From the Beatson Institute for Cancer Research,
Department of Medical Oncology, CRC Beatson Laboratories,
Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD Scotland,
United Kingdom and the § Aaron Diamond AIDS Research Center,
Rockefeller University, New York, New York 10016
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ABSTRACT |
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Chemokines play diverse roles in inflammatory and
non-inflammatory situations via activation of heptahelical
G-protein-coupled receptors. Also, many chemokine receptors can act as
cofactors for cellular entry of human immunodeficiency virus (HIV)
in vitro. CCR5, a receptor for chemokines MIP-1 Chemokines are a family of structurally and functionally related
proteins that play a central role in the regulation of hemopoietic cell
migration during development, immune surveillance, and the establishment of inflammatory and immune responses (1, 2). These
biological effects are mediated through a family of cell surface
G-protein-coupled heptahelical receptors (3). Recently, these receptors
have been implicated in HIV1
pathogenesis with the discovery that a large number of the members of
this family are able to act as cofactors for the entry of HIV into
cells in vitro (for reviews, see Refs. 4 and 5). The ligands
for these receptors are able to compete for binding with the HIV
envelope protein to abrogate viral entry (6-8). While an in
vivo role for many of these receptors has yet to be demonstrated in the context of HIV infection, CCR5, a receptor for RANTES (regulated on activation, normal T cell expressed and secreted), MCP2 (monocyte chemotactic protein-2), and MIP-1 We have been interested in characterizing receptors mediating the
biological effects of MIP-1 The basis for this marked selectivity has not previously been
addressed, however, it is notable that, while the murine genome contains only one copy of MIP-1 Using full-length and truncated variants of LD78 Chemokines--
MIP-1 Receptor Binding Studies--
CHO cells expressing chemokine
receptors were prepared, and binding assays performed, as outlined
previously (23). In short, binding assays were performed using
125I-labeled murine MIP-1 Ca2+ Flux Assays--
HEK 293 cells stably
expressing human receptors CCR1 and CCR5 were derived by subcloning the
cDNAs into pcDNA3 and transfection using Transfectam (Promega,
Southampton, UK) according to the manufacturer's methods. Stably
transfected cells were selected in 0.8 mg/ml G418. To detect
ligand-induced calcium ion fluxes, cells were loaded with Fura-2-AM,
then, ~6 × 106 cells were incubated at 37 °C in
a continuously stirred cuvette in a Perkin-Elmer LS50 Spectrometer (340 nm ( HIV Entry Assays--
5 × 104 CEMx174-CCR5
cells, pretreated with chemokine for 30 min, were incubated for 4 h with luciferase virus (5 ng of p24), pseudotyped by either JR.FL or
ADA according to a previously described protocol (17). Medium was then
changed and the luciferase activity measured 3 to 5 days
post-infection. The extent of inhibition of HIV entry was determined by
comparing luciferase activity of chemokine-treated cells, with
untreated cells. For studies with the replication-competent SF162
virus, protocols were similar to those described elsewhere (30).
Briefly, CEMx174-CCR5 cells, or PHA/IL-2-activated PBMC from HIV1
seronegative donors, were pretreated with chemokine then infected
overnight at 37 °C with SF162 virus (m.o.i. ~0.04). The medium was
then changed, and the p24 concentration subsequently determined after 5 days at 37 °C using enzyme-linked immunosorbent assay. Percentage
inhibition of HIV entry was calculated relative to chemokine-untreated controls.
Statistical Methods--
Analysis of the dose-response
relationship between the MIP-1 In our recent studies of MIP-1
(LD78
), MIP-1
, RANTES, and MCP2, is of particular importance
in vivo as polymorphisms in this gene affect HIV infection
and rate of progression to AIDS. Moreover, the CCR5 ligands can prevent
HIV entry through this receptor and likely contribute to the control of
HIV infection. Here we show that a non-allelic isoform of human
MIP-1
(LD78
), termed LD78
or MIP-1
P, has enhanced receptor
binding affinities to CCR5 (~6-fold) and the promiscuous
-chemokine receptor, D6 (~15-20-fold). We demonstrate that a
proline residue at position 2 of MIP-1
P is responsible for this
enhanced activity. Moreover, MIP-1
P is by far the most potent
natural CCR5 agonist described to date, and importantly, displays
markedly higher HIV1 suppressive activity than all other human MIP-1
isoforms examined. In addition, while RANTES has been described as the
most potent inhibitor of CCR5-mediated HIV entry, MIP-1
P was as
potent as, if not more potent than, RANTES in HIV-1 suppressive assays.
This property suggests that MIP-1
P may be of importance in
controlling viral spread in HIV-infected individuals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and -
(macrophage inflammatory protein-1
and -1
) appears to be critical in HIV pathogenesis as
individuals who are homozygous or heterozygous for a CCR5 null mutation
are relatively resistant to HIV infection and progression to AIDS,
respectively (9-11). Moreover, the CCR5 ligands are produced by
activated HIV-specific cytotoxic and helper T cells, whereby they are
likely to play an important role in controlling viral spread (12-16).
Current data show RANTES to be the most effective antagonist of
CCR5-mediated HIV entry, with MCP2 and MIP-1
and -1
being less
effective (17-19).
, a member of the
subfamily of
chemokines able to induce chemotaxis of many mature leukocyte types (1,
2) and a potent inhibitor of hemopoietic stem cell proliferation (20,
21). Mice lacking this gene display dramatic alterations in responses
to several infectious agents (22) likely due to leukocyte chemotaxis
abrogation. We have recently cloned several receptors for this
chemokine from mouse and human sources (23, 24) and found a number of
discrepancies with respect to their interaction with murine MIP-1
and its presumed human homologue, LD78
. Thus, while the murine and
human forms of the promiscuous
-chemokine receptor D6 (23, 24) bind
murine MIP-1
with high affinity, LD78
interacts poorly with these
receptors. Similarly, murine CCR5 binds murine MIP-1
with high
affinity but does not recognize the putative human homologue.
and -
, in humans these two genes have been duplicated and mutated to produce two different non-allelic isoforms which still retain >90% homology (25,
26).2 For MIP-1
, these
have been called LD78
and -
, with LD78
being the predominant
experimentally used isoform and the one shown to interact poorly with
the cloned chemokine receptors. Both forms are transcribed (26) and can
be secreted from mammalian cells (6, 21, 27, 28). Interestingly,
consideration of differences in the putative signal sequences of the
two LD78 isoforms by predictive algorithms,2 suggests that
there is variation in the site of signal peptidase cleavage between the
two isoforms during secretion, and that LD78
is in fact produced
without the four anticipated amino-terminal amino acids, ASLA (Fig. 1).
Indeed, human MIP-1
has been shown to be produced naturally by CD8+
T cells as a
4 variant with the amino-terminal sequence ADTPT (6,
27). We have worked extensively with the LD78
isoform (21, 28), and
numerous amino-terminal sequencing exercises have consistently
revealed a "full-length" amino terminus APLAADTPT.
and -
, we show
here that the murine/human MIP-1
binding discrepancies are resolved
by studying the properties of LD78
which consistently behaves more
like murine MIP-1
than does LD78
. It appears therefore that
LD78
more accurately represents the functional human homologue of
murine MIP-1
and that LD78
should be considered to be a related but functional distinct chemokine. Importantly, we also show that LD78
is the most potent natural CCR5 agonist described to date. Furthermore, LD78
exhibits a much greater ability to antagonize HIV
entry through CCR5 than other forms of human MIP-1
. In fact, LD78
is consistently better at HIV1 antagonism than RANTES, previously described as the most potent CCR5-dependant HIV1 entry inhibitor. We
demonstrate that the enhanced activity of LD78
is solely due to the
presence of a proline residue at position 2 of the mature protein. We
propose renaming LD78
and -
to MIP-1
S and MIP-1
P, respectively, to reflect the importance of this residue in the functional differences between these two proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S is "human MIP-1
" purchased from
Peprotech, London, United Kingdom. It is derived from the LD78
cDNA and has the amino-terminal sequence of ASLAADTPT. MIP-1
S-4
is human MIP-1
purchased from R&D Systems, Abingdon, UK. It is
derived from the LD78
cDNA and starts as ADTPT. MIP-1
P is
derived from the LD78
cDNA and was prepared as described
previously (28). Sequencing revealed APLAADTPT at the amino-terminal.
MIP-1
P-4 was produced from a modified LD78
cDNA in
bacteria3 with the amino
terminus of ADTPT.
at a constant concentration of
600 pM (for D6), 3 nM (for CCR1), or 9 nM (for CCR5), while varying the concentration of unlabeled
human MIP-1
competitor protein. Remaining radioactivity bound after
90 min, and three ice-cold phosphate-buffered saline washes, was
determined. Each point was done in triplicate, the average taken, and
converted into a percentage of radioactivity bound in the absence of
any unlabeled competitor chemokine. Data were analyzed using the LIGAND
software (29).
ex); 500 nm (
em)) and fluorescence
emission recorded every 100 ms. After 2 min, ligand was added to a
defined concentration and fluorescence recorded every 100 ms for a
further 2 min. To control for day-to-day experimental variation, a full
dose-response curve for LD78
was performed each time a different
ligand was tested.
isoforms and other chemokines in
binding, calcium flux, and HIV suppressive assays were tested by "log
likelihood" methodology essentially as described previously (31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors (23, 24) that revealed
discrepancies between the binding of murine and human MIP-1
, we used
the LD78
isoform of human MIP-1
with the amino acids ASLAADTPT at
the amino terminus (Fig. 1). The close
sequence similarity between LD78
and murine MIP-1
led us to
examine the possible amino acid residues that may be responsible for
the discriminatory binding of these ligands. The only consistent
feature present in the mCCR5 and hD6 ligands but absent in human
LD78
is a proline residue at position 2 of the mature protein (Fig.
1). The serine residue at position 2 of the LD78
we have used may
therefore be preventing optimal receptor interaction. The alternative
human MIP-1
isoform, LD78
, however, does have a proline residue
in position 2, with two reciprocal serine/glycine swaps in the region between cysteines 3 and 4 being the only other differences between the
LD78
and -
proteins (Fig. 1) (25, 26). To simplify the nomenclature, and emphasize the serine/proline residue difference at
position 2 in the two isoforms, we henceforth refer to LD78
and -
as MIP-1
S and MIP-1
P, respectively.
View larger version (19K):
[in a new window]
Fig. 1.
Alignment of the predicted protein sequences
of mature -chemokines. Human and murine
sequences are prefixed with h and m,
respectively. The second residue of each protein is enlarged,
enboldened, and underlined. Ability to bind to human D6 and murine CCR5
is indicated with a "+" to the right of the
alignment. Non-binding chemokines are labeled with a "
." Binding
information is from Refs. 23 and 24 and Graham (G. J. Graham and
R. J. B. Nibbs, unpublished observations).
MIP-1P Interacts with High Affinity with Both Murine and Human
CCR5 and D6--
To test the importance of these isoform differences,
we have now compared the ability of MIP-1
P and MIP-1
S to interact
with known murine MIP-1
receptors stably expressed on CHO cells
(Table I). In addition, we have tested
the
4 presumed naturally secreted form of MIP-1
S (see
Introduction) as well as the corresponding
4 variant of MIP-1
P to
examine the impact of this truncation on receptor interactions. Our
results demonstrate (Table I) that in contrast to MIP-1
S and
MIP-1
S-4 (Kd values of 136 and 133 nM, respectively), MIP-1
P binds with high affinity to murine CCR5 (Kd of 1.1 nM). The
importance, for this high affinity binding, of the proline residue at
position 2 of MIP-1
P is indicated by the fact that MIP-1
P-4
behaves like MIP-1
S and MIP-1
S-4 on murine CCR5
(Kd 127 nM). Murine MIP-1
, which
carries a proline residue at position 2 of the mature protein, binds to
murine CCR5 in a manner similar to that seen with MIP-1
P, suggesting
that this human variant behaves more like the murine MIP-1
on CCR5
than does the more common MIP-1
S isoform.
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Similarly, the presence of the proline residue at position 2 of
MIP-1P allowed it to bind with high affinity to murine D6 (Kd 5.5 nM) in contrast to the other
three isoforms tested (Kd > 200 nM).
Again the comparison with murine MIP-1
indicates that the MIP-1
P
isoform binds to D6 with an affinity that more resembles murine
MIP-1
than does any of the other human variants tested.
In contrast to the data obtained with murine CCR5 and D6, studies with
murine CCR1 expressing cells indicate that the proline at position 2 is
not essential for high affinity binding of the chemokines to this
receptor, indeed, the Kd observed for the
interaction of full-length proteins with murine CCR1 was markedly
higher than that seen with the 4 truncated isoforms. Additionally,
MIP-1
P demonstrates a substantially higher binding affinity to that
seen with murine MIP-1
indicating that the apparent equivalence of
MIP-1
P and muMIP-1
on murine CCR5 and D6 does not extend to muCCR1.
CHO cells expressing human receptors were also tested and showed
similar results (Fig. 2 and Table II).
Thus, with human D6, while the MIP-1P isoform binds with high
affinity (Kd 5.5 nM), the
4 variants
of the two isoforms bind only weakly (Kd 77 nM for MIP-1
S-4 and Kd 124 nM for MIP-1
P-4) and MIP-1
S does not exhibit an
enhanced binding affinity over its
4 variant (Fig. 2A).
Again, as seen with the murine receptors, the Kd
of MIP-1
P for D6 is more similar to that of murine MIP-1
than any
of the other isoforms tested (Table II).
The differences between MIP-1
S and MIP-1
P are, however, somewhat
less stark than those seen with the murine receptor. With human CCR5,
there is an approximately 4-6-fold higher binding affinity of
MIP-1
P (Kd 6.2 nM) than is seen for
the
4 MIP-1
proteins (Kd 25 and 36 nM), and the addition of the ASLA amino acids to
MIP-1
S-4 results in a further reduction in binding affinity (Fig.
2B). Curiously, muMIP-1
binds in a manner more similar to
that seen with either of the
4 variants than that seen with
MIP-1
P. The clear requirement for the proline residue in position 2 for enhanced binding of human MIP-1
to human CCR5 suggests that the
simple presence of the analogous residue in murine MIP-1
is
insufficient in the context of the other evolutionary changes in this
protein, to mediate high affinity binding to human CCR5. Again, in a
manner similar to that observed for murine CCR1, the proline at
position 2 of MIP-1
P appears to be unnecessary for the interaction
of this ligand with human CCR1, with the full-length versions of MIP-1
S and MIP-1
P, showing a consistently lower affinity
interaction with human CCR1 than either of the
4 variants (Fig.
2C). Again, in common with the data obtained using murine
CCR1, murine MIP-1
binds more like the human
4 variants than
either of the two full-length human MIP-1
isoforms (Table II).
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It is important to note that while all the above binding data was
obtained from displacement studies utilizing radiolabeled murine
MIP-1 and the relevant cold competitor, very similar dissociation constants have been obtained from direct binding studies using radiolabeled human MIP-1
isoforms.
MIP-1 Isoform Binding Variations Are Reflected in Signaling
Potency--
To test whether the observed isoform binding variations
are also reflected in the dose response for signaling through human CCR5 and CCR1, ligand-induced mobilization of Ca2+ was
studied. The transfected CHO cell lines used in the above binding
studies are inefficient at fluxing Ca2+ and for this reason
we have used stably transfected HEK cells in the Ca2+
fluxing studies reported in this paper. Binding analyses using these
cell lines indicates that the tested ligands for CCR1 and CCR5 bind
with equivalent affinities to the receptors expressed in either CHO
cells or HEK cells suggesting the likely equivalence of the receptors
expressed on these different heterologous cells types. With full-length
proteins on CCR5, half-maximal signaling potency is seen at
approximately 10 nM for MIP-1
S and at approximately 500 pM for MIP-1
P (Fig.
3A). The dose-response curves
obtained for these two full-length chemokines are highly significantly different (p < 0.0002) indicating, as was seen with
the binding studies, that MIP-1
P is a markedly better ligand for
CCR5 than MIP-1
S. The MIP-1
S-4 variant shows a slight, but
significant (p < 0.001), increase in signaling potency
through CCR5 compared with MIP-1
S, while removal of the terminal 4 amino acids from the MIP-1
P isoform significantly reduces its
activity approximately 10-fold (p < 0.0002).
Dose-response curves obtained using the two
4 variants were not
significantly different and half-maximal activity was observed for both
at concentrations of approximately 5 nM. The potency of
signaling with MIP-1
P is significantly higher (approximately
10-fold; p < 0.001) than that seen with RANTES (half-maximal activity seen at 2.5 nM) and ~100-fold
higher than that seen with MIP-1
(p < 0.0002).
MIP-1
P is therefore the most active natural CCR5 agonist described
to date. We have detected Ca2+ fluxes with MCP2, which has
recently been identified as a CCR5 ligand (32), but this ligand is not
as potent as MIP-1
in this assay (data not shown). Murine MIP-1
signaling potency through human CCR5 accurately reflected the binding
data with a half-maximal activity being observed at 5 nM, a
concentration that was indistinguishable from that seen with either of
the
4 variants (data not shown).
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Signaling through human CCR1 again reflects the results from the
binding studies and indicates no significant differences in signaling
potency between the full-length isoforms of human MIP-1 which both
display half-maximal fluxing capacity at approximately 2.5 nM (Fig. 3B). The
4 variants, however, while
they are not different from each other in calcium fluxing ability, have
a significantly higher signaling potency than the full-length peptides
(p < 0.02) with half-maximal activity being observed
at approximately 750 pM. In our hands, RANTES demonstrates
a half-maximal fluxing potency at approximately 15 nM on
human CCR1 and is thus significantly less potent than any of the
MIP-1
isoforms tested (p < 0.001). Murine MIP-1
mediated a half-maximal flux at approximately 600 pM, again
reflecting the similar binding potency of this ligand to the two
4
variants on human CCR1.
We have so far been unable to demonstrate a signaling role for human D6
(24) and, despite initial encouraging data (23), have been unable to
reproducibly confirm a signaling role for murine D6. Thus the
functional consequences of the high affinity binding of MIP-1P to
either the murine or human orthologues of this receptor remain to be determined.
In summary, these results show that the proline residue at position 2 of MIP-1P is responsible for the enhanced binding of this isoform to
murine and human D6 and CCR5, and for its strong activation of human
CCR5. Conversely, interaction with CCR1 is lessened in the presence of
APLA or ASLA at the NH2 terminus of human MIP-1
. These
conclusions have been supported using
1,
2, and
3 forms of
MIP-1
S with NH2 termini of SLAADTPT, LAADTPT, and
AADTPT, respectively. In short, on human CCR1 and -5, and D6, the
2
and
3 forms behave like the
4 variants, while -1MIP-1
S acts like
full-length MIP-1
S protein (data not shown). Truncation beyond the
4 position reduces CCR1 and -5 activation properties further (not shown).
MIP-1P Is a Potent Natural Antagonist of HIV Interactions with
CCR5--
Given the strong affinity of MIP-1
P for CCR5, we
investigated the potency of this chemokine as an HIV suppressive
agent. Studies were carried out to investigate the potency of
full-length and
4 variants of both isoforms of human MIP-1
in
suppressing entry of JRFL envelope pseudotyped virus into CEMx174-CCR5
cells. These studies demonstrated MIP-1
P to be a significantly
(p < 0.002) more potent suppressor of HIV entry than
either MIP-1
S or the
4 variants (Fig.
4A). Greater than half-maximal
inhibition of JRFL pseudotyped viral entry was achieved with
concentrations of MIP-1
P as low as 5 ng/ml while 10-20-fold higher
concentrations of the other human MIP-1
variants were required
before 50% inhibition of viral entry was achieved. Similar results
were seen with virus pseudotyped with the envelope of the ADA HIV1
strain, although all the variants were less able to inhibit entry (Fig.
4B).
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RANTES has been reported to be a much more potent antagonist of HIV
entry through CCR5 than MIP-1S, MIP-1
, and MCP2 (17-19). Therefore, further experiments were performed using
replication-competent virus entry into CEMx174-CCR5 cells, and
peripheral blood mononuclear cells (shown in Fig. 4, C and
D, respectively), to compare MIP-1
P to RANTES. These
studies suggest that in addition to being by far the most effective
human MIP-1
variant in suppressing HIV entry into target cells,
MIP-1
P is also at least as potent as RANTES. In fact in repeated
experiments, MIP-1
P was consistently better than RANTES as an HIV
suppressive chemokine, however, these differences did not reach levels
of statistical significance. Again, while MIP-1
P was consistently
less active as an HIV suppressive chemokine than the
NH2-terminal modified form of RANTES, AOP-RANTES (33), this
difference did not reach levels of statistical significance (Fig. 4,
C and D, and see "Discussion"). Note that
RANTES and MIP-1
S-4 can enhance HIV entry into peripheral blood
mononuclear cells at the lower concentrations tested, an effect not
seen with MIP-1
P or AOP-RANTES (Fig. 4D). In contrast to
the CCR5-dependent viruses, no inhibition of
CXCR4-dependent viral entry was observed using any of the
MIP-1
isoforms tested thus confirming the CCR5 dependent selectivity
of the MIP-1
P inhibition (data not shown). These results present
MIP-1
P as one of the most potent naturally occurring inhibitors of
HIV1 entry through CCR5.
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DISCUSSION |
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Studies from our own and other laboratories have demonstrated the
curious inability of human MIP-1 to bind with high affinity to CCR5
and D6 to which the closely related (74% identity at the amino acid
level) murine MIP-1
protein binds strongly (23, 24, 34-36). We now
reveal that the basis for this is the absence of a proline residue at
position 2 of the most commonly used isoform of human MIP-1
(MIP-1
S/LD78
). Our studies on the naturally occurring non-allelic
variant of human MIP-1
(MIP-1
P/LD78
) which has a proline at
position 2, reveal this to be active as a potent CCR5 and D6 ligand. It
therefore more closely resembles murine MIP-1
than do any of the
other human MIP-1
isoforms, suggesting that MIP-1
P may be a
closer functional human homologue of murine MIP-1
than MIP-1
S. It
appears, therefore, that despite the similar levels of identity between
MIP-1
S and P and murine MIP-1
, MIP-1
S is a functionally
evolving variant of MIP-1
. Our results suggest that MIP-1
S should
be regarded as a structurally related but functionally distinct
chemokine which has substantially lost its ability to interact with the
CCR5 and D6 receptors but which has apparently enhanced binding to CCR1
due to the proposed truncation of MIP-1
S by 4 amino acids during
release from the cell. Neither murine MIP-1
nor MIP-1
P are
released from mammalian cells as
4 variants (20, 28,
37)4 and thus this putative
alternative signal peptidase cleavage may be regarded as a component of
the functional evolution of this human chemokine. The importance of the
amino terminus to chemokine function is well documented (32, 38-41)
and it is therefore an ideal position for evolutionary modulation of function.
A major consequence of the enhanced interactions between MIP-1P and
CCR5 is that this chemokine variant is now identified as being as
potent as RANTES and AOP-RANTES in antagonizing CCR5-mediated cellular
entry by HIV1. It is important, however, to point out that MIP-1
P
while not reaching levels of significance, was a consistently more
effective HIV suppressive agent than RANTES. This coupled with the
markedly and significantly lower ability of RANTES compared with
MIP-1
P to flux Ca2+ following CCR5 binding, may suggest
that the inability to demonstrate statistical significance in HIV
suppression is a limitation of the assay system used and this is
currently being investigated in more detail in our laboratories.
Intriguingly, and as shown in Fig. 4, A and B,
while it is consistently difficult to achieve higher than 50%
inhibition of entry by JRFL or ADA pseudotyped viruses using other
MIP-1
variants, MIP-1
P can mediate near 100% inhibition with
reasonable ease. Similar effects have been demonstrated for the
synthetic NH2-terminal variant of RANTES, AOP-RANTES, with
recent data suggesting this to be a consequence of alternative
subcellular deposition of internalized receptors, and associated
impairment of recycling of receptors (42). This may suggest that
MIP-1
P, in contrast to MIP-1
S, alters receptor trafficking
post-ligand binding and this is currently being investigated in our laboratory.
These results attesting to the importance of a proline residue at
position 2 of MIP-1 are likely to have implications for our
understanding of the interactions between other chemokines and their
receptors. For example, it is likely that proline 2 in other
-chemokines is necessary for high affinity binding to D6. This,
however, is not sufficient for D6 interaction, as SDF1 also contains a
proline residue at position 2 yet shows no potential to bind to D6
(data not shown). Thus, proline 2 must be presented in the context of a
-chemokine to permit high affinity D6 interaction, while with CCR5
other domains are likely important in restricting the ligands for this
receptor to MIP-1
, -1
, RANTES, and MCP2. Interestingly, the
NH2 terminus Xaa-Pro in chemokines has been demonstrated to
be a target of dipeptidyl peptidase IV (CD26) (41, 43, 44). We would
predict that MIP-1
P, but not full-length nor
4 forms of MIP-1
S,
would be cleavable by this protease with dramatic changes in its
properties, specifically a near total loss of interaction with D6, a
reduced signaling capacity (and inhibition of HIV entry) through CCR5,
and enhanced CCR1 activity. In fact, studies we have performed on a
2
form of MIP-1
S (not shown) suggest that these properties will be
seen with CD26-cleaved MIP-1
P.
The role of the proline residue in CCR5 activation is of interest in
light of recent data indicating the crucial role of proline 2 in SDF1
activation of its receptor CXCR4, which acts as an entry cofactor for
T-tropic HIV1 strains. Indeed, mutation of this residue to glycine
generates a high affinity CXCR4 antagonist (45). It is intriguing that
both major HIV entry co-receptors have a strong preference for a
proline residue at position 2 during receptor activation. Moreover, as
proposed above for MIP-1P, SDF1 has been demonstrated to lose its
anti-HIV and chemotactic activities upon removal of the first two amino
acids by CD26 cleavage (43). Targeted reduction of CD26 activity
in vivo may therefore enhance the activity of these HIV
suppressive chemokines.
In addition to being of general interest to workers in the chemokine
and HIV fields, our data also open up a number of potential therapeutic
avenues of research. For example, earlier studies have demonstrated
that the MIP-1P copy number varies between individuals, and this
gene can in fact be absent from some individuals (25, 26).
-Chemokine production has been reported to be associated with a
number of inflammatory and autoimmune diseases (1, 2), and deletion of
the MIP-1
gene in mice dramatically alters responses to several
infectious agents (22). In addition,
-chemokine production is
clearly of importance in regulating the pathogenesis of AIDS (6,
12-16). Thus, given the ease with which MIP-1
P is transcribed and
translated (21, 26, 28)5
MIP-1
P gene dosage may alter an individuals' response in these pathological situations. In particular, in light of the potent HIV
entry inhibition by MIP-1
P described here, it would be of interest
to test whether gene copy number affects the rate of progression to
AIDS in HIV-infected individuals.
The demonstration of the potent HIV suppressive action of MIP-1P
also indicates that it is possible that, as has been demonstrated with
the chemokine RANTES (33, 39), amino-terminal variants of this protein,
such as AOP-linked MIP-1
P, may exhibit enhanced HIV1 entry
inhibition and/or receptor antagonism and have potential as HIV1 therapeutics.
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ACKNOWLEDGEMENTS |
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We thank Prof. John Wyke and Dr. Paul Clapham for critical reading of the manuscript. R. J. B. N. thanks Dr. Amanda Wilson for manuscript review and support services.
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FOOTNOTES |
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* This work was supported by the Cancer Research Campaign (to G. J. G), National Institutes of Health Grants CA72149, AI36057, and AI1384 (to N. R. L.), and AmFar Grant 02580-23-RGV (to J. Y. and N. R. L).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Elizabeth Glaser Scholar of the Pediatric AIDS Foundation.
** To whom correspondence should be addressed. Tel.: 44-141-330-3982; Fax: 44-141-942-6521; E-mail: g.graham{at}beatson.gla.ac.uk.
2 R. J. B. Nibbs, unpublished data.
3 A. N. Parker, G. J. Graham, A. R. Sim, S. C. Clark, and I. B. Pragnell, manuscript in preparation.
4 G. J. Graham, unpublished observations.
5 G. J. Graham and R. J. B. Nibbs, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
HIV, human
immunodeficiency virus;
MCP2, monocyte chemotactic protein-2;
MIP-1
and -
, macrophage inflammatory protein-1
and -
;
CHO, Chinese
hamster ovary.
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REFERENCES |
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