Download Structure/activity studies of anti-inflammatory

Glycobiology vol. 6 no 8 pp. 831-836, 1996
Structure/activity studies of anti-inflammatory peptides based on a conserved peptide
region of the lectin domain of E-, L- and P-selectin
John B.Briggs, Robert A.Larsen, Robert B.Harris1,
Kumara V.S.Sekar1 and Bruce A.Macher2-3
Glycomed, Inc. (a wholly owned subsidiary of Ligand Pharmaceuticals),
860 Atlantic Avenue, Alameda, CA 94501, USA, 'Viriginia Commonwealth
University, Department of Biochemistry and Molecular Biophysics, Box
980108, Richmond, VA 23298, USA, and Commonwealth Biotechnologies,
Inc., Richmond, VA 23219, USA, and 2Department of Chemistry and
Biochemistry, San Francisco State University, 1600 Holloway Avenue, San
Francisco, CA 94132, USA
'To whom correspondence should be addressed
Previously, it was established that the peptide YYWIGIRK-NH2 inhibits both myeloid cell adhesion to selectins
in vitro and neutrophil influx into inflammatory sites in vivo
(Briggs et aL, 1995). Initial structure/activity studies revealed that at least one Y residue at the N-terminus of the
peptide was essential for these bioactivities but that the
C-terminal K residue was unnecessary for inhibitory activity. We have now synthesized a new series of peptides
which contain single residue substitutions at each position
of the reference peptide, YYWIGIR-NH2, and have tested
these peptides for inhibitory activity in a selectin cell binding assay. In addition, peptides containing single D-amino
acids at selected positions, or an all D-configured reference
peptide sequence, or the retro-inverso version (rigjwyyNH2) of the reference peptide sequence have also been analyzed for inhibitory activity in the same assays. Finally, the
ability of the reference peptide and a specifically designed
control sequence (YYCAIB^GIR-NHj) to discriminate between potential synthetic saccharide ligands, including sialyl-Lewis x, Lewis x, and sialyl-N-acetyl-lactosamine, was
investigated using isothermal titration calorimetry. The results of these studies demonstrate that whereas many single
amino acid substitutions are tolerated in the peptide without complete loss of inhibitory activity, substitution at some
positions (e.g., the W residue) results in relatively inactive
compounds, clearly pointing to the importance of these
residues in making critical contacts with the appropriate
saccharide ligand. Titration calorimetry revealed that the
reference peptide does not discriminate between Lewis x or
sialyl-Lewis x in vitro, but binds these saccharides with
nearly 40-fold higher affinity (KD 25 uM) than the nonfucosylated trisaccharide, sialyl-N-acetyl-lactosamine. We
can infer from these studies that the presence of a sialyl
group, per se, is not a requisite for complex formation
between the reference peptide and its saccharide ligand.
Substitution of single D-amino acid residues at various positions in the reference peptide sequence reduces or eliminates all inhibitory properties. However, the all Dconfigured peptide or the retro-inverso peptide sequence
have greater activity than the all L-configured reference
peptide in the in vitro biological assays, and each was an
effective inhibitor of neutrophil infiltration in a thioglycollate-induced mouse peritonitis model. These results, com> Oxford University Press
bined with the results of titration, allow us to conclude that
binding between the reference peptide and its saccharide
ligand, which affords its inhibitory properties, is mediated
by the presence of a contiguous, nonpolar surface, or face,
presented at the N-terminus of the reference peptide, likely
encompassing the sequence YYWI. Furthermore, the W
plays a critical role in binding, probably through formation
of an essential hydrogen bond with a suitably juxtaposed
group carried on the saccharide ligand.
Key words: selectin/peptides/inhibition/saccharide ligand
Introduction
Discovery of the selectins and demonstration of their role in the
inflammatory response (see Lasky, 1995, and Rosen and Bertozzi, 1994, for recent reviews) has potentially provided new
opportunities for treatment of a host of diseases. Several drug
candidates are being evaluated based on the interaction of selectins with their saccharide ligands, including antibodies directed against selectin proteins and against recombinant forms
of the 'lectin-like' binding domain of the selectins (reviewed in
Rosen and Bertozzi, 1994). Other molecules are being evaluated as potential human therapeutics, including putative antagonists of saccharide binding that are mimics of a defined
portion of the relevant saccharide (e.g., see Brandley et aL,
1993; Nelson et aL, 1993; Yuen et aL, 1994) and synthetic
peptides which incorporate sequences found within the selectins or other carbohydrate binding proteins at the saccharide
binding sites (e.g., see Geng et aL, 1992; Heerze et aL, 1995).
We previously identified and described a selectin-based peptide sequence (YYW1GIRK-NH2; Briggs et aL, 1995) that inhibits selectin-mediated binding of myeloid cells to surfaces in
vitro and also inhibits neutrophil infiltration into inflammatory
sites in vivo. Initial structure/activity analyses of this sequence
showed that both inhibitory activities were partially retained in
the sequence YWIGIR-NH2, but shorter-chain peptides, particularly those that lacked at least one N-terminal Y residue,
were not inhibitors in either assay system. Thus, we concluded
that the C-terminal K residue was dispensable for bioactivity,
but that the N-terminal end of the peptide was essential to
maintain inhibitory potency. In the present study, a much wider
range of analogs of the reference peptide sequence Y YWIGERNH2 were evaluated to determine features of this peptide that
are necessary for biological activity. In addition, analogs containing single D-amino acids, or an all D-configured peptide, or
the retro-inverso version of the peptide sequence were also
evaluated for inhibitory activity as compared to the reference
sequence.
ITC affords simultaneous determination of all thermodynamic parameters relevant to ligand binding by macromolecules, including KA the association constant, and the enthalpic
(AH) and entropic (AS) contributions to the Gibbs free energy
831
J.B.Briggs el aL
of association. Although titration calorimetry is now routinely
used to assess binding between relatively high molecular
weight macromolecules and their ligands, we have successfully
applied the technique to study binding interactions between
relatively short-chain peptides, between short-chain peptides
and protein ligands, or between short-chain peptides and saccharide ligands (You et aL, 1993; Tyler-Cross et aL, 1993,
1994; Page et aL, 1994). In the present study, the ability of the
reference peptide to discriminate between potential synthetic
saccharide ligands, including sLex, Lex, and sLN (Figure 1),
was investigated using ITC. Taken together, our results suggest
that binding between the reference peptide and its saccharide
ligand, which affords its inhibitory properties, is mediated by
the presence of a contiguous, non-polar surface, or face, presented at the N-terminus of the reference peptide, likely encompassing the sequence YYWI-.
Results
Previous analyses established that the peptide YWIGIR-NH2
was a moderate inhibitor (IC J0 5 p,M) of myeloid cell binding
to recombinant forms of E-selectin (Briggs et aL, 1995). We
found that removal of the N-terminal Y residue (Y1, see below)
completely abolished inhibitory activity, but the relative contribution of the other residues to inhibitory activity was not
established. Thus, an extensive series of peptides has now been
prepared and evaluated to establish the importance of particular
chemical functional groups, including amino acid side-chains
and non-amino acids, at each residue position. We also evaluated whether removal or addition of residues altered the inhibitory properties of the peptide.
For ease of reference, the residue positions of the all Lconfigured reference peptides are designated numerically.
Hence, Y° Y1 W2 I 3 G4 I5 R6-NH2 is the reference peptide
sequence.
In vitro binding assays (Table I)
Group A compounds contain single D-amino acid substitutions
at positions 1, 2, or 6, which either increased the IC 50 value by
a factor of 10 (compound Al) or completely abolished the
peptides' inhibitory properties.
Group B compounds contain amino acid substitutions at
position 1. Substitutions with a heterocyclic ring substituent
(see Figure 2 for structures) show -3-6 fold higher I C ^ values
than the reference peptide, whereas those with a charged substituent show dramatically poorer inhibitory potencies (compounds B3 and B4 are -50 fold poorer inhibitors).
Group C compounds (Table I) contain atypical amino acid
substituents at position 2 that are not commonly found in proteins. Substitutions with a bicyclic functional group (Cl and
C2) produced moderately good inhibitors with I C ^ values that
were 4- to 6-fold higher than the reference peptide, whereas
compounds C3 and C4, which contain a monocyclic ring structure, were much poorer inhibitors with IC 50 values that were 30
times the IC 50 of the reference peptide.
The compounds in Group D and F contain substitutions for
I3 and I5, respectively. Tertiobutylalanine (t-BuAla) was an
acceptable substitution for either I residue, but peptides that
contained t-BuGly at these positions were 20- to 40-fold poorer
inhibitors. Substitution of V or L produced very poor inhibitors, whereas substitution of NLeu or NVal produced moderately good inhibitors with I C ^ values 5- to 8-fold higher than
832
the reference peptide. Peptides containing aminobutyric or
aminoisobutyric acid at either position were not inhibitors.
Replacement of G4 with either D- or L-Tyr produced excellent inhibitors (Group E), and substitution with other polar
(E3), or charged (E4, E5) L-amino acids produced good inhibitors. In contrast, peptides containing D-Glu or D-Arg were
poorer inhibitors and substitution of L- or D-Pro resulted in the
elimination of inhibitory properties of the peptide. The peptide
lacking G4 (E10) altogether and the peptide containing an additional G residue (El 1) were poor inhibitors.
Substitution of DAPA or citrulline for R6 produced excellent
inhibitors of cell binding (compounds Gl and G2), but surprisingly, substitution of K for R significantly decreased the inhibitory potency of the peptide.
HyPro was an acceptable replacement for Y° (HI), as was a
replacement of Y° with a heterocyclic ring substitution (H2).
However, replacement with R or K produced much poorer
inhibitors. Two other 7-residue peptides were evaluated as inhibitors. II is entirely D-configured, and 12 is the retro-inverso
peptide sequence. In contrast to the compounds in Group A
that contained a single D-amino acid residue replacement
which rendered the peptide essentially inactive as inhibitors, II
and 12 were excellent inhibitors with IC 50 values that were less
than that of the reference compound (Figure 3).
Taken together these results suggest that presentation of a
nonpolar 'face' or surface at the N-terminal end of the reference compound is essential for inhibitory activity, and that
residues which did not disrupt this nonpolar surface were permissive replacements. Furthermore, W appears to be essential
for biological activity.
To test these ideas further, additional peptides were prepared
and evaluated in which the proposed non-polar surface was
disrupted by inclusion of strictly polar residues (SGRDGEKNH2) or in which the proposed nonpolar surface was disrupted
and residues found to be nonpermissive replacements at positions 4 and 5 were included (SGFAPSR-NH2). Finally, another
peptide was prepared (YY(AIB)IGIR-NH2) in which the essential W2 residue was replaced with aminoisobutyric acid, a
residue that does not mimic the physiochemical properties of
W and that cannot participate in hydrogen bond pairing. As
expected, none of these control peptides were inhibitors in the
cell binding assay, and produced only limited activity (<20%
inhibition) at the highest concentration (500 (JLM) tested.
Isothermal titration calorimetry binding studies
Titration calorimetry was used to directly assess solution-phase
complex formation between YYWIGIR-NH2 and three potential synthetic saccharide ligands, sLe", Le", and sLN, or between YY(ALB)IGIR-NH2 and the same saccharides. We have
previously shown the utility of using ITC to assess the thermodynamic parameters mediating solution-phase binding between short-chain peptides and their carbohydrate (Tyler-Cross
et aL, 1993, 1994), or peptide, or protein ligands (You et aL,
1993; Page et aL, 1994).
As shown (Table II), YYWIGIR-NH2 does not discriminate
between sLe* or Le\ but binds these two ligands with nearly
40-fold higher affinity (KD = -25 pM) than it binds sLN, a
nonfucosylated trisaccharide ligand. Binding is thermodynamically favored (negative AG values of about 6 kcal/mol), and
enthalpically driven. In marked contrast, YY(AIB)IGIR-NH2
did not bind any of the saccharide ligands (Table II), again
pointing to the importance of the Tip residue in saccharide
recognition.
Selectin-based anti-inflammatory peptides
OH
Table I. Inhibition" of HL-60 cell binding to E-selecdn by various analogs
of the reference peptide Y°Y'W2I3G4I5R6-NH2b
OH
ICJQ (JJIM)
Reference peptide
Group A—substitutions with D-amino acids
Al
y'
r6
A2
w2
A3
Group B—substitutions for Y1
Bl
HyPro
3-PyrAla
B2
R
B3
B4
K
Group C—substitutions for W2
Cl
(3-BzThi)Ala
C2
2-NaphAla
C3
3-PyrAla
C4
(2-Thi)Ala
Group D—substitutions for I3
Dl
t-BuAla
D2
NorVal
D3
V
D4
t-BuGly
D5
L
D6
ABA
D7
AIB
Group E—substitutions for G4
El
Y
E2
y
E3
s
E4
Q
E5
R
E6
q
E7
r
P
E8
E9
P
E10
Ell
GG
Group F—substitutions for I5
Fl
t-BuAla
F2
NorLeu
V
F3
F4
t-BuGly
F5
L
F6
ABA
F7
AIB
Group G—substitutions for R6
Gl
Cit
G2
DAPA
K
G3
Group H—substitutions for Y°
HI
HyPro
H2
3-PyrAla
H3
K
R
H4
Group I—others
11
yywigir-NH2
12
rigiwyy-NH2
10
100
280
>500
30
60
500
>500
O:
HO
• ivy
HO
\
/
40
60
300
320
y
Oh
Sialyl Lewis X
5
80
250
380
>500
>500
>500
5
10
10
15
15
100
160
>500
>500
85
480
10
50
200
200
320
>500
>500
OH
OH
>—o
OH
0=
OH \
O
(
OH
\
HO
(
HN
^
O
(
HO
y
HO
\
/
"OH
Hti
OH
^O
T
Sialyl LacNAc
5
10
200
25
25
45
80
<5
<5
"Peptides listed in groups H and I are peptides with amino acid substitutions
to the seven amino acid sequence YYWIGIR-NH2. Peptides listed in groups
A through G are pepudes with substitutions to the sequence YWIGIR-NH2.
b
All compounds were screened in the cell binding assay, as described in
Materials and methods.
c
Lowercase letters denote D-amino acids. Other abbreviations used include:
ABA, a-aminobutyric acid; AIB, ot-aminoisobutync acid; (3-BzThi)Ala,
B-(3-benzothienyl)alanine; Cit, citmlline; DAPA, diaminopropionic acid;
HyPro, 4-hydroxyproline; 2-NaphAla, 2-naphtylalanine; 3-PyrAla,
3-pyridylalanine; (2-Thi)Ala, B-(2-thienyl)alanine; t-BuAla,
tertiobutylalanine; t-BuGly, tertiobutylglycine; (-) denotes the absence of an
amino acid at this position (i.e., YWUR); GG indicates the insertion of an
additional residue at this location (i.e., YWIGGIR). See Figure 2 for
structures of atypical amino acid substituents not commonly found in
proteins.
HO
OH
O
HN
Lewis X
Fig. 1. Structures of sialyl Lewis x, sialyl N-aceryl-lactosamine, and Lewis x.
833
J.B.Briggs et aL
H2N
NH2
NH
2-NaphAla
t-BuGly
AIB
Cit
t-BuAla
ABA
r
NH,
DAPA
p-2-ThiAla
NorVal
NorLeu
3-PyrAla
Fig. 2. Structures of peptides with atypical amino acid substituents tested in the HL-60/selectin chimera binding assays The top line represents the peptide Y1
W I3 G4 I3 R6-NH2. Some of the substitutions represented in Table I are shown below this peptide
In vivo studies
A direct correlation has previously been established between
inhibition in the cell adhesion assay and inhibition of neutrophil influx into sites of inflammation in a murine thioglycollate-induced peritonitis model. To establish a similar correlation with compounds tested in this study, two compounds were
evaluated for inhibitory activity in a mouse model of thioglycollate induced peritonitis. As demonstrated in Figure 4, the all
D-amino acid version of the reference peptide inhibited neutrophil influx into the peritonea] cavity to a degree similar to
that observed with reference peptide, YYWIGIR-NH2. In preliminary studies, the retro-inverso peptide also demonstrated
the ability to inhibit neutrophil influx in the mouse model of
thioglycollate induced peritonitis model (data not shown).
Discussion
YYWIGIRK is a highly conserved sequence within all cloned
selectins and in initial studies, it was found that a synthetic
834
peptide encompassing this sequence was a good inhibitor or
E-selectin mediated cell adhesion (Briggs et aL, 1995). It was
further shown that this reference peptide sequence inhibited
neutrophil influx in a mouse peritonitis model.
Thus, to further complete structure/activity studies of this
sequence, a series of analogs of this compound have now been
analyzed. These studies highlight permissive substitutions at
particular sites within the peptide, demonstrate allowed replacements of D- for L-amino acid, and point to the overall
structural requirements for inhibitory properties.
Some residue substitutions were tolerated at each position,
but some positions were more tolerant of changes than others.
For example, G4 can be replaced with a variety of amino acids,
including D-amino acids and amino acids with completely different physicochemical properties. In marked contrast, W2 is
absolutely required for biological activity and can only be replaced with amino acids or amino acid analogs which contain
side chains similar to that of Tip and which can participate in
SeJectin-based anti-inflammatory peptides
....
100 -I
25000 -
i
90 -1$
•
•
•
80 -
s
:
|
§
^
70^
60 :
o
50-
3
40
\
YYWIGIR-NH2
yywigir-NH2
rigiwyy-NH2
20000 -
15000 -
o
-;
10000 -
30 -:
\
\
20 -
T
10 '-_
|
1
1
,
10
,
1
,
,
,
20
,
1
.
1
1
1
30
40
Peptide Concentration
Fig. 3. Inhibition of HL-60 cell binding to recombinant E-selectin chimera
by YYWTGIR-NH2 (circle), the all D- peptide yywigir-NH2 (square), and
the retro-inverso all D- peptide rigiwyy-NH2 (triangle).
l.pJl.v. TREATMENT
hydrogen bond pair formation. In the peptide YY(AIB)IGIRNH2, W2 is replaced with aminoisobutyric acid, resulting in a
peptide that is completely inactive in the cell binding assays
and that was shown by ITC not to bind any of the sacchande
ligands. We can thus safely conclude that Trp participates in
saccharide binding by forming a productive hydrogen bond
with a suitably juxtaposed saccharide functional group.
Saccharide binding to YYWIGIR-NH2 appears to be mediated by interactions that take place between the saccharide
ligand and residues that constitute a nonpolar face encompassing the N-terminal segment of this peptide. Residues that disrupt this surface are nonpermissive replacements, and residues
that change the nature of this surface to a polar sequence are
also nonpermissive replacements. Consistent with this hypothesis, the all D-residue configured peptide and the retro-inverso
versions of this sequence are excellent inhibitors of selectin
mediated cell adhesion and leukocyte trafficking. Clearly, it
makes no difference if the surface is formed on opposing
'sides' of the peptide backbone, or whether the surface is
Table II. Thermodynamics of saccharide binding by YYWIGIR-NH2
and YY(AIB)IGIR-NH2"
AGb
(kcal/mol)
Peptide
Ligand
KD
(u.M)
AH
(kcal/mol)
AS
(eu)
YYWIG1R-NH2
sLe*
Le»
sLN
sLe*
Lex
sLN
24.8
30.6
1165.5
-73.7
-18.6
-3.2
No binding
No binding
No binding
-249
-6.38
^5.9
-6.25
+ 1.6 -4.06
observed
observed
observed
YY(AIB)IGIR-NH2
"All experiments were done at 30°C in 30 mM phosphate buffer, pH 7 01
For all experiments, the indicated peptide was placed in the calorimeter
cuvette at 1.0 mM and each saccharide was placed in the dropping syringe
at an initial concentration of 10 mM. All isotherms were corrected by
subtraction for heat of mixing and dilution following injection of ligand into
buffer alone.
b
AG = - R T inK.
Fig. 4. Inhibition of leukocyte trafficking into thioglycollate-treated
peritoneum by YYWIGIR-NH2 and yywigir-NH2 peptides. Mice were
treated as described in Materials and methods.
formed at the C-terminal, instead of the N-terminal, end of the
peptide. The results of the binding studies were substantiated
by in vivo studies which clearly demonstrated that the two
different D-amino acid forms of the peptide were fully functional in the mouse peritonitis model. This exciting result has
significant ramifications in that the all D-configured peptide
and the retro-inverso peptides represent a new class of antiinflammatory agents.
Titration calorimetry reveals that the mode of binding between YYWIGIR-NH2 and tri- (Lex) or tetra-(sLex) saccharide
ligands is enthalpically driven and thermodynamically favored.
The recognition of complex oligosaccharides by proteins, including lectins, antibodies, and enzymes is accomplished primarily through interactions with particular hydroxyl groups on
the carbohydrate, but van der Waals interactions also occur in
most instances primarily through stacking of the underface of
pyranose residues with aromatic amino acids (Khare et ai,
1985). Many of the interactions occur through formation of
hydrogen bonds between the sugar hydroxyls and side chains
of suitably opposed amino acids. However, the presence of
sialic acid, per se, on the saccharides tested does not seem to be
involved in stabilizing the peptide/saccharide complex (i.e.,
ITC, YYWIGIR-NH2 did not discriminate with respect to
binding between Le" or sLe"). Given that the N-terminal end of
the peptide contains no charged residues, we can further infer
that electrostatic interactions cannot play a major role in binding. Nonetheless, we have previously demonstrated that YYWIGIR-NH2 functions in vivo to effectively suppress neutrophil infiltration and have shown that the peptide competes with
E-selectin for saccharide binding in the cell adhesion assay.
Thus, we can infer that in vivo, the peptide does bind to the
physiologically relevant saccharide ligand of E-selectin.
835
J.B.Briggs el aL
Eliminating G4 or inserting an additional G residue resulted
in peptides that were only marginal inhibitors. Thus, the chain
length, or more likely, the appropriate spacing and spatial orientation of the residues of the amino acids within the peptide
is critical for optimal inhibitory properties. A few additional
peptides that contained additional amino acids at the Nterminus of the reference sequence were prepared and tested,
but none were better inhibitors than the reference sequence.
Based on this limited set of compounds, it appears that a peptide chain-length of six amino acids is required for inhibitory
activity in the selectin cell adhesion assay.
Materials and methods
Sacchandes
Lex, sLe" and sLN (structures shown in Figure 1) were gifts from Mr. Ken
Wlasichuk (Glycomed, Inc.). The sacchandes were determined to be 100%
pure by NMR and mass spectrometric analyses.
Peptide synthesis
Amidated peptides were purchased from Commonwealth Biotechnologies,
Inc., Richmond, VA, or from Chiron Mimotopes, San Diego, CA.
Prior to use in the biological assays, the peptides were dissolved at the
desired stock concentration in Dulbecco's phosphate-buffered saline (PBS)
containing Ca 2+ and Mg2+. The pH of the peptide solution was adjusted to
neutrality by the addition of NaOH or HC1 and the solution was sterilized by
filtration. In some cases, DMSO was added prior to the addition of the PBS to
facilitate the solubilization of the peptide. When DMSO was used its final
concentration in the peptide solution was 1% (v/v)
HL-60/selectin chimera binding assays
The ability of the test peptides to prevent E-selectin mediated, cell adhesion to
microtiter plate wells was assessed by direct assay, essentially as described
previously (Briggs et al, 1995). Briefly, wells of plastic microuter plates were
coated with E-selectin/IgG chimera protein (Erbe et al, 1992, 1993) by transferring a solution containing the chimera (125 ng in 50 mM carbonate buffer,
pH 9 5) to each well and allowing the plate to stand overnight at 4°C. HL-60
cells grown in RPMI-1640 medium containing 10% fetal calf serum were
fluorescently labeled with BCECF-AM (Molecular Probes, Inc., Eugene, OR)
in PBS and then preincubated with test peptide solution for 30 min al room
temperature. The solution was then transferred to the microtiter wells precoated with E-selectin IgG chimera. After 1 h at 37°C, the nonadherent cells
were aspirated from the wells and the wells were washed with PBS containing
Ca2+ and Mg 2+ . The number of adherent cells was quantified by measuring the
amount of fluorescence released from the cells following detergent lysis (2%
(v/v) Triton X-100 in 0.1 M Tris, pH 9.5).
Thioglycollate induced peritonitis
The ability of the test peptides to suppress neutrophil influx in vivo was
determined using a munne thioglycollate peritonitis model. Hence, female
Swiss Webster mice (7-9 weeks old) (Simonsen Labs, Gilroy, CA) were
injected in the tail vein with 200 u.1 of 3 mM peptide solution or PBS. Immediately following the peptide injection, the mice were injected lntraperitoneally
with I ml of fluid thioglycollate medium (BBL/Becton Dickinson, Cockeysville, MD) prepared as instructed by the manufacturer. Three hours after
thioglycollate injection, the mice were sacrificed by CO2 asphyxiation and the
cells in the peritoneum were harvested with a 5 ml lavage of hepannized (5
U/ml), 0 9% sodium chloride containing 0.1% bovine serum albumin (Sigma.
St. Louis, MO). Peritoneal cell counts were determined with a Coulter counter
as follows. The lavage fluids were diluted 1:50 in Isoton II counting fluid and
the cells lysed by adding S/P Lysing and Hemoglobin Reagent (1:100 final
dilution) (Baxter Scientific Products, Hayward, CA). The nuclei were counted
in a sized window with the lower limit set at 3.9 u.m and the upper limit at 5.7
u.m. Cell counts from peptide treated animals were compared to untreated,
thioglycollate-stimulated controls (Watson et al, 1991).
ITC measurements
ITC was used to assess binding between the test peptides and various synthetic
sacchandes. All experiments were done using an Omega titration calorimeter
(Microcal, Inc) as previously described (You et al, 1993: Page et al. 1994.
836
Tyler-Cross et al, 1993, 1995). Details for the analyses are given in the
footnote to Table II.
Acknowledgments
We thank Mr. Ken Wlasichuk for providing the sacchandes used in this study,
and Dr. James Gilbert, Ms. Mary Schaefer, and Ms. Mane Casabonne for
performing the in vivo studies This work was supported by Glycomed Inc., a
wholly owned subsidiary of Ligand Pharmaceuticals, Alameda, CA, and by a
grant from Commonwealth Biotechnologies, Inc., Richmond, VA , to R.B.H.
Abbreviations
FTC, isothermal titration calonmetry; Le\ Lewis X (Gaip 1,4[Fuca 1,3]GlcNAc);
sLe*, sialyl Lewis x (NeuAca2,3Gaipi,4[Fucal,3]GlcNAc); sLN, sialyl-Nacetyl-lactosamine (NeuAcot2,3GalBI,4GlcNAc); single letter abbreviations
are used to denote amino acids (small letters designate D-amino acids).
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Oda.Y. and Hasegawa,A. (1993) Structure-function studies on selectin carbohydrate ligands. Modifications to fucose, sialic acid and sulphate as a
sialic acid replacement. Glycobiotogy, 3, 633-639.
BriggsJ.B., Oda,Y., GilbertJ.H., Schaefer.M.E. and Macher,B.A. (1995) Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx
into inflammatory sites in vivo. Glycobiology, 5, 583-588
Erbe.D.V., Wolitzky.B.A., PrestaX.G., Norton.C.R , Ramos.R.J., Burns.D K.,
RumbergerJ.M., Rao.B.N.N , Foxall,C, Brandley.B.K. and Lasky,L.A.
(1992) Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion. J. Cell Biol, 119, 215-227.
Erbe,D.V., Watson.S.R., Presta,L.G., Wolitzky.B.A., Foxall.C., Brandley.B.K
and Lasky.L.A. (1993) P- and E-selectin use common sites for carbohydrate
ligand recognition and cell adhesion. J Cell Biol, 120, 1227-1235.
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Received on Max 14. 1996; revised on Julx 12, 1996, accepted on Juh 22,
1996