From the
Interleukin-8 (IL-8) has at least two binding regions for both
the A and the B type IL-8 receptors. This study defines an important
region between Cys
IL-8
We now report that
mutation of amino acids 13 and 15 from the rabbit (His-Ser-Thr) to the
human sequence (Tyr-Ser-Lys) confers the high affinity of human IL-8
for the human IL-8 A receptor to this mutated form of rabbit IL-8. The
site of interaction of this region of IL-8 with the IL-8 type A
receptor appears to be in the amino terminus of the receptor as shown
by studies with a chimeric receptor construct.
Since rabbit IL-8 showed a 200-fold lower affinity for the
IL-8 A receptor than its human homologue and since it is 82% identical
to the human sequence, we decided to mutate amino acids that differ in
the two molecules from the rabbit sequence back to the human sequence.
An increase in affinity of these mutants should define amino acids
in the human sequence that are important for high affinity binding to
the A receptor.
In r/h MUT2 His
These mutants were used to assess
their affinity for the transfected IL-8 A receptor in competition
binding experiments with human IL-8. When His
Results obtained
by competitive binding were paralleled by cell function as assessed by
determinations of Ca
The reverse chimera that places the NH
Additional interaction with interhelical domains has not
been evaluated at this point. Understanding the precise nature of the
interactions between IL-8 and its receptors will provide an essential
basis for the development of therapeutic inhibitors of the
IL-8-stimulated reactions in the inflammatory process.
We thank Dr. Jian Yao for assistance with the computer
modeling of the IL-8 molecule.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and Cys
that, together with
the Glu
-Leu
-Arg
sequence of the
NH
terminus, accounts for the high affinity binding of IL-8
to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares
82% sequence identity with human IL-8, but has 200-fold lower binding
affinity for the IL-8 A receptor, residues of the human homologue were
sequentially exchanged into the rabbit molecule. Replacement of rabbit
His
and Thr
with Tyr
and
Lys
of the human molecule converted the low affinity
binding of the rabbit IL-8 to the high affinity binding of human IL-8
as shown by both competitive binding and by Ca
mobilization. As a corollary, replacement of the Tyr
and Lys
of the human IL-8 with His
and
Thr
of the rabbit IL-8 reduced binding activity of this
mutated human IL-8 200-fold. The site of interaction on the IL-8
receptor type A for the Tyr
and Lys
sequence
was found to be in the NH
-terminal region of this receptor.
A structural pattern of the binding between IL-8 and the A type IL-8
receptor is proposed.
(
)
is a stimulatory peptide for
neutrophils that appears to play an important role in the inflammatory
process
(1, 2) . It shows high affinity binding for two
receptors on neutrophils
(3, 4, 5) : the
specific A-type receptor, which has a K
of 10
M for human IL-8 but binds
rabbit IL-8, MGSA, and NAP2 with poor affinity (>3
10
M in all cases)
(6, 9) and the less selective B-type receptor, which binds human
and rabbit IL-8 and MGSA with nanomolar affinities
(6, 9) and NAP2 with 10
M affinity. It
has been shown that IL-8 binds to both A and B receptors in part
through a Glu
-Leu
-Arg
sequence of
the NH
terminus
(7, 8) . The binding of IL-8
to its two receptors is, however, more complex. While the
amino-terminal Glu-Leu-Arg sequence is necessary for high affinity
binding, it is not sufficient. Both rabbit IL-8 and MGSA contain this
sequence but bind poorly to the A receptor
(6, 9) ,
showing an affinity that is 150- and 200-fold lower than that of human
IL-8. Additional binding sites have been postulated; for the IL-8 B
receptor the carboxyl-terminal
-helix appears to be involved
(8, 9) , and for the IL-8 A receptor a second site has
been postulated to be located between amino acids 7 and 50
(9) .
Several locations in this area, the loop between amino acids 8 and 16,
and the turns at residues 17-21 and 30-35 are
surface-exposed
(10, 11) , and as such they are possible
regions of interaction with the receptor.
Materials
The cDNA representing the IL-8 A
receptor was obtained by PCR amplification of human neutrophil RNA and
cloned into the EcoRI site of the stable mammalian expression
vector pSFFV.neo as described previously
(9, 12) . The
cDNA that encodes the human IL-8 B receptor was kindly provided by
Philip Murphy (NIH) and similarly cloned into pSFFV.neo
(9) .
Reagents and enzymes for the manipulation of DNA were from New England
Biolabs, Life Technologies, Inc., Boehringer Mannheim, or U. S.
Biochemical Corp. Oligonucleotides were purchased from Operon.
I-IL-8 was prepared as described previously
(9) .
Construction of Rabbit IL-8 Mutants
Rabbit and
human IL-8 were expressed in Escherichia coli as fusion
proteins with glutathione S-transferase as described
previously
(9) . r/h MUT1, r/h MUT2 and the h/r mutants
1-3 were generated by introducing the respective base changes
into the oligonucleotide used for PCR amplification and verified by
sequencing with the Sequenase version 2.0 kit (U. S. Biochemical
Corp.). r/h MUT3 was introduced by the method of Kunkel
(13) using the rabbit IL-8 cDNA cloned into M13p19.
Single-stranded DNA template was prepared in the dut ung
strain of E. coli CJ 236.
Mutagenesis was conducted in the E. coli strain DH5aF`,
selected by DNA sequencing and subcloned into the pGEX-2T plasmid
(Pharmacia Biotech Inc.). The mutants described show the sequences
shown in Fig. 1.
Figure 1:
Sequence comparison between human and
rabbit IL-8 and mutants.
Amino acids differing from the rabbit IL-8
sequence are underlined. Protein was purified and thrombin
cleaved as described
(14) . IL-8 was quantified by enzyme-linked
immunosorbent assay using a goat anti-rabbit IL-8 polyclonal antibody
that cross-reacts with human IL-8 in a sandwich enzyme-linked
immunosorbent assay using biotinylated second antibody
(15) .
Construction of IL-8 Receptor A/B and B/A
Chimerae
The chimeric receptors were constructed by using the
natural CelII site in the first transmembrane domain shared by
both the A and the B type IL-8 receptors, as described previously for a
rabbit/human receptor chimera
(16, 17) . Polymerase
chain reaction-amplified cDNA was digested with CelII and
EcoRI and ligated into pSFFV.neo, resulting in a receptor that
switches from the A to the B or from the B to the A receptor sequence
at Asn in the IL-8 A receptor.
Receptor Expression
HL60 cells were transfected
with ScaI-cut receptor DNA by electroporation
(18) ,
and mouse L-cells were transfected with lipofectamine (Life
Technologies, Inc.). Both cells were selected in the presence of 0.6
mg/ml of G418 (Life Technologies, Inc.). Receptor expression was
verified by fluorescence-activated cell sorting analysis using a
polyclonal rabbit antibody raised against a fusion protein of the first
44 amino acids of the IL-8 A receptor with glutathione
S-transferase. The antibody was produced by biweekly
injections of 100 µg of this antigen. 200 µl of 10 cells/ml in 2% fetal calf serum/phosphate-buffered saline were
incubated with a 1:100 dilution of antibody for 30 min on ice. After
three washes, the cells were incubated for 30 min with a 1:250 dilution
of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Tago).
The cells were washed once more in 2% fetal calf
serum/phosphate-buffered saline and analyzed on FACSCAN.
Binding Studies
I-IL-8 binding,
competition with mutant proteins, and Ca
mobilization
were performed as described previously
(9) . Untransfected mouse
L-cells or HL60 cells showed no Ca
response when
stimulated with 10
M IL-8.
and Thr
in
the rabbit IL-8 were replaced with Tyr
and Lys
of the human sequence; in r/h MUT3, Val
of the
rabbit sequence was converted to Ser
of the human
sequence, and in r/h MUT1 the last four differing amino acids starting
with Gln
in the carboxyl-terminal
-helix were
replaced by the human sequence.
and
Thr
in the rabbit IL-8 were replaced with Tyr
and Lys
of the human sequence (r/h MUT2), the
binding affinity rose to that of human IL-8 for the IL-8 A receptor as
shown in Fig. 2 A. The other two mutants, r/h MUT1 and
r/h MUT3 behaved like wild-type rabbit IL-8 in competition binding
experiments with this receptor (Fig. 2 A).
Figure 2:
Competition binding between
I-IL-8 and various mutants in L-cells expressing the IL-8
A receptor.
I-IL-8 binding and competition with mutant
proteins were performed as described previously (9). A,
competition binding with humanized rabbit mutants.
, human IL-8;
, rabbit IL-8;
r/h MUT1;
, r/h MUT2; ▾ r/h
MUT3. The mean ± S.D. of three experiments in duplicate is
shown. B, competition binding with human IL-8 mutated to
rabbit sequence.
, human IL-8;
, h/r MUT1;
, h/r MUT2,
, h/r MUT3
In order to
verify this observation we replaced the Tyr and Lys
in the human sequence with the respective amino acids in the
rabbit molecule (h/r MUT3). To assess the relative importance of each
amino acid, Tyr
(h/r MUT2) and Lys
(h/r MUT1)
were mutated individually, and the competition experiment with
I-IL-8 was repeated. The double mutation had the low
affinity of rabbit IL-8 (Fig. 2 B). While replacement of
Lys
with Thr (h/r MUT1) only resulted in a 12-fold
decrease in affinity compared with human wild-type IL-8, replacement of
Tyr
with His
of the rabbit sequence decreased
the affinity 150-fold (Fig. 2 B). Thus a single amino
acid substitution caused a major loss of affinity.
mobilization in indo-1-labeled
cells (Fig. 3). As in the binding experiments r/h MUT2 behaved
like human wild type IL-8 in L-cells expressing the IL-8 A receptor,
while neither one of the other two rabbit mutants, r/h MUT1 and r/h
MUT3, increased the capacity to mobilize Ca
.
Figure 3:
Comparison of Ca
mobilization in indo-1-labeled mouse L-cells carrying the IL-8 A
receptor after stimulation with rabbit IL-8 or r/h MUT2. Ca
mobilization was measured on an SLM 8000 fluorometer as described
previously (9). The stimulus was added at 10 s
( arrow).
Two
areas of interaction between the IL-8 A receptor and IL-8 have been
described. These are the third extracellular loop and the amino
terminus of the receptor. The third extracellular loop of the A
receptor, specifically Asp and Arg
are
necessary for high affinity ligand binding
(19) . This area is
conserved throughout the receptor family
(3, 4, 20) and has been proposed to interact with the Glu-Leu-Arg
sequence of IL-8. Domain swapping between the IL-8 A and B receptors
has shown that the amino terminus determines whether the receptor
behaves like the A type receptor, which specifically binds human IL-8,
or like the B type IL-8 receptor, which interacts with high affinity
with MGSA as well as with IL-8
(17, 18) . Systematic
mutations of the first 30 amino acids of the IL-8 A receptor failed to
define any consensus sequence involved, but individual substitution by
alanines of Thr
, Pro
, and Tyr
decreased the affinity, and the presence of Cys
is
absolutely necessary for ligand interaction, presumably because of its
function in maintaining structure
(21) . A high affinity for
rabbit IL-8, as described previously for MGSA, depended on the presence
of the amino terminus of the IL-8 B receptor; when we exchanged the
first 57 amino acids of the human IL-8 A receptor with B receptor
sequence, rabbit IL-8 replaced human IL-8 with high affinity at a
K
of 8.1
10
M, which is close to the K
of the B receptor for human IL-8 (5
10
M).
terminus of the IL-8 A receptor onto the B receptor sequence at
the same location in the first transmembrane spanning domain behaves
like the IL-8 A receptor. This A/B IL-8 receptor chimera shows poor
binding competition for rabbit IL-8 but high affinity binding when r/h
MUT2 with Tyr
and Lys
was used
(Fig. 4). These results suggest that the region of amino acids
13-15 in human IL-8 interacts with the amino terminus of the IL-8
A receptor, which indicates that there are at least two areas of
interaction between IL-8 and the A receptor.
Figure 4:
Competition binding between human
I-IL-8 and human, rabbit, or r/h MUT2 and MGSA in HL60
cells expressing an A/B receptor chimera. The mean ± S.D. of
three experiments in duplicate is shown.
, human IL-8;
,
rabbit IL-8;
, r/h MUT2 and MGSA.
In Fig. 5the
regions on IL-8 important for high affinity binding to the IL-8 A
receptor are highlighted. Amino acids 4-6 of IL-8 (Glu-Leu-Arg)
bind to the receptor, presumably to Glu and Arg
in the third extracellular loop, while the region in the IL-8
molecule including Tyr
, Ser
, and Lys
interacts with the amino terminus of the IL-8 A type receptor.
Alternatively, the Tyr
and Lys
sequence may
affect the conformation of IL-8 in a way that increases the affinity
for the amino terminus of the A receptor. The structural analysis of
IL-8
(11) shows that the area of amino acids 8-18 forms a
loop in a series of nonclassical turns and that the structure of MGSA
differs significantly from that of IL-8 in that area. In agreement with
this, r/h MUT2 had no effect on binding competition for the IL-8 B
receptor, suggesting that this region is of little importance to that
receptor. A 6-fold decrease in affinity for the combination of the two
IL-8 receptors as expressed on neutrophils has recently been described
for a human IL-8 molecule in which Tyr
was replaced with
Thr
(22) . Alternatively a threonine substitution in this
location may have a smaller effect on the IL-8 A receptor individually.
The one amino acid substitution from Tyr to His in this location had a
much greater effect in our system where we looked specifically at IL-8
type A receptor binding. This specific effect of a single amino acid
may explain why systematic substitution of all amino acids of the
NH
terminus of the IL-8 A receptor with alanines failed to
define a receptor ligand binding site
(21) . As shown in
Fig. 5
both Tyr
and Lys
of the IL-8
molecule are surface-exposed. These two areas, i.e. the
Glu-Leu-Arg sequence and the loop including
Tyr
-Ser
-Lys
form opposite ends
on the same face of the IL-8 molecule. The two regions lie 23 Å
apart, which would allow the molecule to span the distance between the
amino terminus and the adjacent third extracellular loop of the
receptor. While the NMR structure of IL-8 shows a dimer, IL-8 is
functional in its monomeric form at physiological concentrations
(23, 24) . Both the NH
-terminal Glu-Leu-Arg
sequence and the Tyr
-Lys
region would be
surface-exposed independently from the monomeric or dimeric state of
the molecule.
Figure 5:
Schematic representation of the two areas
of human IL-8 important for high affinity binding to the IL-8 A
receptor: the ELR consensus sequence (amino acids 4-6) at the
amino terminus and the region around amino acids 13 and 15. The precise
location of Glu was not assigned in the data
bank.
Complex interactions with seven-membrane-spanning
receptors have been described previously for small hydrophobic peptide
ligands
(25) , amines
(26) , and the photon-induced
cis-trans conversion of retinal in the rhodopsin system
(27) .
These ligands interact primarily with residues in the hydrophobic
transmembrane domains. While the larger, hydrophilic IL-8 molecule
seems to interact with more surface-exposed amino acids on the
receptor, the interaction clearly involves more than one area of
importance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.