentration
(10 µM) that completely blocks the conversion of EGF to
des-Arg
-EGF in MDCK cells (Fig. 2). Under these
conditions, EGF maintained its mitogenic effect and was still equally
as potent as des-Arg
-EGF, which was used to control for
any possible nonspecific effects of MGTA (Fig. 6).
-EGF on MDCK cells. Bars represent percent
of [
H]thymidine incorporation compared to control
cells incubated in buffer alone under the same conditions. Values are
given as the mean ± S.E. (n = 10-12). Open bars, des-Arg
-EGF; dark bars,
EGF.
-EGF on MDCK cells. Bars represent percent
of [
H]thymidine incorporation compared to control
cells incubated in buffer alone under the same conditions. Values are
given as mean ± S.E.; n = 11-13 for the
10-ng dose; n = 5 for the 100-ng dose. Dark
bars, EGF; dark slashed bars, EGF in the presence of 10
µM MGTA; open bars, des-Arg
-EGF; light slashed bars, des-Arg
-EGF in the presence
of 10 µM MGTA.
-EGF was identified as an active,
naturally occurring metabolite(2) . However, whether EGF was
converted by an enzyme on renal tubular cells or in urine was unknown.
The present investigation shows that exogenous EGF is converted to
des-Arg
-EGF by renal tubular epithelial cells as well as
in urine, indicting that this conversion may take place in both
locations in vivo. We previously purified and characterized
the major B-type carboxypeptidase in human urine, prior to the
discovery of CPM(12) , and later noted that the two enzymes had
similarities. The present studies show that this urinary enzyme is
indeed a soluble form of CPM. CPM protein and mRNA are also present in
human kidney, showing that the protein is synthesized
there(9, 10) . It can thus be concluded that CPM, both
in its membrane-bound and soluble forms, is likely responsible for the
appearance of des-Arg
-EGF in urine.
-EGF, attributable to hydrolysis by CPM in this
fluid. Whether this conversion influences the physiological effects of
EGF during gestation has not been investigated, although the specific
binding of
I-des-Arg
-EGF was reported to be
18-20% higher than that of
I-EGF to placental
membrane receptors(16) . Although the present data would argue
against an effect on mitogenesis, EGF has other effects that might be
influenced by conversion to des-Arg
-EGF, such as its
ability to increase the level of prostaglandin H
synthetase
and prostaglandin E
production in amnion cells or to
stimulate the production of placental lactogen, human chorionic
gonadotropin, and progesterone in placental cells(1) .
-EGF at the cell surface or in early endosomes. The
second and third steps, occurring in late endosomes, involve
endoprotease cleavage after Lys
followed by basic
carboxypeptidase hydrolysis to produce
EGF
(3, 4, 5, 6) .
Subsequent to these steps, EGF is completely degraded to
trichloroacetic acid-soluble products in lysosomes. The enzymes
carrying out the initial processing steps were not identified and,
although the processing is sequential in cells, it is not known whether
removal of the COOH-terminal Arg of EGF is required before further
cleavage at Lys
can occur. The present study shows that
CPM in MDCK cells efficiently converts EGF to des-Arg
-EGF.
Because CPM is widely
distributed(7, 10, 11, 13, 15) ,
it is likely involved in this conversion on other cell types as well.
Studies on truncated EGF indicate that receptor binding is decreased
only after removal of Lys
and especially Leu
(100-fold decrease in
affinity)(16, 28, 29) . However, it has never
been determined whether the initial processing is required for the
mitogenic activity of EGF. Because EGF is originally released from a
larger precursor protein, it is not unreasonable to speculate that the
release of the COOH-terminal arginine is a final prohormone processing
step as is the case for most neuropeptide
hormones(7, 18) . Some support for this hypothesis is
provided by the finding that des-Arg
-EGF binds to the EGF
receptor with an equal or greater affinity than native
EGF(5, 16, 17) . However, as demonstrated
here, the mitogenic potency of des-Arg
-EGF is equal to
that of EGF on MDCK cells, and the conversion of EGF to
des-Arg
-EGF is not a required step for this activity to be
expressed.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.