From the Department of Biochemistry and Molecular Biology, St.
Louis University School of Medicine, St. Louis, Missouri 63104
CKS-17, a synthetic heptadecapeptide homologous
to a conserved domain in retroviral envelope protein p15E, mimics the
immunosuppressive properties of p15E in vitro and in
vivo, but the mechanisms are not understood. Here we report that
a synthetic pentacosapeptide designated CKS-25, a longer version of
CKS-17 that contains a functional transforming growth
factor-
3 (TGF-
3) active-site motif
(RXXD), inhibits 125I-labeled
TGF-
1 (125I-TGF-
1) binding to
cell-surface TGF-
receptors in cultured epithelial cells. Multiple
conjugation of CKS-25 to bovine serum albumin and carbonic anhydrase
enhances the 125I-TGF-
1 binding inhibitory
activity and confers a partial TGF-
agonist activity (growth
inhibition but not transcriptional activation). Since TGF-
is a
potent immunosuppressive factor, these results suggest that the
immunosuppressive properties of CKS-17-bovine serum albumin conjugate
and p15E are mediated at least in part by their TGF-
agonist
activities.
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INTRODUCTION |
Although retroviral infections frequently cause immunosuppression,
the molecular mechanisms of retrovirus-induced immunosuppression are
not understood (1, 2). Increasing evidence suggests that p15E, the
transmembrane envelope protein of feline and murine leukemia viruses,
possesses immunosuppressive activities (1, 2). A hydrophilic 26-amino
acid region of the p15E envelope protein is conserved among the
transmembrane envelope proteins of animal and human retroviruses,
including human T-cell leukemia virus and human immunodeficiency virus
(1-4). CKS-17, a heptadecapeptide whose amino acid sequence is derived
from this conserved region, is immunosuppressive in vitro
and in vivo after conjugation with bovine serum albumin
(BSA)1 (1-4). The mechanisms
by which p15E and CKS-17-BSA conjugate cause immunosuppression are not
known (1, 2). Here we show that CKS-25, a pentacosapeptide and a longer
version of CKS-17 that possesses a putative TGF-
3
active-site motif RXXD (5), inhibits
125I-labeled TGF-
1 binding to cell-surface
TGF-
receptors. Evidence is presented that the RXXD motif
is essential for the inhibition of TGF-
binding. We also demonstrate
that CKS-25-BSA and CKS-25-carbonic anhydrase (CA) conjugates possess a
TGF-
agonist activity.
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EXPERIMENTAL PROCEDURES |
Materials--
Na125I (17 Ci/mg) and
[methyl-3H]thymidine (67 Ci/mmol) were
purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). High molecular mass protein standards (myosin, 205 kDa;
-galactosidase, 116 kDa;
phosphorylase, 97 kDa; bovine serum albumin, 66 kDa), BSA, human CA,
and other chemical reagents were obtained from Sigma. Disuccinimidyl
suberate (DSS) was obtained from Pierce. Human TGF-
1 was
purchased from Austral Biologicals (San Ramon, CA).
125(41-65), a specific TGF-
peptide
antagonist whose amino acid sequence is derived from and corresponds to
the 41st to 65th amino acid residues of TGF-
1, was
prepared as described previously (5). Mink lung epithelial cells were
grown in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum.
Preparation of CKS-25, CKS-25 RA/DA, and CKS-25 Protein
Conjugates--
CKS-25, a pentacosapeptide whose amino acid sequence
corresponds to the 62nd to 86th amino acid residues of a murine
leukemia virus (AKV) (6), and CKS-25 RA/DA, in which both arginine and aspartic acid residues in the RXXD motif are replaced with
alanine residues, were synthesized using tert-butoxycarbonyl
chemistry on an Applied Biosystems model 431A peptide synthesizer and
purified by reverse-phase high pressure liquid chromatography (7). The purity (>95%) of the synthetic peptides was assessed by automated Edman degradation. CKS-25-BSA and CKS-25-CA conjugates were prepared using DSS as the conjugation agent at a molar ratio of DSS:BSA(or CA):peptide (270:8:450) (5). CKS-25-BSA and CKS-25-CA conjugates contained ~5-10 peptides per molecule of protein and were used throughout the experiments. CKS-25-BSA and -CA, which were prepared at
a molar ratio of DSS:BSA (or CA):peptide (27:8:45) contained ~1
peptide per molecule of protein and were not significantly more active
than CKS-25 without conjugation.
125I-TGF-
1 Binding and Affinity
Labeling in Mink Lung Epithelial Cells--
The
125I-TGF-
1 binding and affinity labeling
were carried out as described previously (8, 9). The specific binding
of 125I-TGF-
1 was estimated by subtracting
nonspecific binding from total binding. The nonspecific binding was
obtained in the presence of 100-fold excess of unlabeled
TGF-
1. The 125I-TGF-
1
affinity-labeled cell-surface TGF-
receptors were analyzed by 5%
SDS-polyacrylamide gel electrophoresis and autoradiography.
[methyl-3H]Thymidine Incorporation and RNA
Analysis--
Cells grown on 24-well cluster dishes at near confluence
were incubated with various concentrations of CKS-25, CKS-25-BSA, or
CKS-25-CA in the presence of several concentrations of
TGF-
1 or 10 µM
125(41-65) (5) in Dulbecco's modified
Eagle's medium containing 0.1% fetal calf serum. The assay of
[methyl-3H]thymidine incorporation into
cellular DNA was carried out in triplicate as described previously
(10). For RNA analysis, cells grown on 12-well cluster dishes were
treated with 10 µM CKS-25, 10 µM
CKS-25-BSA, 10 µM CKS-25-CA conjugate, or
TGF-
1 (0.25 and 2.5 pM) at 37 °C for
2.5 h in 0.1% fetal calf serum. The extraction of RNA and
Northern blot analysis were carried out as described previously
(10).
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RESULTS AND DISCUSSION |
We have recently developed three potent synthetic TGF-
pentacosapeptide antagonists (
125(41-65),
225(41-65), and
325(41-65)), which all contain a
(W/R)XXD motif that is essential for their TGF-
antagonist activities (5). Conjugates containing multiple units of
these pentacosapeptides conjugated to BSA or CA have a partial TGF-
agonist activity (5). These results led us to investigate the TGF-
activity of immunosuppressive retroviral envelope proteins that contain
this putative TGF-
3 active-site motif (RXXD)
and have oligomeric structures (1, 2). As shown in Fig.
1, the amino acid sequences for a
conserved region of retrovirus transmembrane envelope proteins contain
a RGLD motif (1, 2). This is homologous to the
motif (RSAD) of
325(41-65) peptide, the amino acid sequence
of which corresponds to the 41st to 65th amino acid residues of human
TGF-
3 (5). The other motif
(LAVE) in human immunodeficiency virus, type 1 gp41 is homologous to those of
125(41-65)
and
225(41-65) peptides
(WSLD and WSAD) (5).
The amino acid sequences of
125(41-65) and
225(41-65) peptides correspond to the 41st
to 65th residues of human TGF-
1 and
TGF-
2, respectively (5). In structural models of TGF-
molecules, the motif (W/R)XXD is located in a loop
structure, which is accessible to solvents (11, 12). Although the RGLD motif is located at the end of an
-helix in the three-dimensional structure of a p15E peptide fragment, its configuration in the structure of intact p15E molecule is not established (13, 14). However,
it seems likely that the side chains of the arginine and aspartic acid
residues in the RGLD motif of p15E protein are exposed to solvents (13,
14).

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Fig. 1.
Amino acid sequences for the conserved region
of retrovirus transmembrane envelope proteins and of human
TGF- 1, TGF- 2, and TGF- 3
peptide antagonists. The putative TGF- active-site motif is
underlined. Human immunodeficiency virus, type 1 gp41 contains two motifs (RAIE and
LAVE). CKS-25, a synthetic
retroviral envelope pentacosapeptide; CKS-17, a synthetic
retroviral envelope heptadecapeptide;
125(41-65), a synthetic
TGF- 1 peptide antagonist with an amino acid sequence
that corresponds to the 41st to 65th amino acid residues of human
TGF- 1;
225(41-65), a synthetic
TGF- 2 peptide antagonist with an amino acid sequence
corresponding to the 41st to 65th amino acid residues of human
TGF- 2;
325(41-65), a synthetic
TGF- 3 peptide antagonist with an amino acid sequence
corresponding to the 41st to 65th amino acid residue of human
TGF- 3; MoMuLV, Moloney murine leukemia virus;
FLV, Friend leukemia virus; AKV, a murine
leukemia virus endogenous to various strains of mice, e.g.
AKR; GLV, Gross leukemia virus; MMCF, mink-cell focus-forming virus of Moloney origin; AMCF, mink-cell
focus-forming virus of AKR origin; FeLV, feline leukemia
virus; BLV, bovine leukemia virus; MPMV,
Mason-Pfizer monkey virus; SRV, simian retrovirus 1;
HTLV-1, human T-cell leukemia virus type 1;
HTLV-2, human T-cell leukemia virus type 2;
HIV-1, human immunodeficiency virus, type 1.
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To test the TGF-
antagonist activity of the immunosuppressive
retroviral envelope proteins (i.e. their ability to block
TGF-
binding to the TGF-
receptors), we synthesized a
pentacosapeptide, designated CKS-25, whose amino acid sequence is
derived from the viral p15E protein of the AKV murine leukemia virus.
It corresponds to amino acid residues 62-86 of the viral protein (6).
CKS-25 includes the whole amino acid sequence of CKS-17 and has
additional amino acid residues at the N and C termini of CKS-17 (Fig.
1). CKS-17 has been shown to comprise the active-site sequence of p15E
(1, 2). The TGF-
antagonist activity of CKS-25 was analyzed by
incubating mink lung epithelial cells with 0.1 nM 125I-TGF-
1 in the presence of various
concentrations of CKS-25 at 0 °C for 2.5 h. As shown in Fig.
2A, CKS-25 at ~75
µM inhibited ~50% of
125I-TGF-
1 binding to cell-surface TGF-
receptors of mink lung epithelial cells. This IC50 (~75
µM) is comparable with that (~20 µM) of
the peptide antagonist,
13(41-65), which
contains the putative TGF-
3 active-site motif (RSAD) (5). Conjugates containing multiple
peptides (~5-10 CKS-25 peptides per molecule of protein) conjugated
to BSA and CA showed enhanced 125I-TGF-
1
binding inhibitory activity (IC50 of ~80-150
nM). The analysis of 125I-TGF-
1
affinity labeling of cell-surface TGF-
receptors revealed that
CKS-25-BSA conjugate (1 µM) almost completely abolished
125I-TGF-
1 binding to all cell-surface
TGF-
receptors including types I, II, III, and V TGF-
receptors
(Fig. 2B, lane 1 versus lane 2). To investigate
the importance of the RGLD motif in the activity of CKS-25, a
structural variant of CKS-25 designated CKS-25 RA/DA (in which the
arginine and aspartic acid residues in the motif were replaced with
alanine residues) was synthesized and tested for its
125I-TGF-
1 binding inhibitory activity.
CKS-25 RA/DA showed no inhibition of
125I-TGF-
1 binding to cell-surface TGF-
receptors in mink lung epithelial cells (Fig. 2A). These
results suggest that the RXXD motif in CKS-25 is important
for its activity.

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Fig. 2.
Inhibition of
125I-TGF- 1 binding (A) and
affinity labeling (B) in mink lung epithelial cells by
CKS-25 and CKS-25-BSA (or -CA). Cells were incubated with 0.1 nM 125I-TGF- 1 in the presence of
various concentrations of CKS-25 or CKS-25-BSA (or -CA). The specific
binding of 125I-TGF- 1 and
125I-TGF- 1-affinity labeling was then
determined. The specific binding of
125I-TGF- 1 in the absence of CKS-25 and
CKS-25-BSA (-CA) was taken as 0% inhibition (12,591 ± 981 cpm/well). The error bars were derived from the standard deviation of
triplicate cell cultures. The figure is representative of seven
experiments, which have comparable results.
125I-TGF- 1 affinity labeling of cell-surface
TGF- receptors was analyzed by 5% SDS-polyacrylamide gel
electrophoresis and autoradiography. The brackets indicate
the locations of types I, II, and III TGF- receptors (T R-I,
T R-II, and T R-III). The arrow
indicates the location of the type V TGF- receptor
(T R-V).
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TGF-
is a potent growth inhibitor for a variety of cell types
including epithelial cells, endothelial cells, and T-cells (2, 15, 16).
To determine the TGF-
agonist and antagonist activities of CKS-25
and CKS-25-protein conjugates, we determined their effects on the
growth of mink lung epithelial cells as measured by
[methyl-3H[thymidine incorporation into
cellular DNA. As shown in Fig. 3A, CKS-25 (10 µM) blocked the inhibition of
[methyl-3H]thymidine incorporation into
cellular DNA induced by 0.25 pM TGF-
1. In
the absence of TGF-
1, CKS-25 did not affect DNA
synthesis. By contrast, the protein conjugates of CKS-25, CKS-25-BSA,
and CKS-25-CA inhibited DNA synthesis ~40-50% in mink lung
epithelial cells at 1 µM (Fig. 3B). The
CKS-25-BSA-induced DNA synthesis inhibition was blocked in the presence
of 10 µM
125(41-65), a
specific TGF-
1 peptide antagonist, suggesting that the
inhibition is mediated by TGF-
receptors (Fig. 3B).
125(41-65) also blocked the
CKS-25-CA-induced DNA synthesis inhibition (data not shown), and both
BSA and CA conjugated without peptide did not have growth inhibitory
activity as reported previously (5). These results indicate that
multiple conjugation of CKS-25 peptides to proteins produces a
multivalent ligand that has TGF-
activity inducing growth inhibitory
response though much higher concentrations are required to exhibit this
activity (IC50 = ~0.1-0.3 µM).

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Fig. 3.
CKS-25 blocking of
TGF- 1-induced inhibition of DNA synthesis
(A) and CKS-25-BSA (or -CA) conjugate-induced inhibition of
DNA synthesis (B) in mink lung epithelial cells.
A, cells were incubated with various concentrations of
TGF- 1 (0, 0.25, and 2.5 pM) in the presence
and absence of 10 µM CKS-25. The
[methyl-3H]thymidine incorporation into
cellular DNA was determined. The [methyl-3H]thymidine incorporation in the
presence and absence of 10 pM TGF- 1 was
taken as 100 and 0% inhibition (434 ± 50 and 25,349 ± 1,412 cpm/well), respectively. The error bars were derived
from the standard deviation of triplicate cell cultures. The figure is
a representative of six experiments, which gave comparable results.
B, cells were incubated with various concentrations of CKS-25-CA or CKS-25-BSA conjugate, as indicated, in the presence and
absence of 10 µM 125(41-65).
The [methyl-3H[thymidine incorporation in the
absence of peptide and peptide-protein conjugate was taken as 0%
inhibition (23,192 ± 582 cpm/well). The error bars
were derived from the standard deviation of triplicate cell cultures.
The figure is a representative of six experiments, which gave
comparable results.
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The transcriptional activation of plasminogen activator inhibitor 1 (PAI-1) and fibronectin is the other characteristic response of TGF-
binding to receptors (15, 16). Thus, we investigated the effect of
CKS-25-BSA on the transcriptional expression of PAI-1. At 10 µM, CKS-25-BSA did not have a significant effect on PAI-1
expression (Fig. 4), indicating that
CKS-25-BSA acts as an agonist in the growth inhibition assay but not in
the assay that measures transcriptional regulation of PAI-1.
Nonetheless, CKS-25-BSA could compete for binding to the TGF-
receptors and function as an antagonist for
TGF-
1-induced PAI-1 expression (Fig. 4).

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Fig. 4.
PAI-1 expression in mink lung epithelial
cells treated with TGF- 1 and CKS-25-BSA. Cells were
treated with TGF- 1 (0 and 2.5 pM) and
CKS-25-BSA (10 µM) at 37 °C for 2.5 h. The
Northern blot analysis of PAI-1 and glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) was then carried out as described in
the text.
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An interesting question raised by the growth inhibitory activities of
CKS-25-protein conjugates reported here is which if any of the known
TGF-
receptor types mediate their growth inhibitory activities. Mink
lung epithelial cells, T-cells, and other normal cell types express
types I, II, III, and V TGF-
receptors (7, 8, 17, 18). The types I,
II, and V TGF-
receptors are Ser/Thr-specific protein kinases and
mediate signaling that leads to cellular responses (7, 8, 17, 18). The
type III TGF-
receptor is a proteoglycan membrane protein, which is
believed not to participate directly in signaling (17, 18). The type V
TGF-
receptor has been recently shown to mediate a growth inhibitory response though the types I and II TGF-
receptors are required for
the maximal growth inhibitory response (8). The CKS-25 monovalent
ligand competes for TGF-
binding to all the receptors. It is thus
possible that the growth inhibitory response stimulated by CKS-25
protein conjugates could be mediated by any of the known receptors or
some combination. The type V TGF-
receptor was suggested as a
candidate by the finding that insulin-like growth factor protein 3, which also contains a WCVD motif (19), is a specific ligand for the
type V TGF-
receptor and exhibits partial TGF-
agonist activity
(growth inhibition but not transcription activation) (10). Like the
CKS-25 protein conjugates, insulin-like growth factor protein 3 exerts
a maximum of ~50% inhibition of DNA synthesis (10).
Since TGF-
is one of the most potent immunosuppressive polypeptides
known (2) and since we show here that CKS-25 protein conjugates can
function as partial TGF-
agonists, we hypothesize that the
transmembrane envelope protein p15E of retroviruses suppresses immune
responses at least in part by their TGF-
agonist activities. This
hypothesis is supported by several lines of evidence. 1) Although
TGF-
is more potent than p15E and CKS-17-BSA conjugate, p15E,
CKS-17-BSA conjugate, and TGF-
have similar immunosuppressive activities (2). 2) CKS-17 or CKS-25 comprises the active-site sequence
of p15E (1, 2). 3) CKS-25 and CKS-25-protein conjugates compete with
125I-TGF-
1 for binding to cell-surface
TGF-
receptors (Fig. 2). 4) CKS-25-protein conjugates by themselves
exhibit growth inhibitory activities that can be blocked by a
monovalent TGF-
-specific peptide antagonist,
125(41-65) (Fig. 3). 5) Multivalency
appears to be required for the TGF-
agonist activity. Similarly, it
is known that the dimeric structure of TGF-
is required for its
biological activities (11, 12). The heterodimerization or
hetero-oligomerization of the TGF-
receptors is known to be
important for the biological responses to TGF-
(20). Also, p15E is a
trimeric protein and present in many copies in the viral envelope (1,
2, 13). CKS-17-BSA/CKS-25-BSA has multiple valences of the putative
active-site motif (1, 2, 5). The multivalency of all of these
structures may allow them to activate TGF-
receptors by inducing
heterodimerization or hetero-oligomerization, which can explain their
TGF-
agonist-like activity in a growth inhibition assay
system.
An interesting and unresolved question is how these multivalent
conjugates show selectivity in their TGF-
agonist activity. It will
be very interesting to determine how the TGF-
receptors respond
selectively with the growth inhibition response without inducing the
other prominent response, producing a significant effect on
transcriptional regulation of PAI-1.
We thank Drs. William S. Sly and Frank E. Johnson for critical review of the manuscript. We also thank John H. McAlpin for typing the manuscript.