(Received for publication, April 28, 1995; and in revised form, June 28, 1995)
From the
E-selectin is an inducible cell adhesion molecule which mediates
rolling of neutrophils on the endothelium, an early event in the
development of an inflammatory response. Inhibition of
selectin-mediated rolling is a possible means for controlling
inflammation-induced diseases, and several classes of compounds have
been tested for this use. We describe here the use of recombinant
peptide library screening for identification and optimization of novel
ligands which bind to E-selectin. Several of these peptides bind with K values in the low nanomolar range and
block E-selectin-mediated adhesion of neutrophils in static and
flow-cell assays. Administration of the peptide to mice undergoing an
acute inflammatory response reduced the extent of neutrophil
transmigration to the site of inflammation, demonstrating the utility
of this compound as a potential therapeutic. The identification of a
peptide ligand for E-selectin suggests that the complete natural ligand
for this adhesion molecule may include protein as well as carbohydrate
moieties.
E-selectin is a cell adhesion molecule which is induced on the
surface of endothelial cells in response to inflammatory
cytokines(1, 2) . Binding of E-selectin to its ligand
expressed on the surface of circulating neutrophils initiates rolling,
an early step in the recruitment of these cells to a site of injury or
inflammation(3, 4) . The sequence of E-selectin (2, 5, 6) shows that it is a member of the
mammalian C-type lectin family(7) , and its three-dimensional
structure shows strong similarity to mannose-binding
protein(8) . The carbohydrate structure sialyl Lewis x
(sLe;
Neu5Ac
2-3Gal
1-4[Fuc
1-3]GlcNAc)
has been identified as a ligand which binds to the lectin domain at the
N terminus of E-selectin (9, 10, 11, 12) . However, natural
ligands may also contain protein (13, 14) or other
carbohydrate structures. Inhibition of neutrophil adhesion to
endothelium is an attractive approach to controlling
inflammation-mediated diseases such as rheumatoid arthritis or
psoriasis(15) . Several potential therapeutics have been tested
for their ability to inhibit the E-selectin-neutrophil adhesion event,
including carbohydrate-based molecules(16, 17) ,
antibodies(18) , soluble E-selectin(19) , and
selectin-Ig chimeras(20) . While these molecules have been
useful to show the utility of selectin blockers for treating
inflammation, each has significant drawbacks as a therapeutic,
including short in vivo half-life, potential immunogenicity,
high cost, and other possible side effects. A further limitation of
these approaches is the lack of an efficient means to improve the
pharmaceutical properties of these molecules.
In the past few years, several methods for creating and screening vast libraries of recombinant peptides have been developed(21, 22, 23, 24) . These libraries have been used to discover novel peptide ligands for several proteins, including antibodies(21, 24, 25) , receptors(26, 27) , and lectins(28, 29) , as well as novel enzyme substrates(30, 31, 32, 33) . We report here the use of recombinant peptide display technology (21) to identify and optimize peptide ligands for E-selectin. Members of this peptide family block E-selectin-mediated neutrophil adhesion in vitro and reduce influx of neutrophils to a site of inflammation in vivo.
Phage libraries were constructed in the phage vector fAFF1 as described (21) . Mutagenesis libraries used the phagemid vector pAFF2, which was constructed by cloning the pIII gene from fAFF1 into the phagemid expression vector pBAD18 (a gift from L. Guzman and J. Beckwith). Expression of the pIII gene is under the control of the araB promoter and begins with an ATG start codon. The peptides-on-plasmids analog library was constructed and screened as described(24, 32) . The 70:10:10:10 mutagenesis strategy indicates use of a mixture of 70% correct nucleotide with 10% each of the other three nucleotides during synthesis of the oligonucleotide used for construction of the library. For panning, phage were incubated in microtiter wells coated with mAb179 at 5 µg/well followed by a 1:50 dilution of the PI-PLC harvest of E-selectin extracellular domain. Phage were incubated in wells at 4 °C for 2 h, washed for 30 min with cold PBS, and eluted with 0.1 M HCl adjusted to pH 2.2 with glycine and containing 0.1% BSA. Eluted phage were amplified, and screening was repeated over three, four, or five rounds. Individual phage clones were picked from agar plates and amplified, and receptor-specific clones were identified using a phage ELISA (40) in microtiter wells coated with mAb179 (5 µg/well) followed by a 1:50 dilution of E-selectin harvest (Fig. 1). Negative control wells contained mAb179 alone or an unrelated PIG-linked receptor (TNF receptor p55) immobilized on the mAb. DNA was prepared from clones found to be positive on E-selectin-coated wells but negative on control wells, and DNA sequencing was carried out as described previously(21) .
Figure 1:
Phage ELISA assay used
to determine specificity for E-selectin. Microtiter wells were coated
with mAb179, a high-affinity antibody which recognizes the epitope
contributed by the PI-glycan signal sequence. This epitope is present
on the C terminus of all receptors expressed in the PIG vector, so
mAb179 is a generic reagent for immobilizing a variety of receptors in
this format. E-selectin or other receptors were immobilized using an
amount of receptor found to saturate the available mAb179 binding sites
(see ``Experimental Procedures''). Individually amplified
phage clones (10
phage particles/well) were added to
wells in which E-selectin or a control receptor was immobilized. Phage
were allowed to bind and were detected with rabbit anti-phage antibody
directly conjugated to horseradish peroxidase (HRP) (as shown)
or with rabbit anti-phage followed by horseradish peroxidase-conjugated
goat anti-rabbit Ig (not shown). Signals were generated by a
colorimetric reaction mediated by the horseradish
peroxidase.
The flow assay was conducted in a parallel plate flow
chamber as described previously(46) . A HUVEC monolayer was
mounted on the flow chamber, and PBS with Ca and
glucose was perfused over the monolayer for 2 min. Peptide AF11677 was
added at various concentrations to this perfusion buffer. AF11793, an
unrelated peptide which does not bind to E-selectin
(H
N-NTCKDGWCTVGGGGS-CONH
), was used as a
negative control at 100 µM, and F(ab`)
fragments of the monoclonal anti-E-selectin antibody CL2/6 were
used at 20 µg/ml. The neutrophil suspension was diluted to 10
cells/ml in PBS with Ca
and glucose, the
peptide was added and the suspension was passed over the HUVEC
monolayer at a wall shear stress of 1.85 dyne/cm
. The
interaction was observed for 11 min under a phase-contrast microscope
(Diaphot-TMD, Nikon Inc., Garden City, NY) and videotaped. OPTIMAS, an
imaging program (Bioscan Inc., Edmonds, WA), was used to determine the
average number of neutrophils that rolled on the HUVEC monolayer in
five different fields of view. Data were tested for statistical
significance using an unpaired one-tailed Student's t test.
The initial mutagenesis library (M1) was constructed such that individual nucleotide bases were replaced at a rate which would lead to an amino acid change at approximately half of the positions, i.e. 70% correct nucleotide, 10% each of the other three nucleotides during synthesis of the peptide-encoding oligonucleotide. Libraries in which a portion of the sequence was fixed while other residues were randomized were designed to reveal the minimum size of the epitope involved in binding to E-selectin. Clones isolated from seven mutagenesis libraries (M2-M8) showed strong sequence consensus, with four of the 12 positions conserved in every clone isolated. No E-selectin-specific clones were isolated from two of the nine libraries, probably for technical reasons. A library (M9) which contained extensions of four amino acids on both the N- and the C termini and also contained a mutagenesis of the core sequence yielded phage which bound to E-selectin, thus demonstrating that an N-terminal extension did not abolish binding. However, no consensus sequence was recognized outside the core 12-amino-acid sequence. A library (M10) that contained these four conserved positions and randomized the other eight again showed strong preferences for related amino acids at several positions. Based on analysis of all E-selectin-binding peptide sequences selected from the mutagenesis libraries, it appears that the structural motif which is responsible for binding to E-selectin spans at least eight amino acids and requires tryptophan at positions 4 and 8, leucine at position 7, and methionine at position 11; hydrophobic amino acids are preferred at position 10, and serine or threonine is always found at position 3 in these phage clones.
A mutagenesis library (M11, Table 1) was constructed in a peptides-on-plasmids vector, in which peptides are displayed at the C terminus of the lac repressor protein(24) . E-selectin-binding clones were identified in this library, thus demonstrating that a free N terminus is not required for binding and that a free C terminus is tolerated. The characteristics of peptides isolated from this library were somewhat different from those expressed at the N terminus of the phage pIII protein. Most striking was the finding that peptides-on-plasmids clones were much more likely to encode positively charged amino acids at the N terminus (Table 1), while phage clones more frequently had a negatively charged N-terminal amino acid. This may reflect biological biases against positive charges at the pIII N terminus (47) or differences in the binding requirements for a peptide with a free C terminus. Further, greater diversity was observed in the peptides displayed as a C-terminal fusion in positions 1, 2, and 3; serine and threonine were often seen at position 3, as in phage clones, but other amino acids were also found.
Figure 2:
Competition binding assays. A,
radiolabeled peptide binding assay was performed on immobilized
E-selectin as described under ``Experimental Procedures.''
Nonspecific background was determined by preincubating immobilized
receptor with 10 µM unlabeled AF10166. Representative
curves are shown from assays used to determine the IC of
synthetic peptides binding to E-selectin.
, AF10166;
,
AF10172;
, AF10181. B, calcium is not required for
peptide binding to E-selectin. Competition assays were carried out as
described under ``Experimental Procedures'' with radiolabeled
AF10166 as the tracer. Assays were performed in buffer containing
either 10 mM EDTA (
) or 424 µM calcium
and 406 µM magnesium (
). AF10185
(H
N-DITWDQLWDLMK-CONH
) was added to wells at
the concentrations shown (8
10
to 2.1
10
M).
A series of peptides was synthesized to test the effects of truncating one or two amino acids from the N and C termini of the highest affinity peptide, AF10166 (Table 3). Removal of the N-terminal aspartic acid reduced binding affinity by about 3-fold; removal of a second N-terminal residue diminished affinity by an additional 220-fold. Truncation of the C-terminal lysine residue decreased binding affinity by 120-fold, and further truncation abolished receptor binding. Amidation at the C terminus of the full-length peptide did not alter binding affinity, while acetylation at the N terminus caused a small decrease. The truncation series shows that 11 or 12 amino acids provide the optimal structural features for high-affinity binding to E-selectin.
Figure 3:
Binding of peptide to human E-, L-, and
P-selectins. A, phage expressing the peptide sequence
QITWAQLWNMMKpIII (same sequence as synthetic peptide AF10172,
IC
16 nM) were tested for binding to each human
selectin or human nerve growth factor receptor (p75) immobilized on
mAb179 using a phage ELISA as shown in Fig. 1(40) .
Bound phage were detected using rabbit anti-phage antibody as
described. B, radiolabeled peptide binding assay was performed
on immobilized receptors as described under ``Experimental
Procedures.'' Total input was 50,000 cpm. Nonspecific background
was determined by preincubating immobilized receptor with 10 µM unlabeled AF10166.
Figure 4:
E-selectin-binding peptide inhibits cell
adhesion in static assays. A, HL-60 cell adhesion on
immobilized E-selectin. , AF10166;
, scrambled peptide
(AF11678);
, adhesion to unrelated receptor (TNF-R
p55). B, normal human granulocytes adhering to
immobilized E-selectin.
, AF10166;
, scrambled peptide
(AF11678). C, HL-60 cells adhering to IL-1-stimulated HUVEC.
, AF10166;
, adhesion to HUVEC with no cytokine
stimulation;
, adhesion in the presence of blocking
anti-E-selectin (BBA-2). D, HL-60 cells adhering to murine
TNF-
-stimulated mouse brain endothelial cells.
, AF10166;
, adhesion to mouse EC with no cytokine
stimulation.
To confirm
that this peptide family is effective at blocking the interaction of
neutrophils with E-selectin, one compound was tested for its ability to
block rolling of normal neutrophils on LPS-stimulated HUVECs in a flow
assay. Neutrophil rolling in this assay format is known to be mediated
by E-selectin and L-selectin(46) . Addition of the
high-affinity peptide at concentrations of 2.5 µM and
higher inhibited leukocyte rolling (Fig. 5). Lower peptide
concentrations inhibited rolling to a lesser extent in a dose-dependent
manner. The apparent IC for this assay was approximately 5
µM, similar to that determined in the static adhesion
assays. The peptide decreased rolling to a greater extent (>90% at
high concentrations) than did the blocking anti-E-selectin antibody
CL2/6 (
40%). Since multivalent peptide expressed on phage was
found to interact with L-selectin (Fig. 3A), it is
possible that at high concentrations the peptide binds to L-selectin on
the neutrophils and thus blocks rolling more completely than does
anti-E-selectin alone. Addition of a different peptide of similar size
which does not bind to E-selectin (AF11793) did not reduce rolling.
Thus this family of E-selectin-binding peptides is effective at
preventing E-selectin-mediated neutrophil rolling on cytokine-activated
endothelial cells in both static and flow assays.
Figure 5:
Reduction of neutrophil rolling on
LPS-stimulated HUVEC by E-selectin-binding peptide. Normal human
neutrophils were mixed with peptide solution, then introduced into the
flow cell, and passed over LPS-stimulated HUVEC at a wall shear stress
of 1.85 dyne/cm. Columns represent the percent inhibition
of rolling neutrophils/square millimeter compared to the control, as
determined by video image analysis. Standard errors were calculated
from seven replicate experiments; asterisks indicate a
statistically significant decrease in number of rolling leukocytes (p < 0.05). AF11677, E-selectin-binding peptide (a
minor modification of AF10166: Ac-DITWAQLWDLMK-CONH
,
approximately 3 nM binding affinity); AF11793,
unrelated peptide without detectable affinity for E-selectin
(H
N-NTCKDGWCTVGGGGS-CONH
, inactive at 1 mM in the E-selectin binding assay); CL2/6, blocking
anti-E-selectin antibody, used as F(ab`)
fragments at 20
µg/ml.
The murine analog
of E-selectin has recently been identified, and sequence analysis
showed that this receptor has a high degree (73%) of amino acid
homology to human E-selectin in the lectin and EGF domains(6) .
To determine whether the peptides isolated by affinity for human
E-selectin would cross-react with the mouse homologue, we tested
whether AF10166 would inhibit binding of HL-60 cells to a
cytokine-stimulated mouse brain endothelial cell line. Titration of
AF10166 into wells containing TNF-
-stimulated MBEC inhibited
adhesion of HL-60 cells in a dose-dependent fashion, with an IC
of approximately 10 µM (Fig. 4D), a
dose-response similar to that found for human endothelial cells (Fig. 4C). Radiolabeled AF10166 bound specifically to a
mouse E-selectin-Fc fusion protein (6) immobilized on protein
A, but did not bind to a human type I IL-1 receptor-Fc fusion (data not
shown). Thus AF10166 appears to bind to mouse E-selectin with high
affinity.
E-selectin is up-regulated during the early stages of an
inflammation reaction, and blocking E-selectin with
antibody(18, 55, 56) , soluble E-selectin-Ig
chimeras(20) , soluble E-selectin(19) , or
sLe-like carbohydrates (16, 17, 57) prevents some acute inflammation
reactions. Because AF10166 was able to bind to mouse E-selectin, we
tested whether administration of an E-selectin-binding peptide to
glycogen-injected mice would diminish the influx of neutrophils into
the chemically irritated peritoneum(58, 59) . Mice
which had been given intraperitoneal glycogen received an intravenous
injection containing 2 mg of AF10185 3 h after the glycogen injection,
and peritoneal neutrophils were harvested and counted 1 h later.
Peptide treatment significantly reduced the number of neutrophils in
peritoneal lavage fluids (Fig. 6A). Control experiments
administering the same dose of a closely related peptide sequence, Affy
9, which did not bind to E-selectin (Table 2), showed no decrease
in neutrophil influx relative to buffer-injected mice (Fig. 6B). Thus the E-selectin-binding peptide can
block an acute inflammation reaction in vivo when administered
intravenously. The requirement for this relatively high dose of peptide
may reflect a short in vivo serum half-life of this peptide. A
complete study of the pharmacokinetic properties of derivatives of this
peptide is in progress.
Figure 6:
Neutrophil influx in chemical peritonitis. A, data from 10 mice in each group are shown. AF10185 is
carboxyamidated AF10166 (HN-DITWDQLWDLMK-CONH
),
with a binding affinity of 4 nM. Statistical analysis using a
nonparametric unpaired t test gave a two-tailed P value < 0.002. B, data from five (Affy 9) and
seven (vehicle) mice are shown. Affy 9 is
H
N-HITWDQLWNVMLRRASLG-COOH, with no detectable binding at
11 µM (Table 2). There is no statistically
significant difference between the two
groups.
We have used peptide display libraries to identify novel ligands for E-selectin, which was previously known to bind only to carbohydrates. Peptides in this family bind with high affinity to the selectin and block cell adhesion in both static and rolling assays. Intravenous administration of peptide blocks neutrophil influx into the glycogen-stimulated peritoneal cavity of mice. Thus peptides in this family can serve as lead structures for the development of E-selectin-blocking therapeutics.
The peptide family which was
identified in these experiments has very clear structural relationships
and suggests that the requirements for binding to E-selectin are
stringent. Of more than 10 analogs of the original sequence
which were tested in mutagenesis libraries, only a tiny fraction bound
to E-selectin. All active peptides contained four conserved residues,
which appear to be required for binding with high affinity, but many
phage which retained these four residues failed to bind to E-selectin (e.g. mutagenesis libraries M10 and M11). Truncations
demonstrated that the highest affinity interaction occurs with 11 or 12
amino acids, though smaller fragments bind with lower affinity. These
structural requirements suggest that the peptides in this family may
take on a defined structure in solution, despite their small size.
Circular dichroism and NMR studies are in progress to address this
possibility.
Binding of AF10166 to human E-selectin blocks
neutrophil adhesion in both static and flow-cell assays, demonstrating
that the peptide blocks the binding of the natural ligand present on
the surface of neutrophils. The carbohydrate sLe has been
identified as a critical part of the ligand structure which binds to
E-selectin (10, 11, 12) , and thus it is
possible that AF10166 may mimic a carbohydrate structure. Peptides have
been identified by phage display technology which bind to the lectin
concanavalin A and which block binding of its carbohydrate ligand,
methyl
-D-mannopyranoside (28, 29) .
However, our data suggest that the peptides described here are not
acting as glycomimetics. AF10166 and sLe
do not compete for
binding to E-selectin, and AF10166 binding does not require calcium as
does sLe
. Thus AF10166 may bind to a different site on the
receptor which is involved in recognition of the natural ligand,
perhaps a protein moiety. Alternatively, binding of the peptide might
cause a conformational change in the receptor which then prevents it
from recognizing the ligand on the neutrophil surface. It is clear that
sLe
is not the entire ligand for E-selectin. sLe
is found on many cells which do not adhere to
E-selectin(60, 61) , and many
sLe
-containing glycoproteins do not bind to P-selectin or
E-selectin(14, 62) . Further, sLe
alone is
a poor inhibitor of cell adhesion in vitro, even at
concentrations up to 1 mM(17) . A glycoprotein has
been characterized as the E-selectin ligand on mouse myeloid cells by
affinity purification of cell extracts on murine
E-selectin(14, 63) , and a glycoprotein P-selectin
ligand, PSGL-1, binds to E-selectin as well(64) . This and
other evidence suggest that additional elements, possibly complex
carbohydrate structures or protein determinants, make up the complete
E-selectin ligand(14) . However, there is no direct evidence to
date that any selectin actually interacts with a natural protein
determinant.
In addition to the possible E-selectin ligands described above, several groups have shown evidence that L-selectin presents carbohydrate determinants to E-selectin and thus acts as an E-selectin ligand(46, 65, 66) . The peptide family described here binds to L-selectin as well as to E-selectin, albeit with low affinity (Fig. 3). The dramatic inhibition of rolling observed in the flow cell assays suggested that some binding to L-selectin may have occurred at the high concentrations used for these experiments and led to more complete inhibition of the interaction between neutrophils and endothelial cells. While it is formally possible that this peptide interacts with some other surface structure on the activated endothelial cells to inhibit adhesion under conditions of shear flow, this seems unlikely since the peptide was isolated on purified E-selectin and was shown to have no detectable binding to ICAM-1, VCAM-1, or several other cytokine receptors. Addition of the peptide to a P-selectin-mediated static adhesion assay had no effect, and neutrophils were not directly activated by the peptide. Thus the most likely explanation for the strong inhibition of neutrophil rolling is that the peptide is bound by both E-selectin and L-selectin, resulting in a level of inhibition similar to that seen with antibodies against both adhesion molecules. While L-selectin-dependent adhesion mechanisms are clearly important for rolling on endothelial cells under conditions of shear flow, experiments with E-selectin-transfected L cells suggested E-selectin interaction with other yet undefined ligands might be important in the initial steps of rolling(46) . Whether this is true for endothelial cells as well as L cells has not been established. In contrast to the rolling assays, peptide inhibition of static adhesion assays on activated HUVECs was about the same as anti-E-selectin (Fig. 4C), suggesting that the mechanism responsible for rolling differs from that for static adhesion.
While the complete natural ligand(s) for E-selectin has not been fully characterized, several mucin-like glycoproteins have been identified as ligands for L- and P-selectins(63, 64, 67, 68, 69, 70) . Notably, the first four residues of the mature PSGL-1 sequence, which binds to both P-selectin and E-selectin, are identical to four residues in the core of AF10166 (Gln-Leu-Trp-Asp). It is not known whether this part of PSGL-1 is critical for selectin binding, but one may speculate that this homology suggests a role of the protein in cell recognition by selectins. There is no homology of AF10166 with the primary structures of the L-selectin ligands GLYCAM-1 (69) or CD34(71) .
The adhesion of neutrophils to vascular endothelium is an early step in the inflammatory process and is mediated by selectins(3, 4) . Much effort has gone into demonstrating the importance of E-selectin-mediated cell attachment in inflammation, using a variety of methods to block the activity or expression of this cell adhesion molecule. While redundancy in adhesion pathways exists in the form of related selectins and multiple steps that are required for diapedesis (72) , blocking one adhesion molecule with antibodies, Fc fusions, or soluble extracellular domains has proven effective in reducing inflammation in experimental systems(18, 19, 20, 55, 56, 73, 74, 75, 76, 77) . Thus it has been established that blocking E-selectin can reduce inflammation induced in various vascular beds by different inflammatory stimuli. While it is unlikely that blocking any one adhesion molecule will completely inhibit inflammation, particularly in chronic disease, a substantial reduction in pathology has been achieved in several model systems.
E-selectin's role in different models of inflammation may vary. For example, the ability of E-selectin to target memory T lymphocytes which bear the cutaneous lymphocyte antigen to sites of chronic inflammation in the skin (78, 79, 80) suggests that dermal inflammation may be particularly sensitive to inhibition of E-selectin. Various animal models of inflammation are differentially sensitive to blockage by inhibitors of selectins and other cell adhesion molecules (for example, (74, 81) ), and there may be differences in the ability of different vascular beds to express adhesion molecules. The relative importance of different adhesion steps in diverse sites will be clarified as specific tools become more broadly available for use.
The identification of high affinity inhibitors of E-selectin-mediated cell adhesion is one of the first demonstrations of the utility of recombinant peptide display technology to discover ligands for an important therapeutic target. As drug discovery tools, these highly diverse peptide libraries offer many distinct advantages over traditional chemical synthesis approaches to the identification of receptor antagonists, notably the inexpensive and rapid identification and optimization of novel ligands. The peptides described here provide excellent leads for development of anti-inflammatory therapeutic agents. While the pharmacokinetic properties of L-amino acid peptides are unlikely to be optimal for use in treatment of inflammation-mediated diseases, these peptides can provide information that confirms the importance of E-selectin in various types of inflammation. Further, improved characteristics of stability and bioavailability can be conferred by modifications of the peptide, including use of nonnatural amino acids and terminal protecting groups. It is expected that the use of combinatorial synthetic analog technology (82) coupled to rapid screening methods will allow us to identify improved antagonists of E-selectin with greatly enhanced therapeutic potential.