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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1738-1748
Cell-Specific Peptide Binding by Human Neutrophils
By
Luca Mazzucchelli,
James B. Burritt,
Algirdas J. Jesaitis,
Asma Nusrat,
Tony W. Liang,
Andrew T. Gewirtz,
Frederick J. Schnell, and
Charles A. Parkos
From the Department of Pathology, Brigham and Women's
Hospital and Harvard Medical School, Boston, MA; the Department of
Pathology and Laboratory Medicine, Emory University, Atlanta, GA; and
the Department of Microbiology, Montana State University, Bozeman, MT.
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ABSTRACT |
Analysis of peptide binding to human neutrophils (PMN) using phage
display techniques has revealed cell-specific motifs reactive with the
PMN surface. Phage libraries displaying either linear 9-mer or cyclic
10-mer and 6-mer peptides were incubated with normal human neutrophils
followed by elution of bound phage with low pH (pH 2.2) and non-ionic
detergent. Three rounds of selection generated several related peptide
sequences that bound with high avidity to PMN. Using the linear 9-mer
library, PMN-binding phage expressed peptides with the motif
(G/A)PNLTGRW. The binding of phage bearing this motif was highly
specific since no binding was observed on lymphocytes, fibroblasts,
epithelial, or endothelial cells. Functional assays revealed that phage
bearing the sequence FGPNLTGRW induced a pertussis toxin-sensitive
increase in PMN cytosolic calcium analogous to that observed with
G i coupled receptors. Other prominent motifs identified
included phage bearing the consensus DLXTSK(M/L)X(V/I/L), where X
represents a non-conserved position. Phage with this motif bound
exclusively to a sub population of human PMN that comprised
approximately 50% of the total and did not elicit a calcium response.
The binding of such phage to PMN was prevented by co-incubation with
competing peptides displaying identical or similar sequences
(IC50 range from 0.6 µmol/L to 50 µmol/L for DLXTSK and
GPNLTG, respectively). We speculate that these techniques will be
useful in identifying functional cell-specific binding motifs and
contribute to the development of new therapeutic and diagnostic
strategies in human disease.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE IDENTIFICATION of specific cell types
has become a crucial component in the diagnosis and treatment of
disease and in experimental work requiring strict definition of cell
populations. In general, specific cell types have been defined through
the identification of individual or groups of protein/carbohydrate(s) that reside on the cell surface through the use of monoclonal antibodies (MoAbs). The ability to specifically identify individual or
groups of specific cell surface structures has greatly increased our
understanding of both normal and pathologic processes. For example, by
labeling specific cell surface structures, it has been possible to
hypothesize pathways of differentiation in both normal and malignant
cells. It has also been possible to define states of activation in a
variety of cell types through the identification and quantification of
specific cell surface structures. In neutrophils, for example, this
approach has demonstrated that cellular activation with physiologic
agonists results in the mobilization of internal stores of CD11b/CD18
to the cell surface along with shedding of cell surface
L-selectin.1,2 Furthermore, cellular activation, as
dictated by conformational changes in cell surface proteins such as
 2 integrins3 is readily detectable by this
general approach.
Recently, others have taken a new approach to identify ligands on cells
and whole organs by using phage display techniques.4-9 Through these observations, it has become apparent that cell-type specific recognition might be achieved by scanning whole cells with
phage display peptide libraries. To date, however, few cell-type specific binding peptide sequences have been identified using phage
display techniques. Furthermore, little information exists on library
constructs that preferentially interact with cellular targets and the
selection procedures used to identify cell-specific phage.
Here we show the identification of specific, cell-binding peptide
sequences using phage display libraries. We demonstrate that affinity
purified phage-bearing these peptide sequences bind specifically to the
membrane surface of human neutrophils (PMN) or monocytes. We also show
that phage bearing these peptide sequences can induce receptor-mediated
functional responses in PMN as indicated by intracellular calcium
measurements. Our results demonstrate that the use of both linear and
structurally constrained libraries is complementary and may be crucial
for the identification of high affinity ligands. Finally, we show that
numerous, unique cell-binding motifs can be identified through the use
of different phage affinity purification procedures. Taken together,
these data indicate that the phage display approach is likely to have broad applications in diagnosis and management of human disease.
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MATERIALS AND METHODS |
Reagents and buffers.
Oligonucleotides for library construction as well as synthetic peptides
were purchased from Macromolecular Resources (Fort Collins, CO).
Peptides were synthesized by solid phase synthesis (Macromolecular
Resources) and analyzed by high performance liquid chromatography
(>90% purity) and mass spectrometry. HBSS consisted of (in g/L):
0.185 CaCl2, 0.094 MgSO4, 0.4 KCl, 0,06 KH2PO4, 8 NaCl, 0.048 Na2HPO4, 1 glucose, and HEPES added to 10 mmol/L (pH 7.4). HBSS( ) was prepared as HBSS but without
CaCl2 or MgSO4. Saline HEPES consisted of 150 mmol/L NaCl and 10 mmol/L HEPES (pH 7.4). TBS buffer consisted of 150 mmol/L NaCl and 50 mmol/L Tris/HCl pH 7.4.
Random peptide phage display libraries.
Three random peptide phage display libraries were used in these
studies. J404, a linear random nonapeptide M13 phage library with
kanamycin resistance, has been successfully used in the identification of several epitopes recognized by MoAbs and interactive regions of
proteins.10-13 As detailed below, two structurally
constrained libraries displaying hexa- and decapeptide loops,
respectively, were constructed essentially as previously
described14 using a vector, M13KBstX,15 which
has kanamycin resistance. A schematic diagram of the modified pIII coat
protein for the constrained library construction is shown in Fig
1.
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| Fig 1.
PIII protein construct for phage libraries. The glycine
and two proline residues at the carboxyl end of the random regions
function as a flexible linker and may serve to discourage association
of the random peptide with the native pIII protein. Structural
constraints in the CL10 and CL6 libraries were imposed by disulfide
bonds between the cysteine residues flanking the variable region. CL10
and CL6 also include at the amino terminus of the mutated pIII protein
wild-type residues (glutamic acid and alanine) and may help to preserve
the signal peptidase cleavage site allowing cleavage of the peptide
backbone at the correct site on the pIII pre-protein.
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M13KBstX replicative form (RF) DNA was extracted from infected K91
Escherichia coli, purified on a equilibrium CsCl
gradient,16 cleaved with BstXI, further purified on
a potassium acetate gradient,15,17 followed by
dephosphorylation with calf intestinal phosphatase (Pharmacia
Biotech, Piscataway, NJ) as described by the manufacturer. A synthetic DNA construct containing the ligation-compatible
BstXI ends and the random inserts with the designed flanking
residues was prepared by annealing a collection of degenerate
oligonucleotides with two "half-site" oligonucleotides as
described.14 The coding strand of this DNA construct
contained (NNK)6 or (NNK)10 codons, where N
corresponds to equimolar amounts of A, T, G, or C and where K
corresponds to equimolar amounts of G or T. Following ligation and
fill-in reaction, the DNA product was transfected into electrocompetent
MC 1061 cells18 with a Bio-Rad Gene Pulser. The decapeptide
library termed "CL10" was produced by 108 separate electroporations using a total of 440 µg of ligated vector DNA resulting in a diversity of approximately 1.04 × 109
unique clones. To produce the hexapeptide library termed "CL6," a
total of 136 µg of ligated vector DNA was transfected in 34 separate
electroporations yielding a library containing 9 × 108
unique clones. Libraries were propagated in LB containing kanamycin, harvested and resuspended in TBS buffer containing 0.03% sodium azide
at a final concentration of 1 × 1013 Pfu/mL as previously described.19 To replenish depleted stocks of the J404
linear library, an additional 9-mer linear library was constructed
using the original degenerate oligonucleotide constructs10
as above. For this library, termed LL9, 450 µg of ligated vector DNA
was transfected in 106 separate electroporations yielding a library containing 3.66 × 109 unique clones. Analysis of the LL9
library revealed a random distribution of the frequency of codons in
the insert with no duplicate occurrences of peptide sequences as has
been described for the J404 library10 (data not shown).
PMN isolation.
PMN were isolated from whole blood (anticoagulated with
citrate/dextrose) obtained from healthy volunteers, using a gelatin sedimentation technique previously described.20 PMN were
resuspended in modified HBSS devoid of Ca2+ and
Mg2+ (HBSS()) at a concentration of 4 × 107 cells/mL (4°C) and used for subsequent experiments.
Selection of PMN-binding phage.
Freshly isolated PMN (2 × 107) were incubated with
20-µL aliquots of phage library (~5 × 1010 phage)
in 1 mL HBSS containing 0.1% bovine serum albumin (phage buffer) for 2 hours at 4°C or for 1 hour at 20°C in a 1.5-mL microcentrifuge
tube. PMN were then washed five times with phage buffer and bound phage
were eluted for 5 minutes in 2 mL of 0.1 mol/L glycine (pH 2.2)
followed by addition of phage buffer containing 0.5% Tween 20 to the
remaining cell pellet. After neutralization with Tris buffer pH 8.1, the titer of phage was determined in each fraction by plaque assay according to standard procedures.21 Phage eluates were then amplified in "starved" K91 E. coli19 on solid
LB agar containing 75 µg/mL kanamycin,10 purified by
precipitation with polyethylene glycol, and resuspended in 600 µL
saline/HEPES buffer. An aliquot (20 µL) of purified phage was
subsequently re-applied to newly isolated PMN for a total of three
affinity purification and two amplification steps. Individual phage
clones were then isolated, amplified, and DNA prepared (Sequenase
version 2.0 kit; Amersham, Cleveland OH). The random peptide sequences
were deduced after sequencing the unique nucleotide region of the pIII
protein by the Sanger method. As a control for PMN-specific selection,
parallel phage affinity purification steps were carried out in a 1.5-mL microcentrifuge tube in the absence of PMN, and a sample of the recovered phage was analyzed by sequencing of the DNA insert.
ELISA for detecting phage binding to PMN.
Suspensions of 2.5 × 105 PMN in 100 µL HBSS were
allowed to settle and bind to wells of a microtiter plate
(Limbro-Titertek; Flow Laboratories, Irvine, CA) for 30 minutes (20°C). After gentle washing, plate wells were blocked with
0.5% bovine serum albumin (BSA) in HBSS for 30 minutes (blocking
buffer). Amplified phage clones (2.5 × 1010 phage
particles) were added to each well and allowed to bind for 20 minutes.
Unbound phage were subsequently removed by gentle washing. Bound phage
were fixed to PMN by addition of paraformaldehyde (3.7%) for 10 minutes followed by washing and incubation in blocking buffer for 45 minutes. Bound phage were assayed after incubation of wells with a
1:1,000 dilution of biotin-sheep anti-phage antibody (5prime-3prime,
Boulder, CO) followed by incubation with alkaline
phosphatase-streptavidin (Pierce). After substrate addition, wells were
analyzed in a microtiter plate reader at 405 nm.
Immunofluorescence.
For immunofluorescence studies, suspensions of 1 × 105
PMN in HBSS were allowed to spread on glass coverslips for 30 minutes at room temperature followed by washing and blocking with 0.5% BSA in
HBSS for 30 minutes. PMN were then incubated with individual phage
clones for 20 minutes (109 phage/coverslip). PMN were then
washed, fixed with 3.7% paraformaldehyde, and permeabilized with 0.5%
Triton X-100 in HBSS for 30 minutes at 20°C. After blocking reactive
groups with 0.5% BSA in HBSS, PMN were incubated with biotinylated
sheep anti-phage antibody as described above and followed by incubation
with FITC-Streptavidin (1:1,000; Jackson Laboratories, Bar Harbor, ME).
PMN were viewed with a BioRad MRC-600 confocal fluorescence microscope.
Flow cytometry studies.
Suspensions of PMN (5 × 106) in 1 mL HBSS containing
0.5% BSA were incubated with individual phage clones for 2 hours at
4°C or for 1 hour at 20°C followed by washing and fixation with
3.7% paraformaldehyde. PMN were then stained with anti-phage antibody and FITC-streptavidin as described above. Cells were analyzed for bound
phage with a FACSscan flow (Becton-Dickinson Immunocytometry Systems,
Mountain View, CA). Identical experiments were performed with other
human cell types including monocytic cells (U937), epithelial cells
(T84 and HT29), an endothelial cell line (ECV 304), fibroblasts (VA2),
and peripheral blood lymphocytes. The latter were isolated using Ficoll
density purification from heparinized blood of healthy volunteers.
Isolated lymphocytes were then cultured with Phytohemagglutinin
(GIBCO-BRL, Gaithersburg, MD) in RPMI 1640 medium supplemented with
10% fetal bovine serum and 2 nmol/L recombinant human IL-2 (Boehringer
Mannheim) and used after 1 week of culture.
PMN binding assays with selected clones were also performed in the
presence of competing peptides or of control scrambled peptides. The
inhibitory effect of added peptide was quantified by evaluating the
percentage of maximal phage binding as defined by flow cytometry
studies above.
Measurement of cytosolic [Ca++] in PMN.
PMN were loaded with the calcium indicator Indo-1 and cytoplasmic
[Ca++] measured as previously described22,23
using a Hitatchi F-4500 spectrofluorimeter. PMN, 2 × 106,
were suspended in 960 µL of HBSS and stimulated by the adding 40 µL
of purified phage (4 × 1010 PFU) from a stock solution containing 1012 PFU/mL in saline/HEPES buffer. Controls
included buffer vehicle alone, irrelevant phage, and the well
characterized agonists fMLF (100 nmol/L) and IL-8 (14 nmol/L). In a
subset of experiments, PMN were pre-incubated with 1 µg/mL pertussis
toxin (Sigma, St Louis, MO) for 2 hours (37°C) in HBSS() before
calcium experiments.22,23
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RESULTS |
Library construction.
Two random peptide phage libraries displaying cyclic hexapeptides or
decapeptides were produced with the M13KBst vector and used in addition
to a previously characterized linear library (J404) with the intent to
characterize PMN-binding phage peptides. The two circular libraries,
CL10 and CL6, were analyzed by nucleotide sequencing of the 5' end of
the pIII protein. All of the 112 different phage clones sequenced were
found to contain the random insert flanked by the correct invariant
sequences. Approximately 3% of the sequences coded for random peptides
shorter than the intended 10 or 6 residues, indicating rearrangements
occurring during the assembly of the DNA construct.
The amino acid sequences of random peptides deduced from the nucleotide
sequence of 48 clones from the CL10 and 64 clones from the CL6 library
indicated a random distribution of the frequency of codons in the
insert. Duplicate occurrences of peptide sequences were not found.
Codons for Lys and Ala were found to be slightly over-represented,
whereas those for Tyr and Cys were somewhat under-represented. Cysteine
residues in the displayed peptides were almost exclusively found in
pairs. Phage with odd numbers of cysteine residues were not found
suggesting negative biological selection. Thus, no relevant bias in the
CL10 and CL6 libraries was observed, in agreement with other reports of
phage library construction.14,15,24
Affinity purification of PMN-binding phage bearing linear motifs.
Experiments were performed to affinity isolate phage binding to human
PMN isolated from whole blood obtained from normal human volunteers. In
parallel experiments, PMN were resuspended in HBSS containing BSA and
coincubated with phage library at either 20°C or 4°C. The two
different binding temperatures were used to evaluate the effects of
temperature and potential phage internalization on binding motif
patterns. Phage binding to human PMN were initially selected with the
linear nonpeptide phage library. Bound phage were first eluted under
acidic conditions (pH 2.2) followed by PMN incubation in
detergent-containing buffer to recover any remaining cell-associated
phage. The acid-eluted and cell-associated phage fractions were then
amplified separately and re-applied to fresh PMN. After three rounds of
such selection, an amplification of approximately 104 was
achieved for affinity purification's either at 4°C or at 20°C
(data not shown). No significant increase of nonspecific phage binding
was observed.
The random insert region was sequenced from 44 phage selected in the
third round of affinity purification from experiments performed at
4°C and 47 phage derived from experiments performed at 20°C (Fig
2). Sequence similarity was present in all
but two sequences (95%) from phage selected at 4°C and 35 of the 47 sequences (74%) from phage recovered at 20°C. A consensus,
DLXTSK(M/L)X(V/I/L), where X represents a non-conserved position, was
particularly apparent in the cell-associated (detergent lysis)
fractions (Fig 2). Here, powerful selective pressure of the PMN-phage
peptide interaction is apparent with exact matches observed in seven
residue sequences in some of the recovered phage (peptide sequences
such as DLVTSKLQV and DLSTSKLQI). The most conserved residues displayed at the amino terminus of peptide inserts were Asp and Leu followed by
Thr and Lys. Similarity between phage peptides was increased by
considering conservative substitutions between amino acids.
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| Fig 2.
Neutrophil-binding phage peptide sequences isolated from
the linear (X9) phage display library. The unique region of phage DNA
recovered from the third round of affinity purification was sequenced
and the motifs aligned to form the consensus DLXTSK(M/L)X(V/I/L) and
GPNLTGRW, respectively. Identical residues and conservative
substitutions are shown in bold. Following sets are considered to
contain homologous amino acids (I, L, V), (K, R), (D, E), (S, T), (Y,
F). The number of clones encoding the same peptide is shown in
parentheses. The linker sequence at the amino terminus (GPP, not shown)
was correctly displayed in all clones. Abbreviations for the amino acid
residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R,
Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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The analysis of phage eluted with low pH buffer at 20°C revealed an
additional consensus sequence, GPNLTGRW. While this motif was present
in only three different phage clones, there was an eight residue match
between sequences, indicating a very strong selection. The DNA insert
corresponding to the random peptides displayed by the three
consensus-bearing phage showed different triplets encoding for Pro,
Leu, and Arg, indicating uniqueness of each selected phage. Such eight
residue matches provide an alternative estimate of the true amino acid
sequence diversity of the library which is much larger than the initial
estimate of ~3.6 × 108. Finally, a small group of phage
peptides binding to PMN at 20°C and recovered by acid elution showed
a relative abundance of Tyr residues without displaying an obvious consensus.
Affinity purification PMN-binding phage bearing circular motifs.
Affinity isolation experiments using two circular libraries, CL6 and
CL10, were performed as above with the linear library. An amplification
of approximately 103 to 104 was achieved after
three rounds of affinity purifications for both cyclic libraries at
either 4°C or 20°C (data not shown). The deduced amino acid
sequences of phage selected from the CL10 library after three rounds of
affinity purification are shown in Fig 3.
Nine of the 13 (69%) distinct phage clones identified in the 4°C
experiments had the same linear consensus DLXTSK(M/L)X(I/V/L) that
was identified with the J404 library. Conservative substitutions indicated an even greater homology of the phage peptides to this consensus sequence. However, as can be seen in Fig 3, none of these
clones displayed the linker sequences as designed by the library
construction (Fig 1). In particular, the Cysteine residues flanking the
random region were absent, suggesting a linear conformation instead of
a constrained loop. Phage clones bearing a Cys residue in either the 5'
or 3' portion of the linker sequence always displayed an
additional Cys within the random region, suggesting the
formation of a short, structurally constrained sequence within the
displayed peptide. Conversely, the analysis of the PMN binding phage
selected after three rounds of affinity purification performed at
20°C revealed different consensus motifs that were mostly cyclic. In particular, 14 of the 17 phage clones (82%) isolated from the cell-associated fraction displayed peptide sequences related to the
consensus CXXGXFLGXWLC. The residues Leu and Gly were absolutely conserved and occupied same position within the random region in all of
the phage clones. Analysis of phage clones recovered from the acid
eluted fraction showed additional consensus motifs that are grouped in
Fig 3. Interestingly, two phage clones, CLVEGWCNPWV and CLVKDWCGTMR,
contain a short loop displaying the consensus CLVXXWC and were
identical to phage clones recovered from the acid eluted fraction at
4°C and from the cell-associated fraction at 20°C, respectively.
Finally, phage bearing the cyclic peptide CPFGPDLQGKWC showed a 6 residue match with the peptide FGPNLTGRW selected with the linear J404
library under the same conditions.
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| Fig 3.
Neutrophil-binding phage peptide sequences isolated from
the cyclic (CL10) phage display library. Selection and sequencing of
phage was performed as described in the text. The peptides displayed by
the phage recovered from the experiments performed at 4°C are aligned
to the consensus motif DLXTSK(M/L)X(V/I/L) that was previously
identified with the linear nonapeptide library. Identical residues and
conservative substitutions are shown in bold. Underlined residues
represent the putative linker sequences which, in several clones,
diverge from the linker sequences designed by the library construction
(see Fig 1) due to the absence of the cysteine residues. The phage
clones recovered from the cell-associated fraction at 20°C are
aligned to form the consensus motif CXXGXFLGXWLC. One phage in the
eluate fraction at 20°C (first from the top) shows a high degree of
homology with the FGPNLTGRW displaying phage recovered with the J404
library under the same experimental conditions, representing a
structurally related sequence. Other possible consensus motifs are
tentatively grouped and shown in bold.
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Analysis of the random region of PMN-binding phage selected from the
CL6 library revealed similarities to the motifs obtained from the
libraries with longer random inserts. Twenty-eight phage clones
sequenced from the acid eluted and cell associated fractions affinity
purified at 4°C displayed 12 distinct peptides. Eight of them (66%)
confirmed the strong selection for a phage subpopulation displaying an
unconstrained linear form similar to the main consensus identified with
the J404 library. In particular, the phage clone bearing
AESDLLTNR LGPP,
recovered nine times in the acid eluted fraction, did not contain Cys
residues in the linker sequences (underlined) and displayed a four
residue match (bold) to the DLXTSK(M/L)X(V/I/L) motif. Analysis of
41 phage affinity purified at 20°C showed, in the acid eluted and in
the cell-associated fractions, 19 distinct phage clones. Eleven of them
(58%) contained homologies to the consensus WLGXW, which is similar to
the motif CXXGXFLGXWLC identified with the CL10 library.
Binding studies with affinity purified phage.
Phage bearing consensus-related sequences were tested for their ability
to bind to adherent PMN in a microtiter plate. As shown in Fig
4A, three phage clones bearing different
consensus-related sequences bound to PMN when compared with an
irrelevant control phage. OD values for phage displaying the peptides
FGPNLTGRW and CKDGLFLGSWLC were consistently higher than that for the
phage clone bearing the peptide DLVTSKLQV suggesting either higher
affinity or more binding sites per PMN. In Fig 4B, the effect of
peptide sequence variability on phage binding to PMN was explored.
Here, related phage displaying different grades of homology to the
consensus DLXTSK(M/L)X(V/I/L) were tested for binding to adherent
PMN. As expected, peptide sequences with fewer residue matches showed diminished binding.
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| Fig 4.
Phage attachment assay. Purified phage clones were
incubated in microtiter plates coated with PMN freshly isolated from
peripheral venous blood. Bound phage were detected with polyclonal
anti-phage antibody and quantified by light absorbance as described in
Materials and Methods. (A) Distinct PMN binding phage clones selected
from different experiments. The DLVTSKLQV phage consistently showed a
weaker detection signal as compared with the other binding phage. (B)
The binding affinity of phage clones displaying different grades of
homology to the consensus motif DLXTSK(M/L)X(V/I/L). Compared with the
DLVTSKLQV-displaying phage, clones with fewer residues matching to the
consensus motif show a decreased binding affinity. Control phage
displayed irrelevant sequences (AQPQVRPIG or GPRPGPPKL). Optical
density at 405 represents phage incubated wells after subtraction of
the wells without phage incubation. Phage clones incubated on BSA
coated wells (not shown) yielded OD value between 0.1 and 0.2. Values
representative of one of two experiments showing the mean ± SD of
quadruplicate determinations.
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Immunofluorescence studies were performed in an attempt to localize
PMN-bound phage. Adherent phage were visualized by confocal microscopy
after staining with biotinylated anti-phage antibody and FITC-avidin
(Fig 5). As shown in the figure, phage
bearing DLVTSKLQV or FGPNLTGRW motifs showed a comparable multifocal
staining pattern restricted to the PMN surface, whereas control phage
failed to label. Intracellular fluorescence staining patterns
indicative of internalized phage particles were not observed in the
permeabilized PMN preparations. Interestingly, a considerable
proportion of the PMN did not show any detectable fluorescent signal
(approximately 50%). No morphological difference between labeled and
unlabeled PMN were observed to account for the difference in labeling.
Additional fluorescence studies with the CKDGLFLGWLC bearing phage
clone yielded a similar focal staining pattern (data not shown).
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| Fig 5.
Localization of PMN-binding phage clones by
immunofluorescence. PMN isolated from peripheral blood were incubated
with phage clones, fixed with paraformaldehyde, permeabilized with
Triton X-100, and labeled with biotinylated polyclonal antibodies as
described in Materials and Methods. There was a bright membrane
staining after incubation with the FGPNLTGRW (A) or DLVTSKLQI (C)
displaying phage. Incubation of PMN with an irrelevant phage resulted
in no detectable immunofluorescence (E). Cytoplasmic staining
suggestive of phage internalization was not detected. For orientation,
the corresponding Nomarski images are shown in (B), (D), and (F). As
can be seen, phage incubation labeled only part of the neutrophils.
Incubation with the CKDGLFLGSWLC-displaying phage yielded staining
patterns comparable with those found with the other PMN-binding phage
(data not shown).
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Because of the focal nature of the phage staining observed by confocal
microscopy, the PMN binding properties of phage bearing consensus-related sequences were examined by flow cytometry.
Interestingly, as shown in Fig 6, flow
cytometric analysis revealed binding of phage bearing consensus-related
sequences to a subpopulation of PMN. In particular, phage with the
DLVTSKLQV peptide, as well as other phage clones bearing the same
consensus motif (data not shown), exclusively stained a subpopulation
of PMN comprising approximately 50% of the cells. While an identical
pattern of staining was observed at 4°C and 20°C, the fluorescence
signal from cells labeled at 20°C was substantially brighter than
that obtained at 4°C. In contrast to this bimodal staining pattern, phage displaying the FGPNLTGRW motif labeled all PMN and produced a
particularly bright signal when the binding reactions were performed at
20°C. Temperature sensitive labeling was observed for one of the
cyclic motifs identified. As shown in the figure, there was no
detectable labeling of PMN at 4°C with phage bearing the CKDGLFLGSWLC peptide sequence. However, all of PMN were labeled with the phage when
binding was performed at 20°C. This finding is consistent with the
affinity purification results, since phage bearing CKDGLFLGSWLC-related sequences were not recovered from selection procedures performed at
4°C. As can be seen in Fig 6, labeling PMN at 4°C with phage bearing the peptide sequence CKDGLFLGSWLC did result in a small population of highly fluorescent cells (approximately 4%). However, analysis of orthogonal light scatter revealed that this small population of cells most likely represented contaminating monocytes. Additionally, such binding profiles were not affected by stimulation with phorbol myristate acetate or by chelation of extracellular calcium
(data not shown).
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| Fig 6.
Staining of isolated PMN with affinity purified phage
clones and analysis by flow cytometry. PMN were incubated with affinity
purified phage clones or irrelevant control phage followed by
biotinylated anti-phage antibody and labeled with FITC conjugated to
streptavidin. The distinct staining pattern of the analyzed phage
suggests binding to different target molecules. All phage clones, but
not the control phage, showed brighter signals by incubation at 20°C.
The DLVTSKLQV displaying phage (or other homologous phage, data not
shown) consistently labeled approximately 50% of the cells. Incubation
of cells with the CKDGLFLGSWLC-displaying phage at 4°C resulted in
staining of a small cell subpopulation that could be identified as
monocytes on the orthogonal light scatter (data not shown) but not in
labeling of the main cell population consisting of PMN. Histograms
represent specifically stained cell numbers on the vertical axis
(labeled counts) plotted against fluorescence on a log scale from 5,000 cells per condition. These results were obtained in four separate
analyses.
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To examine cell specificity of phage clones bearing consensus-related
sequences, the binding of selected phage clones to various cell types
was examined. In Table 1 is a summary of
the results of phage binding to a number of different cell lines
analyzed by flow cytometry. As summarized in the table,
DLVTSKLQV-displaying phage bound exclusively to PMN, whereas the
FGPNLTGRW and the CKDGLFLGSWLC bound to both PMN and monocytic (U937)
cells. Interestingly, phage bearing a sequence related to the former
monocyte-reactive one, CPFGPDLQGKWC, retained reactivity with PMN but
failed to label U937 monocytic cells. No phage binding was observed on
fibroblasts, endothelial cells, epithelial cells, and peripheral blood
lymphocytes.
Synthetic peptide binding and inhibition of phage-PMN interactions.
To confirm the binding specificity of phage-bearing consensus-related
peptide sequences, competition assays were performed with synthetic
peptides (Fig 7). Bound phage were then
quantitated by flow cytometric techniques as above. As can be seen,
binding of DLVTSKLQV-displaying phage to PMN was markedly inhibited in the presence of synthetic peptides containing identical or similar sequences of DLVTSKLQV and DLETSKMQV. A control peptide with a scrambled sequence KQLSEMVTD had no effect. Half maximal inhibition (IC50) for both peptides was seen at 0.6 µmol/L. Similarly, as shown
in Fig 7B, addition of the peptide GGPNLTGRW resulted in marked
inhibition of binding of FGPNLTGRW containing phage to PMN. Here, the
IC50 was estimated at 50 µmol/L, and phage binding was unaffected by
a control scrambled peptide (RPGNGWLGT). Interestingly, even high
concentrations of the GGPNLTGRW peptide (1 mmol/L) had no effect on the
PMN binding of the phage displaying the cyclic CPFGPDLQGKWC motif.
These results suggest that the cyclic motif may either recognize a
different receptor/receptor configuration, or that it binds with higher
affinity to the putative receptor for the linear nonpeptide.
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| Fig 7.
Phage binding competition assays. Effect of the synthetic
peptides was evaluated as described in Materials and Methods. (A)
Inhibition of PMN binding of the DLVTSKLQV displaying phage by the
DLVTSKLQV and DLETSKMQV peptides. The scramble peptide KQLSEMVTD had no
effect. (B) Inhibition of PMN binding of the FGPNLTGRW displaying phage
by the GGPNLTGRW synthetic peptide. Phage binding was unaffected by the
control scramble peptide RPGNGWLGT. The data points represent the mean ± SD from three experiments.
|
|
Cell specificity of peptide binding was further confirmed in
experiments using a photo-activatable derivative of FGPNLTGRW. Using
the biotin-conjugated photo-activatable reagent,
Biotin-GF(Benzoyl)GPNLTGRW, we compared the labeled protein profiles of
PMN and HT29 epithelial cells (Fig 8).
Cells were incubated with 100 µmol/L labeled peptide alone or in the
presence of excess (1 mmol/L) unlabeled peptide followed by photolysis.
As shown in the figure, probing Western blots of the biotinylated cell
extracts with avidin-peroxidase revealed significant differences
between PMN and HT29 cells. In particular, labeled protein bands from
HT29 cells were not eliminated by excess unlabeled peptide, whereas
biotin labeling of PMN protein bands in the 30 to 60 kD range was
inhibited by coincubation with unlabeled peptide. These results suggest
that peptides derived from cell binding phage retain cell-type binding
specificity at the protein level. Furthermore, such reagents may be
useful in the characterization of receptors for phage bearing specific
cell binding sequences.
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| Fig 8.
Cell-specific labeling by a synthetic peptide derived
from phage containing a PMN-binding peptide sequence. Suspensions of
1 × 107 HT29 epithelial cells (A) or PMN (B) in
HBSS containing 0.5% BSA were incubated at 4°C for 1 hour in the
presence (+) or absence () of 1 mmol/L FGPNLTGRW followed by the
addition of Biotin-GF(Benzoyl)GPNLTGRW to a concentration of 100 µmol/L. After a 30-minute incubation in the dark, the samples were
placed on ice and photolyzed for 10 minutes under UV light. The washed
cell pellets were solubilized in 150 mmol/L NaCl, 100 mmol/L KCl, 2 mmol/L EDTA, and 10 mmol/L Hepes pH 7.4 containing 1% N-octylglucoside
and the protease inhibitors DFP, PMSF, aprotinin, bestatin,
chymostatin, and pepstatin. Cell lysates were then incubated with
avidin-Sepharose for 2 hours followed by washing and denaturation of
the avidin beads with reduced sodium dodecyl sulfate (SDS) sample
buffer. Samples were subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) on 6% to 16% polyacrylamide gradient gels
followed by western blot, probed with peroxidase-conjugated
streptavidin, and developed by enhanced chemiluminescence (Amersham
Inc). Each lane represents the biotin-labeled protein profiles of ~3 × 106 cells. Molecular weights in kiloDaltons are shown
to the side. As can be seen, specific labeling of PMN protein bands in
the 30 to 60 kD range was inhibited by coincubation with unlabeled
peptide, whereas no specific labeling is observed for HT29 cells.
|
|
Phage effects on PMN calcium signaling.
To test for phage effects on PMN function, experiments were performed
to determine whether motif-bearing phage could induce changes in
cytosolic [Ca++] in PMN. As shown in Fig 9A, addition of
phage bearing the sequence FGPNLTGRW induced a significant change in
cytosolic [Ca++] when compared with the addition of
control irrelevant phage. In contrast, phage bearing the binding
sequence DLVTSKLQV had absolutely no effect. The magnitude of the
increase in cytosolic [Ca++] induced by the
FGPNLTGRW-displaying phage was approximately one third of that induced
by a saturating dose (10 7 mol/L) of the
well-characterized PMN agonist fmet-leu-phe (fMLF). To test whether the
phage-induced calcium signal might be G-protein mediated, calcium
experiments were performed on pertussis toxin-treated PMN. As shown in
Fig 9B, incubation of PMN for 2 hours with pertussis toxin completely
blocked the phage-induced response, as well as the fMLF-induced
response (positive control, not shown), indicating that the
FGPNLTGRW-displaying phage is indeed activating through a
G i-mediated pathway. As a negative control,
demonstrating that pertussis toxin had not simply blocked all PMN
signal transduction, stimulation with immune complexes that are known
to activate PMN by a pertussis toxin-insensitive Fc
receptor-mediated pathway,22,23,25 still exhibited a large
cytosolic [Ca++] increase (data not shown).
| |
DISCUSSION |
Currently, there are limited approaches that can be taken to identify
peptide binding domains on cells or proteins without prior specific
functional or structural information. While antibody approaches have
proven successful in this regard, extreme variability in the antigenic
response has remained problematic. Recently, phage libraries containing
vast repertoires of random peptide inserts of various lengths and
conformations have been used in the identification of interactive
regions of proteins and other molecules. In addition, with phage
techniques it is possible to identify peptide binding domains on cells
or proteins without having any preexisting information on such domains
or the biology of the target cells. We have used three such phage
display libraries to identify peptides that bind preferentially to
human neutrophils. These include groups of peptide sequences isolated
under different affinity purification procedures that contained both
the linear and structurally constrained consensus motifs
DLXTSK(M/L)X(V/I/L), GPNLTGRW, and CXXGXFLGXWLC, respectively. We have
also shown that one of these peptide motifs, GPNLTGRW, has functional
effects as defined by a Gi-linked rise in intracellular
calcium. These results define, for the first time, functional
cell-specific binding peptides identified with phage display library technology.
A dramatic finding in these studies was the identification of distinct
PMN-binding peptides that were exclusively displayed in a linear or in
a cyclic form by the bearing phage. In general, it has been assumed
that libraries displaying cyclic peptides are more capable of
interacting with targets at higher affinities than libraries displaying
unconstrained oligopeptides free to assume different structural
conformations in solution.9,26-31 In particular, previous
reports have demonstrated that peptides containing cysteine pairs can
be selected in high affinity screens of linear peptide
libraries.26 In our studies, however, we were able to
affinity purify PMN-binding phage containing linear sequences homologous to the unconstrained DLXTSK(M/L)X(V/I/L) motif even by
screening phage libraries constructed to display the random region with
a cyclic configuration. These latter findings suggest an extremely
strong selection from the CL10 and CL6 libraries for a phage
subpopulation displaying the random peptide without the intended
structural constraints, ie, missing one or both Cys residues in the
linker sequences intended to be conserved throughout all of the library
members. These findings are consistent with previous studies on MoAbs
that demonstrated an unpredictable preference for a specific type of
constraint32 but also raise the possibility of nonrandom
selection of particular clones in the unscreened libraries.
Fortunately, such phage variants have never been identified in randomly
selected clones from our library stock solutions or in phage selected
from epitope mapping studies we performed with a number of different
MoAbs (L.M. and C.A.P., unpublished observations, 1996, 1997).
Most likely, these biases result from errors in the DNA encoding for
the random region and the linker sequences. Events such as single base
changes and rearrangements undoubtedly occur during oligonucleotide
synthesis and assembly of the DNA construct. While the majority of
these errors probably result in defective phage particle formation, our
data suggests that the CL10 and CL6 phage libraries contain only a very
small percentage (<1 in 105 or 106) of
members displaying an unconstrained peptide. However, given the large
diversity of these libraries, even this small percentage results in a
considerable phage subpopulation.
In addition to the unusual selection for structural variants contained
in cyclic phage libraries, several other findings indicate that at
least part of the selected peptides bind to PMN with high affinity. For
instance, the selection of certain motifs in the detergent fractions
after low pH treatment implies high affinity binding. We excluded the
trivial explanation of internalization by low temperature experiments
(4°C) and by the lack of evidence by confocal fluorescence microscopy
on permeabilized cells. The specificity of such binding was confirmed
by demonstrating that phage binding to PMN could be competitively
inhibited by coincubation with relatively low concentrations of
synthetic peptides displaying identical or similar sequences. This
latter finding is particularly significant since the competing
synthetic peptides interact with PMN in a monovalent fashion, whereas
the phage binding is likely to occur in a multivalent fashion, and
since each phage carries five copies of the modified pIII protein.
While the nature of the PMN receptors for the peptide sequences
identified by these phage display library studies are unknown, the
results of the calcium signaling experiments indicate that FGPNLTGRW-displaying phage induce a transient intracellular calcium increase that is indistinguishable from those observed with pertussis toxin-sensitive Gi-coupled receptors. While the seven
membrane spanning chemoattractant receptors are classical examples of
this class, we have not detected chemotactic activity in experiments with the synthetic peptide FGPNLTGRW. As shown in Fig 8, cell labeling
experiments using photoaffinity derivatives of this peptide may be
useful in the identification and characterization of the membrane receptor(s).
Analyses of GPNLTGRW, DLXTSK(M/L)X(V/I/L), and CXXGXFLGXWLC using
Gen-Bank/EMBL did not reveal any significant homology with known
membrane proteins/receptors. This is not surprising since phage may
also recognize complex, discontinuous epitopes11 or non-proteinacious determinants, such as sugar groups. Interestingly, several of the analyzed peptide motifs showed homology with various viral coat proteins. In addition, some homology was identified between
the DLXTSK(M/L)X(V/I/L) motif and integrins. Specifically, the Asp and
Leu residues displayed at the amino terminus of this motif are also
present in a number of putative integrin ligand binding
sites33 and integrin recognition sequences.27
In addition, the TSK motif is present in the 10th type-III repeats of
the cell binding region of fibronectin.34 However, the
binding of phage bearing consensus-related sequences to PMN is not
reduced under conditions that inhibit integrin function such as low
temperature and in the absence of divalent cations.
A notable finding in our report was that phage bearing the
DLXTSK(M/L)X(V/I/L) motif recognized approximately 50% of the PMN in
flow cytometry studies and suggests heterogeneity in peripheral blood
PMN. Indeed, there are other reports of MoAbs that label subpopulations
of human PMN.35,36 Interestingly, flow cytometry studies
with these antibodies showed a labeling pattern indistinguishable from
those observed here with DLVTSKLQV-bearing phage. While the protein
antigen for one of these antibodies was identified as a 59-kD membrane
protein, it remains uncharacterized at the molecular level.
The identification of only a few distinct peptide sequences by scanning
whole cells (this report) or even organs7,9 is unexpected.
However, important variables such as membrane receptor number and
affinity might prevent the selection of weakly binding peptides. Phage
particles are relatively large and might be sterically hindered from
interacting with potential target molecules on the PMN membrane that
are of lower affinity or lower density. Additionally, because of the
relatively large size of the phage, higher affinity interactions might
tend to "coat" the cell with phage and effectively form a barrier
for phage bearing lower affinity peptides. In support of this
hypothesis, we found that affinity selections performed in the presence
of competing synthetic peptides (1 mmol/L of DLETSKMQV and GGPNLTGRW)
resulted in total inhibition of recovery of phage with related
sequences. In these experiments, new phage populations were selected
that were enriched in tryptophan residues (L.M. and C.A.P.,
unpublished results, 1996, 1997). Thus, it is likely that
additional cell binding sequences can be identified by inclusion of
competing peptides derived from sequence information obtained from
phage with high binding affinity. Additional factors that might
influence the recovery of specific motifs include nonspecific cell
binding and selection bias for particular sequence-bearing phage. Other
groups have indicated that absorption steps in which the library is
incubated with absorber cells may be very effective to prevent the
amplification of "undesired" sequences.5,37 Lastly,
it is unlikely that the motifs identified here are secondary to
nonspecific phage selection due to some particular property of the
mutated pIII protein. We tested phage displaying DLVTSKLQV, FGPNLTGRW,
and CKDGLFLGWLC motifs for their ability to infect E coli, and
differences between these clones and control phage were not found (data
not shown).
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| Fig 9.
Phage bearing a specific PMN-binding peptide sequence
induce a pertussis toxin-sensitive calcium signal in PMN. (A) As
described in Materials and Methods, INDO-1 loaded PMN were suspended
for 2 minute at 37°C in a Ca++-containing buffer and
cytosolic [Ca++] measured by spectrofluorimetry
before and after the addition of 4 × 1010 of phage
bearing the sequences indicated. (B) PMN were treated for 2 hours with
pertussis toxin or buffer (control), loaded with INDO-1 and treated
with FGDNLTGRW-displaying phage as in (A). Positive and negative
controls, respectively (not shown), included stimulation with
107 mol/L fMLF and immune complexes.
|
|
In addition to the prominent consensus motifs reported, our affinity
selection procedures yielded single clones that were not investigated
further but may be relevant. For instance, no matching clones were
found for the partially constrained sequence CRNCGWDRPMIF that was
recovered from the CL10 library in the acid eluted and cell-associated
fraction at 20°C. In this case, the substitution of the Cys residue
in the linker sequence at the carboxy terminus with a Phe strongly
suggests that the recovering of this phage represents a genuine selection.
In conclusion, we report herein on the identification of
neutrophil-specific binding peptides using random phage display
techniques. Peptide sequences identified by these techniques provide
new information to assist in the identification and characterization of
functional surface receptors that are cell-type specific. While the
selection of relatively few high affinity peptide ligands is favored
with these methods, the number of peptide sequences identified can be
considerably expanded through modification of affinity purification procedures, thus increasing the probability of identifying motifs with
potential biological and medical importance.
| |
ACKNOWLEDGMENT |
Thanks to Claudia Baggi and Lily Hong for expert technical assistance
and James Madara for helpful suggestions and critical review of this manuscript.
| |
FOOTNOTES |
Submitted March 31, 1998; accepted October 21, 1998.
Supported by grants from the Swiss National Science Foundation, the
Swiss Cancer League, and the Bern Cancer League (L.M.) and by grants
from the National Institutes of Health HL54229, HL60540 (C.A.P.),
AI26711, and AI22735 (A.J.J.), and a Biomedical Science grant from the
Arthritis Foundation (C.A.P.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Charles A. Parkos, MD, PhD,
Department of Pathology and Laboratory Medicine, Emory University,
Woodruff Memorial Research Bldg, Room 2309, Atlanta, GA 30322.
| |
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D. L. Jaye, F. S. Nolte, L. Mazzucchelli, C. Geigerman, A. Akyildiz, and C. A. Parkos
Use of Real-Time Polymerase Chain Reaction to Identify Cell- and Tissue-Type-Selective Peptides by Phage Display
Am. J. Pathol.,
May 1, 2003;
162(5):
1419 - 1429.
[Abstract]
[Full Text]
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S. Zitzmann, V. Ehemann, and M. Schwab
Arginine-Glycine-Aspartic Acid (RGD)-Peptide Binds to Both Tumor and Tumor-Endothelial Cells in Vivo
Cancer Res.,
September 15, 2002;
62(18):
5139 - 5143.
[Abstract]
[Full Text]
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H.-C. Wu, C.-T. Yeh, Y.-L. Huang, L.-J. Tarn, and C.-C. Lung
Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A
Appl. Envir. Microbiol.,
July 1, 2001;
67(7):
3201 - 3207.
[Abstract]
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D. L. Jaye, H. A. Edens, L. Mazzucchelli, and C. A. Parkos
Novel G Protein-Coupled Responses in Leukocytes Elicited by a Chemotactic Bacteriophage Displaying a Cell Type-Selective Binding Peptide
J. Immunol.,
June 15, 2001;
166(12):
7250 - 7259.
[Abstract]
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A. ODERMATT, A. AUDIGÉ, C. FRICK, B. VOGT, B. M. FREY, F. J. FREY, and L. MAZZUCCHELLI
Identification of Receptor Ligands by Screening Phage-Display Peptide Libraries Ex Vivo on Microdissected Kidney Tubules
J. Am. Soc. Nephrol.,
February 1, 2001;
12(2):
308 - 316.
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Top
Abstract
Introduction