BMC Evolutionary Biology
BioMed Central
Open Access
Research article
Evolutionary conservation of P-selectin glycoprotein ligand-1
primary structure and function
Bénédicte Baïsse1, Frédérique Galisson1,2, Sylvain Giraud1, Marc Schapira1
and Olivier Spertini*1
Address: 1Service and Central Laboratory of Hematology, Centre Hospitalier Universitaire Vaudois, Bugnon 46, 1011 Lausanne, Switzerland and
Institute of Bioinformatics, Center for Integrative Genomics, UNIL, Dorigny, 1015 Lausanne, Switzerland
2Swiss
Email: Bénédicte Baïsse - Benedicte.Baisse@chuv.ch; Frédérique Galisson - fgalisson@bluewin.ch; Sylvain Giraud - Sylvain.Giraud@chuv.ch;
Marc Schapira - Marc.Schapira@chuv.ch; Olivier Spertini* - Olivier.Spertini@chuv.ch
* Corresponding author
Published: 14 September 2007
BMC Evolutionary Biology 2007, 7:166
doi:10.1186/1471-2148-7-166
Received: 22 March 2007
Accepted: 14 September 2007
This article is available from: http://www.biomedcentral.com/1471-2148/7/166
© 2007 Baïsse et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: P-selectin glycoprotein ligand-1 (PSGL-1) plays a critical role in recruiting leukocytes in
inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal
threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little
information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the
evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail.
Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human,
chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog) and
examined mammalian PSGL-1 interactions with human selectins.
Results: A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14
species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation
site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal
threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between
the conserved N-terminal O-glycosylated threonine (Thr-57 in human) and the transmembrane domain, and
contains a central region exhibiting a variable number of decameric repeats (DR). Interspecies and intraspecies
polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The
moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor
conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human,
bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin. By contrast, pig or rat neutrophils were
much less efficiently recruited than human or bovine neutrophils on human selectins. Horse PSGL-1, glycosylated
by human or equine glycosyltransferases, did not interact with P-selectin. In all five species, tyrosine sulfation of
PSGL-1 was required for selectin binding.
Conclusion: These observations show that PSGL-1 amino acid sequence of the transmembrane and cytoplasmic
domains are well conserved and that, despite a poor conservation of PSGL-1 N-terminus, L- and P-selectin binding
sites are evolutionary conserved. Functional assays reveal a critical role for post-translational modifications in
regulating mammalian PSGL-1 interactions with selectins.
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Background
Leukocyte recruitment in inflammatory lesions is dependent on the sequential interactions of adhesion receptors
with their ligands [1-3]. Leukocyte rolling along inflamed
blood vessels is mediated by selectins [4-6]. L-selectin is
expressed by leukocytes while activated endothelium and/
or platelets express E- or P-selectin [5]. Early in inflammatory reactions [2,3], P-selectin mediates leukocyte rolling
on its major ligand P-selectin glycoprotein ligand-1
(PSGL-1) [7]. PSGL-1 is a homodimeric mucin-like glycoprotein [8,9], which is expressed on leukocyte microvilli
and functions as a common ligand for the three selectins
[10,11]. PSGL-1 interactions with L-selectin strongly
amplify leukocyte recruitment by supporting free-flowing
leukocyte rolling on leukocytes adherent to microvascular
endothelium or leukocyte membrane fragments [11,12].
Moreover, E-selectin interactions with PSGL-1 and CD44
and/or other potential ligands support leukocyte slow
rolling along inflamed endothelium [13-15].
Fucosylated core-2 O-glycans, bearing sialyl Lewis-x (sLex)
and/or Lex determinants, attached to human PSGL-1 Thr57 are required for optimal binding of all three selectins
[16-19]. Sulfation of Tyr-46, -48 and -51 is necessary for
optimal binding of L- and P-selectin to PSGL-1 but not Eselectin [16-18,20-22]. Murine and human PSGL-1 may
differ in their interactions with P-selectin, as sulfation of a
single tyrosine residue is sufficient for optimal binding of
murine PSGL-1 to P-selectin [23].
Human, mouse, rat, bovine and equine PSGL-1 sequences
encode a signal peptide and, except for bovine and equine
PSGL-1, a propeptide, which is predicted to be cleaved by
paired basic amino acid converting enzymes (PACE)
[9,24-26]. These sequences encode a common PSGL-1 primary structure with a N-terminal peptide expressing
potentially sulfated tyrosine residues and a O-glycosylated
threonine [9,24,25], and a mucin-like domain constituted
of a variable number of decameric repeats (DR) [24-26].
Comparison of these mammal sequences shows that the
transmembrane and cytoplasmic domains are highly conserved [9,27,28]. Little information is however available
on the intra- and inter-species evolution of decameric
motives and on the conservation of PSGL-1 N-terminus.
Multiple sequence alignment of a large number of mammalian PSGL-1 sequences is necessary to examine these
points and define motives associated with the core-2 Oglycosylation of the N-terminal threonine, homologous
to Thr-57 on human PSGL-1.
Whether the selectin binding site on mammalian PSGL-1
is evolutionary conserved has not been studied in detail.
As PSGL-1 is an attractive target for anti-inflammatory
therapy [7,29-39], this information might be helpful to
design inhibitors of inflammatory and/or thrombotic
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reactions [40-42]. We therefore compared PSGL-1
sequences of 14 mammals (9 sequences described herein
by us and 5 reported by others; [9,24-26] and performed
flow adhesion assays using neutrophils or CHO cells
expressing mammalian homologues of human PSGL-1.
Despite a poor conservation of the N-terminal amino-acid
sequences, we show that L- and P-selectin binding sites are
evolutionary conserved and that most mammalian PSGL1 bind to human selectins. Importantly, these interactions
are strongly dependent on PSGL-1 glycosylation and sulfation.
Results
Conservation of PSGL-1 sequence
Multiple alignment of mammalian PSGL-1 sequences is
presented in figure 1. A signal peptide (SP) cleavage site is
predicted between residues 17 and 18 in most sequences.
Equine PSGL-1 is an exception with a predicted cleavage
site between residues 18 and 19. Nine sequences including human have a propeptide predicted to be cleaved by
paired basic amino acid-converting enzymes (PACE/furin;
[9,43] at residue 41 (38 for northern tree shrew). By contrast, the PACE consensus sequence, RX [R/K]R is not
observed in bovine, sheep, cat, bat and equine PSGL-1
(Fig. 1).
N-terminal tyrosine sulfate residues and threonine Olinked glycans are high affinity binding sites for P- and Lselectin to human and mouse PSGL-1 [16-18,20,44-46],
which contribute to stabilize leukocyte rolling [16]. A
threonine residue, homologous to human PSGL-1 Thr-57,
is present in the various species studied here (Fig. 1). Thr57 belongs to the consensus sequence T [D/E]PP [D/E] in
12 out of 14 species. The region preceding the conserved
threonine contains 1 to 3 potentially sulfated tyrosine residues in an acid-rich region (5 species contain 3 tyrosines,
6 contain 2, and 3 only 1; Fig. 1).
A mucin-like domain is present in all studied species. It
lies between the conserved N-terminal O-glycosylated
threonine (Thr-57 in human) and the transmembrane
domain, and contains a central region exhibiting decameric repeats (DR). This region was analyzed using the
MEME program, whose parameters were applied to each
sequence individually and/or simultaneously to all
sequences. DR-containing central regions were aligned
considering the intra- and inter-species evolution of decameric motives. The degree of inter-species conservation in
the N- and C-terminal ends of the mucin-like domain
(which sometimes contains traces of mutated decamers)
is low. The mucin-like domain is composed of 247 to 322
residues and the number of DR varies from 7 in pig to 18
in chimpanzee and rhesus monkey (Table 2). The number
of DR varies in human from 14 to 16 repeats [47-50]. We
also observed a polymorphism in rat. One of the three
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Figureacid
Amino
1 sequences of mammalian PSGL-1
Amino acid sequences of mammalian PSGL-1. (A) Multiple alignment of 12 complete PSGL-1 amino acid sequences and
(B) of N-terminal sequences from putative PSGL-1 proteins of cat and sheep, which were inferred respectively from partial
genomic sequence and EST sequences identified through homology searches (EMBL/Genbank/DDBJ respective accession numbers: AANG01098304 and DY506895). The putative signal peptide (SP) and propeptide (Pro) cleavage sites are indicated by
arrows. The consensus sequence for propeptide cleavage (RX [R/K]R), recognized by PACE, is boxed. Threonine homologous
to human Thr-57 is indicated by a black arrowhead. Arbitrary gaps have been added in each sequence in order to isolate and
align separately the mucin-like region containing the decameric repeats, which is surrounded by a frame. The transmembrane
domain (TM) is marked by a bar. Asterisks indicate the amino acids involved in moesin binding.
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Table 2: Length of the mucin-like domain from the conserved
threonine up to the juxta-membrane cysteine
human
chimpanzee
rhesus monkey
mouse
rat
tree shrew
bushbaby
dog
pig
bovine
bat
equine
threonine
position
cysteine
position
length
(aa)
number of
decameric repeats
57
57
57
58
59
55
56
59
58
55
37
41
320
340
340
307
330
332
378
346
315
335
328
288
263
283
283
249
271
277
322
287
257
280
291
247
16
18
18
10
11
13
13
13
7
11
12
12
aa, amino acid
available sequences contains 12 DR [51], whereas only 11
repeats have been observed in the sequences cloned by us
(Fig. 1) and others [25]. This polymorphism suggests a
dynamic intraspecies evolution of this region.
The analysis of the sequences of PSGL-1 mucin-like
regions showed that several constitutive repeats of 10
amino acids can be identified in the center of these
regions, while both ends are made up with unconserved
amino acids. The best permutation motif, which is the
most consistent with the different sequences and which
optimizes the number of repeated units per sequence, is
AATEAQTTQP.
Interestingly, in canine PSGL-1, 3 DR strongly differ in
their sequences from the others (Fig. 1A). These units are
identical to each other and are located every 30 positions.
Combining decamera to form repeats of 30 amino acids
displays a greater consensus between repeats suggesting
that duplication of 30 amino acid units (itself created by
two duplications of 10 amino acid units followed by
mutations in the third one) arose at least twice in the evolution of dog PSGL-1. The same kind of phenomenon is
observed in bat, where the best repeated unit has a length
of 15 amino acids. Similarly, equine repeated units
exhibit a greater consensus when they are formed of 20
residues units instead of 10 [52].
A transmembrane domain of 23 residues is predicted in
all sequences immediately after the conserved cysteine
involved in PSGL-1 dimerization (Fig. 1A). A short extracellular juxta-membrane region is involved in binding
versican G3 domain, whose interaction with PSGL-1 promotes leukocyte aggregation [53]. Interestingly, three
positions in this region are perfectly conserved in all studied species (Asp-313, Val-317, Lys-318). The transmem-
brane domain is followed by a cytoplasmic tail, which is
made up of two highly conserved regions. Over the 31 first
positions of the cytoplasmic domain, 20 are completely
conserved and 5 contain conservative substitutions (Fig.
1A). Among the conserved positions, Ser-346, Arg-347,
Lys-348 and Ser-358 (Fig. 1A) are involved in moesin
binding to the cytoplasmic domain of human PSGL-1
[54]. In all sequences, the C-terminal region is ended by
11 almost perfectly conserved residues.
Human L- and P-selectin interact with human, rat, bovine,
pig or equine CHO-PSGL-1 cells
CHO cells co-expressing human FucT-VII and C2GnT-I
and human, bovine, pig, rat or equine PSGL-1 were prepared. The five transfectants expressed similar levels of
sLex and CLA. PSGL-1 expression was detected using a
mAb reacting with PSGL-1 C-terminal 6 × His tag (Invitrogen). The anti-human PSGL-1 mAbs PL1, KPL1 and PL2
[28,55] did not react with bovine, pig, rat or equine PSGL1 (data not shown). Flow cytometric analysis of human Por L-selectin/μ binding to the various CHO-PSGL-1 transfectants showed that P- and L-selectin/μ bind similarly to
human, bovine, pig, rat or equine PSGL-1 expressed by
transfected CHO cells. As the reactivity of mouse PSGL-1
with human selectins was previously described [23], we
did not repeat these analyses (Fig 2).
Human L-, P- and E-selectin bind heterogeneously to
human, bovine, pig or rat neutrophils
PSGL-1 expressed by CHO transfectants differ in their glycosylation pattern from mammalian neutrophil PSGL-1.
In CHO transfectants, the various mammalian PSGL-1 are
glycosylated by FucT-VII and C2GnT-I of human origin,
while in mammalian neutrophils PSGL-1 is glycosylated
by their own glycosyltransferases. As the glycosylation
pattern may affect PSGL-1 interactions with L- or P-selectin, we examined the reactivity of human selectins with
mammalian neutrophils (Fig. 3). L- and P-selectin/μ chimera strongly reacted with human and bovine PSGL-1,
while a weaker reaction was observed with pig and rat.
The L- and P-selectin carbohydrate ligands sLex and CLA,
identified by CSLEX-1 and HECA-452 mAbs respectively,
were strongly expressed by human neutrophils and also,
surprisingly, by equine neutrophils (mean fluorescence
intensity ± SD: human: 74 ± 1, n = 2 and 79 ± 12, n = 2;
equine: 173 ± 9, n = 2 and 108 ± 34, n = 2). By contrast,
despite significant selectin binding, sLex and CLA were
undetectable on bovine, pig and rat neutrophils (not illustrated). As selectin binding is dependent on cell surface
expression of fucosylated ligands, we examined FucT-VII
mRNA expression by RT-PCR amplification of total RNA
from bovine, pig, rat and equine neutrophils. FucT-VII
mRNA transcripts were detected in all investigated species
(data not shown). Thus, as previously established for
mouse leukocytes [56], the lack of reactivity of mAbs
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Human
Figure L2 and P-selectin/μ chimera cross-react with CHO cells expressing mammalian homologues of PSGL-1
Human L- and P-selectin/μ chimera cross-react with CHO cells expressing mammalian homologues of PSGL1. CHO cells, stably expressing similar levels of human C2GnT-I, FucT-VII and human (h), bovine (b), pig (p), rat (r) or equine
(e) PSGL-1, were incubated with saturating concentrations of human P- or L-selectin/μ chimeras (filled histogram). Chimera
binding was abrogated by 10 mM EDTA (open histogram). Human P- and L-selectin chimera did not bind (< 2%) to mock-transfected CHO cells (not shown). The percentage of positive cells and the mean fluorescence intensity are indicated in each histogram. Histograms are representative of 3–4 experiments.
CSLEX-1 and HECA-452 with most mammalian PSGL-1 is
likely due to the strong specificity of these mAbs for
human oligosaccharides. Moreover, the observation that
mAbs CSLEX-1 and HECA-452 strongly react with equine
neutrophils suggests that human and equine neutrophils
exhibit common carbohydrate structures, which are not
detectable in mouse, rat, pig or bovine.
CHO cells expressing mammalian PSGL-1 efficiently roll
on human L- or P-selectin
The role of PSGL-1 in regulating CHO-PSGL-1 cell rolling
on human L- or P-selectin was assessed under hydrodynamic flow conditions. Human PSGL-1 expressing cells
were less recruited on human P-selectin than CHO cells
expressing bovine PSGL-1. Moreover, on human L-selectin, cell recruitment of CHO cells expressing human
PSGL-1 was less efficient than that of cells expressing
bovine, pig or rat PSGL-1 (Fig. 4A). Surprisingly, CHO
cells expressing equine PSGL-1 did not roll on P-selectin
and were weakly recruited on L-selectin.
Previous studies showed that N-terminal tyrosine sulfate
residues are involved in supporting human PSGL-1dependent rolling on L- and P-selectin [16,20,45].
Human, bovine, rat and pig PSGL-1 exhibit two or three
potential N-terminal tyrosine sulfation sites, whereas
equine PSGL-1 contains only one single site (Fig. 1A). The
contribution of PSGL-1 sulfation to cell rolling was
assessed by comparing recruitment of CHO cells expressing control or desulfated human, bovine, rat, pig and
equine PSGL-1 on L- or P-selectin (Fig. 4B). Inhibition of
PSGL-1 sulfation strongly reduced L- and P-selectindependent rolling. The recruitment of CHO cells expressing human PSGL-1, on P-selectin, was inhibited by 88 ±
5%, whereas the recruitment of cells expressing bovine,
rat and porcine PSGL-1 was almost abrogated (Fig. 4B).
Rolling inhibition induced by desulfation was also seen
on L-selectin (although to a lesser degree than on P-selectin). Thus, as previously described for human PSGL-1, sulfation of bovine, pig, rat or equine PSGL-1 N-terminal
tyrosine residues is required to support PSGL-1-dependent rolling on L- or P-selectin.
Interestingly, multiple sequence alignment of mammalian L- or P-selectin shows partial or complete conservation of amino acid residues that regulate human selectin
binding to PSGL-1 tyrosine sulfate residues [16,18]. Ser47, Lys-112 and His-114 on human P-selectin bind to
human PSGL-1 Tyr-48, while human L-selectin Lys-85
and P-selectin Arg-85 interact with Tyr-51 (Fig. 4C)
[16,18]. In mammalian P-selectins, Ser-47 is conserved,
except for bat and rhesus monkey, and Lys-112 and His114 is either conserved or replaced by arginine, which
may interact with sulfated Tyr-48. Except for pig and
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Figure
Human
bovine, pig
L-,
3 Por or
ratE-selectin/μ
neutrophilschimeras bind to human,
Human L-, P- or E-selectin/μ chimeras bind to
human, bovine, pig or rat neutrophils. Neutrophils
were incubated with saturating concentrations of human L-,
P- or E-selectin/μ chimera (filled histogram). Chimera binding
was abrogated by 10 mM EDTA (open histogram). The percentage of positive cells and the mean fluorescence intensity
are indicated in each histogram. Histograms are representative of 2–3 experiments.
horse, Arg-85, which binds to human PSGL-1 Tyr-51, is
conserved or replaced by lysine (Fig. 4C). L-selectin Ser47, which binds to human PSGL-1 Tyr-48, is conserved or
replaced by a threonine, except for mouse, tree shrew and
cat, while L-selectin Lys-85, which interacts with human
PSGL-1 Tyr-51, is perfectly conserved. Results of Fig. 4B
and alignment of Fig. 4C suggest that, like in human
PSGL-1, tyrosine sulfation of mammalian homologues is
critical for L- and P-selectin interactions. Sulfation of a
unique tyrosine sulfate residue was sufficient to support
equine PSGL-1-dependent rolling on human L-selectin
(Fig. 4B). However, the recruitment of CHO cells expressing equine PSGL-1 on L-selectin was much less efficient
than that of all other CHO cell transfectants (Fig. 4A).
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Mammalian neutrophil recruitment on human L- or Pselectin is heterogeneous
The impact of PSGL-1 glycosylation by mammalian FucTVII and C2GnT-I on PSGL-1-dependent rolling on human
L- or P-selectin was assessed under various shear stresses
(0.5 to 2.0 dynes/cm2; Fig. 5). The recruitment of bovine,
porcine, rat and equine neutrophils on human L- or Pselectin strongly differed from that of the corresponding
CHO-PSGL-1 cells (Kruskal-Wallis test, P < 0.0001; Fig.
4A). At 1.5 and 2.0 dynes/cm2, bovine and human neutrophils rolled similarly on P-selectin. However, at lower
shear stresses, bovine neutrophils were significantly less
recruited than human neutrophils. Human and bovine
neutrophil recruitment on L-selectin was similar at 1.0
and 2.0 dynes/cm2 (Fig. 5). Above 0.5 dynes/cm2, porcine,
rat and equine neutrophils were less recruited on L- or Pselectin than human or bovine neutrophils (Fig. 5). At 1.5
dynes/cm2, recruitment of porcine, rat and equine neutrophils was respectively 4-, 290- and 3-fold lower on Lselectin and 53- and 36-fold lower on P-selectin than that
of human neutrophils. As observed with CHO cells
expressing equine PSGL-1, equine neutrophils did not roll
on P-selectin. At all shear stresses, rat neutrophils poorly
rolled on human L- or P-selectin. These observations are
in agreement with results of human selectin chimera
binding to neutrophils (Fig. 3); both assays showed that
bovine neutrophil PSGL-1 strongly interacts with human
L- or P-selectin whereas interactions are weaker between
human selectins and porcine neutrophil PSGL-1 and
almost absent with rat and equine PSGL-1 (Fig. 5). These
results are in contrast with those obtained with CHO cells
expressing pig or rat PSGL-1, which are much more efficiently recruited on human selectins (Fig. 4A). Interspecies differences in PSGL-1 core-2 O-glycosylation may
explain these observations.
Rolling velocities of CHO-PSGL-1 transfectants and of
mammalian neutrophils on human L- or P-selectin
Rolling velocities of CHO cells and neutrophils expressing
human, bovine, pig, rat or equine PSGL-1 were measured
under constant shear stress (Fig. 6). Velocities significantly
differed among species (Fig. 6A, P < 0.001). CHO cells
expressing human PSGL-1 rolled on P- or L-selectin with
the slowest velocities (median rolling velocity (mrv) on Pselectin: 3.6 μm/s; on L-selectin: 24.1 μm/s, n = 3). The
fastest mrv were exhibited by CHO cells expressing rat
PSGL-1 on P-selectin (36.9 μm/s) and by CHO cells
expressing equine PSGL-1 cells on L-selectin (121.5 μm/
s). Mrv of CHO cells expressing bovine PSGL-1 on P-selectin appeared three times faster than that of CHO cells
expressing human PSGL-1 (11.9 vs. 3.6 μm/s, P < 0.001),
while they were similar on L-selectin (Fig. 6A). Rolling
velocities of CHO cells expressing pig or rat PSGL-1 were
significantly higher than that of CHO cells expressing
human PSGL-1 on both L- and P-selectin (Fig. 6A, P <
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Figureof
Rolling
4 CHO cells expressing human, bovine, pig, rat or equine PSGL-1 on L- or P-selectin
Rolling of CHO cells expressing human, bovine, pig, rat or equine PSGL-1 on L- or P-selectin. (A) CHO-PSGL-1
cells were perfused under constant shear stress (1.5 dynes/cm2) on recombinant human P-selectin or at 1.0 dyne/cm2 on
human L-selectin/μ chimera adsorbed on a coverslip, precoated with goat anti-human IgM antibody, and bound to the bottom
of the flow chamber. Cell recruitment was analyzed by videomicroscopy at 4–5 min of perfusion. Results represent the mean ±
SEM of 3–5 experiments (***, P < 0.001; NR: no rolling). (B) Impact of sulfation on PSGL-1-dependent rolling. Control (black
columns) and desulfated CHO cells (white columns) were pretreated with proteinase K. Desulfated cells were cultured for 72
h in MEMα medium containing 60 mM sodium chlorate and exposed for 60 min to arylsulfatase. Results are expressed as mean
percentage of rolling cells ± SEM of 3 experiments (***, P < 0.001). (C) Amino acid sequence alignments of mammalian homologues of P- and L-selectin lectin domains. Homologues of human residues [16, 18] interacting with sulfated Tyr-48 or -51 are
respectively indicated by asterisks or arrowheads. The percentages of identity between aligned sequences are grey shaded
(dark grey: > 80%, grey: > 60% and light grey: > 40%).
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panel), whereas they rolled, like equine neutrophils, with
the slowest velocities on L-selectin (46.8 μm/s and 48.9
μm/s, respectively P < 0.001, Fig. 6B, right panel).
species-dependent
LFigure
and P-selectin-dependent
5
recruitment of neutrophils is
L- and P-selectin-dependent recruitment of neutrophils is species-dependent. Human, bovine, porcine,
rat and equine neutrophils were perfused under 0.5–2.0
dynes/cm2 on human P-selectin/μ or on human L-selectin/μ
chimera adsorbed on coverslips precoated with goat antihuman IgM antibody. PSGL-1-dependent neutrophil rolling
was abolished in presence of 10 mM EDTA. Cell recruitment
was assessed at 4–5 min of perfusion. Results represent the
mean ± SEM of 3–4 experiments (*, P < 0.05, ***, P < 0.001).
0.001). Compared to CHO cells expressing human PSGL1, increased velocities of CHO cells expressing bovine
PSGL-1 on P-selectin may have resulted in increased cell
recruitment on human selectins (Fig. 4).
The rolling velocities of human and bovine neutrophils
on human P-selectin did not differ significantly (mrv: 4.2
μm/s vs. 4.1 μm/s, n = 3, Fig. 6B, left panel), whereas
human neutrophils rolled slower on L-selectin than
bovine neutrophils (57.2 μm/s vs. 67.1 μm/s, n = 3, P <
0.01, Fig. 6B, right panel). Surprisingly, porcine neutrophils rolled with the fastest velocities on human Pselectin (mrv: 25.3 μm/s, P < 0.001, n = 3; Fig. 6B, left
The stability of rolling velocities was assessed by measuring CHO-PSGL-1 cell and neutrophil displacements on
human L-selectin within successive video frames (0.1 ms).
Peaks represent increases in velocity and valleys decreases
(Fig. 7). The stability of CHO-PSGL-1 cell rolling velocities on human L-selectin was heterogeneous among the
studied species. Although CHO cells expressing human
and bovine PSGL-1 had similar mrv, rolling velocities of
CHO cells expressing bovine PSGL-1 (mean SD ± SD: 21
± 3 μm/s) were less stable than those of cells expressing
human PSGL-1 (11 ± 2 μm/s). CHO cells expressing rat
PSGL-1 were the least stable (48 ± 9 μm/s, Fig. 7A). The
stability of neutrophil rolling velocities was also highly
heterogeneous among the studied species (Fig. 7B).
Human and equine neutrophils exhibited the most stable
rolling velocities (mean SD ± SD: 33 ± 4 μm/s vs. 32 ± 6
μm/s, n = 10, NS, Fig. 7B), whereas rat neutrophils were
the least stable (67 ± 40 μm/s, n = 4). Interestingly, pig
neutrophils exhibited periods of very slow rolling (mean
velocity < 10 μm/s) alternating with sudden acceleration,
rapidly followed by deceleration (Fig. 7B). Bovine and
equine neutrophils had similar behaviors. Despite the
presence of oligosaccharides recognized by HECA-452
and CSLEX-1 mAbs on both CHO cells and neutrophils
expressing equine PSGL-1, transfected CHO cells rolled
significantly faster and less stably than equine neutrophils, suggesting that other structures regulate equine
neutrophil rolling.
Discussion
Selectins and PSGL-1 play a critical role in regulating leukocyte migration in inflammatory lesions [4]. Whether
human selectins can bind to mammalian PSGL-1 had not
been previously studied. As PSGL-1 is an attractive target
for anti-inflammatory therapy [29-33,35,37-39], the identification of conserved PSGL-1 functional regions may be
helpful to design selectin inhibitors mimicking PSGL-1.
We therefore analyzed PSGL-1 amino acid sequences of
several mammals (5 previously described sequences;
[9,24-26] as well as 9 new sequences described herein, 7
complete and 2 N-terminal sequences; Fig. 1A–B) identified by us or others [25,26]. Multiple sequence alignments
(Fig. 1) show that conservation of sequence is not homogeneous along the protein, and that the primary sequence
of the site of interaction of L- or P-selectin [16,18] is not
perfectly conserved. All sequences contain a threonine
homologous to the core-2 O-glycosylated Thr-57 in
human, and a T [D/E]PP [D/E] motif, which is conserved
in all species, except in horse and dog. Nevertheless, even
if the region preceding this threonine always contains at
least one tyrosine residue in an anionic environment (pre-
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Figurevelocities
Rolling
6
of CHO-PSGL-1 transfectants or neutrophils on human L- or P-selectin
Rolling velocities of CHO-PSGL-1 transfectants or neutrophils on human L- or P-selectin. (A) CHO cells expressing human, bovine, pig, rat or equine PSGL-1 or (B) neutrophils were perfused under a constant shear stress on recombinant
human P-selectin (1.5 dynes/cm2) or L-selectin/μ chimera (1.0 dyne/cm2). Cell velocities were measured at 4–5 min of perfusion. Curves were constructed in (A) using 183–755 or (B) 25–381 independent determinations of cell-rolling velocities and
are representative of three experiments. Median rolling velocities, representative of 3 experiments, are indicated.
dicting sulfation; [57] its length in the mature protein, as
well as the number (1 to 3) and positions of the potentially sulfated tyrosine residues are variable. Desulfation
and sulfation inhibition studies suggest that tyrosine sulfation plays a key role in supporting mammalian PSGL-1
interaction with human L- and P-selectin (Fig. 4B). Data
presented here indicate that L- and P-selectin binding sites
on PSGL-1 are evolutionary conserved and emphasize the
role of threonine-linked core-2 O-glycans and tyrosine
sulfate residues in supporting mammalian PSGL-1 interactions with human selectins.
A signal peptide is predicted to be cleaved in all PSGL-1
sequences between positions 17 and 18, except in horse
where cleavage is predicted between Leu-18 and Gln-19.
Nine out of fourteen sequences exhibit a propeptide
sequence ended by a PACE cleavage site, whereas five others (horse, bat, bovine, cat and sheep) do not contain it.
Of note, the cleavage predictions of both the signal peptide and the propeptide have been corroborated in rat by
N-terminal sequencing of PSGL-1.[25] Cleavage predictions suggest that the mature PSGL-1 protein starts at position 42 of the precursor in most studied species, but at
position 18 or 19 in five other species (bovine, bat, horse,
cat, sheep), and that the length of the N-terminal
sequence preceding the O-glycosylated threonine varies
from 14 amino acids in bushbaby to 39 in cat and sheep.
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PACE cleaves PSGL-1 propeptide on human neutrophils.
In contrast, the propeptide remains on CHO-PSGL-1 cells,
which do not express the PACE protease. The lack of
PSGL-1 cleavage by PACE in bovine and equine neutrophils did not prevent PSGL-1 interactions with
selectins. The importance of propeptide cleavage is
unclear: whether it may facilitate tyrosine sulfation or Nterminal O-glycosylation remains to be determined [21].
dependent on the binding of PSGL-1 cytoplasmic domain
to moesin, which serves as adaptor between PSGL-1 and
Syk [54]. Importantly, the moesin binding residues, corresponding to Ser-346, Arg-347, Lys-348, and Ser-358 in
human PSGL-1 [54] are perfectly conserved in all analyzed mammalian sequences. Of note, these amino acids
are located within a group of 31 amino acids, among
which 20 are identical and 5 similar.
The T [D/E]PP [D/E] sequence, which is associated on
human and mouse PSGL-1 with threonine O-glycosylation [9,18,58], is observed in most mammals except dog
and horse, in which it is respectively replaced by TDAPE
and TDLLK sequences. Despite these changes, equine neutrophils rolled on human L-selectin (Fig. 5). By contrast,
neither equine neutrophils nor CHO cells expressing
equine PSGL-1 significantly interacted with human Pselectin (Fig. 4, 5). This suggests that the T [D/E]PP [D/E]
motif may be important for mammalian leukocyte rolling
on human P-selectin.
On L-selectin, rolling velocities of CHO cells expressing
human, bovine, and pig PSGL-1 were similar, whereas the
median rolling velocity of CHO cells expressing rat or
equine PSGL-1 was 4- and 5-fold higher respectively than
that of CHO cells expressing human PSGL-1 (Fig. 6). The
increased rolling velocities of CHO cells expressing
bovine, pig or rat PSGL-1 on P-selectin may partially
explain the preserved cell recruitment on P-selectin (Fig.
4A). As all CHO-PSGL-1 transfectants are glycosylated by
human C2GnT-I and FucT-VII, differences in CHO-PSGL1 cell recruitment and rolling velocities may mainly result
from differences in N-terminal amino-acid residues interacting with the lectin domain of human L- or P-selectin.
Among these residues, tyrosine sulfate residues may critically regulate PSGL-1 interactions with L- or P-selectin,
like in human PSGL-1 [16,18,22,45]. The strong inhibition of CHO-PSGL-1 cell interactions with P- or L-selectin
after desulfation and inhibition of sulfation supports this
possibility (Fig. 4B). In addition, in most studied mammals, the amino acids regulating selectin interactions with
potentially sulfated tyrosine residues are conserved (Fig.
4C). In mouse, Tyr-54 and Thr-58 regulate PSGL-1 interactions with P-selectin.[23] Because only one tyrosine is
used, it was suggested that mouse PSGL-1 binding may
rely more on O-glycans attached to Thr-58 than does
human PSGL-1.[23] This may also occur in other mammals, which exhibit a single tyrosine residue (tree shrew,
bat and horse, Fig. 1).
We observed that the sequence AATEAQTTQP is the best
permutation motif to optimize the number of decameric
units per sequence and that the most similar units lie at
the center of the mucin-like region, while unconserved
amino acids are more frequently present at both ends.
This suggests that decamera located at the center of the
mucin-like domain might be the most recent and that the
evolution of this region might have proceeded by duplications of decameric units, followed by mutations and deletions. This process allowed the conservation of the length
of the mucin-like domain with a 250–280 amino acid
length (except in bushbaby), despite a variable number of
repeated units among species (from 7 DR in pig to 18 DR
in monkeys, Table 2). The preservation of PSGL-1 length
may play a role in supporting the rolling on human
selectins of leukocytes or CHO cells expressing human,
bovine, pig or rat PSGL-1 (Fig. 4).
Transmembrane and cytoplasmic domain sequences are
well conserved (Fig. 1). The juxta-membranous cysteine
residue, involved in human PSGL-1 dimerization and in
stabilizing leukocyte rolling on P-selectin [27,28,59] is
perfectly conserved. A role for PSGL-1 as signaling molecule was indicated by its involvement in activating GTPase
Ras and mitogen-activated protein kinases, as well as in
inducing the secretion of inflammatory molecules [60-62]
or in activating αMβ2 or αLβ2 integrins [63-65]. The high
degree of conservation of the cytoplasmic domain suggests that PSGL-1-mediated intracellular signaling is evolutionary conserved. Human PSGL-1 engagement induces
Syk phosphorylation and recruitment in lipid rafts as well
as the expression of the early-immediate gene c-fos
[54,66]. Syk activity, which is critically involved in regulating PSGL-1-dependent rolling on P-selectin [66], is
Differences in tyrosine sulfation and O-glycosylation may
affect the stability of rolling velocities on L-selectin. Thus,
the patterns of bovine, pig and equine neutrophil displacements differed from those of CHO cells expressing
mammalian PSGL-1. In particular, pig neutrophils, and
also bovine and equine neutrophils, exhibited periods of
very slow rolling velocity, alternating with rapid accelerations and decelerations (Fig. 7). These observations
emphasize the role of post-translational modifications in
regulating PSGL-1 binding to human selectins.
Conclusion
Data presented here indicate that mammalian PSGL-1
share a common primary structure and has evolutionary
conserved interactions with L- and P-selectin. As in
human, PSGL-1-dependent rolling is regulated by core-2
O-glycosylation of a conserved threonine residue and by
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human and mouse PSGL-1 [9,24]. Primers are listed in
Table 1.
Full-length PSGL-1 cDNAs were obtained using primers
specific for each species (Table 1): forward human,
bovine, pig, rat and equine PSGL-1 contain an AflII restriction site and reverse PSGL-1 primers an AgeI and a ClaI
restriction site removing the stop codon. Forty amplification cycles were performed using the Platinum® Pfx DNA
Polymerase (Invitrogen; 30 s at 94°C, 45 s at 54°C, 2 min
at 72°C). PCR products were gel-purified, sequenced,
digested with AflII/AgeI and cloned in the pcDNA5/FRT/
V5-His-TOPO® expression vector containing, C-terminally, 6 × His tag (Invitrogen).
α1–3 fucosyltranferase-VII (FucT-VII) mRNAs from
human, bovine, pig, rat and equine neutrophils were
amplified using the Superscript™ One-Step RT-PCR with
platinum® Taq Kit (Invitrogen). Primers were derived from
human and mammalian FucT-VII sequences (Table 1). βactin transcripts were used as control.
Figure 7of rolling
Stability
mammalian
neutrophils
velocities
on human
of CHO-PSGL-1
L-selectin transfectants or
Stability of rolling velocities of CHO-PSGL-1 transfectants or mammalian neutrophils on human Lselectin. Frame-by-frame rolling velocities of (A) CHOPSGL-1 transfectants or (B) human, bovine, pig, rat or equine
neutrophils on human L-selectin. The velocity of tracked cells
was determined by measuring cell displacements within successive video frames (0.1 ms) in the flow direction under a
shear stress of 1.0 dyne/cm2. Cells were tracked for 4 to 6 s.
Data are representative of 4–10 experiments.
tyrosine sulfation. The high degree of conservation of
PSGL-1 cytoplasmic domain suggests, as for human PSGL1, a potential involvement in signal transduction and in
regulating cell rolling. These results provide additional
insights into the structure and function of PSGL-1 and
may be helpful to design PSGL-1 peptidomimetics.
Methods
Bovine, porcine, murine and equine PSGL-1 and FucT-VII
cDNAs
RNA was extracted from mammalian lymphocytes using
TRIzol® (Invitrogen, Basel, Switzerland). Bovine, pig and
rat homologues of human PSGL-1 cDNAs were generated
from lymphocyte total RNA using GeneRacer™ Kit (Invitrogen), according to the manufacturer protocols. Primer
design was based on sequence homologies between
Cells
Mammalian lymphocytes were isolated by blood centrifugation on Ficoll and polymorphonuclear cells (PMN)
were obtained by dextran sedimentation and erythrocyte
hypotonic lysis [10]. Flp-In™-CHO-K1 cells (Invitrogen)
stably expressing core2 β(1,6)-N-acetyglucosaminyltransferase-I (C2GnT-I) and FucT-VII [16] were transfected
using TransIT®-LT1 (Mirus Corporation, Madison, WI)
with human, bovine, pig, equine or rat PSGL-1 constructs.
CHO cells were cultured in MEMα medium (Invitrogen)
containing 10% fetal calf serum (FCS), 800 μg/mL G418
(Invitrogen) and 700 μg/mL Hygromycin B (CalbiochemNovabiochem, Schwalbach, Germany). CHO cells coexpressing similar levels of sialyl Lewis × (sLex), cutaneous
lymphocyte antigen (CLA) and PSGL-1 terminated by Cterminal polyhistidine (6 × His) tag were isolated by limiting dilution. CHO-P-selectin and 300.19-L-selectin cells
were cultured as described [19].
Inhibition of sulfation
CHO-PSGL-1 cells (107 cells in 1 mL of PBS) were treated
with proteinase K (170 μg/mL; Roche Diagnostics,
Rotkreuz, Switzerland) for 20 min at 37°C.[67] After proteinase K inhibition with phenylmethylsulphonylfluoride
(Sigma-Aldrich, St-Louis, USA), cells were cultured for 72
h in sulfate-deficient MEMα medium containing 10% dialyzed FCS and 60 mM sodium chlorate (Sigma [68]). They
were then further desulfated, for 60 min at 37°C, with
Aerobacter aerogenes arylsulfatase (1 U/ml in PBS, type VI,
Sigma).
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Table 1: Sequences of primers used for RACE and RT-PCR analysis
primer name
Sequence
Ta
GeneRacer™ 5'
Reverse GSP
GeneRacer™ 5' Nested
Reverse Nested GSP
GeneRacer™ 3'
Forward GSP
GeneRacer™ 3' Nested
Forward Nested GSP bovine
Forward Nested GSP pig
Forward Nested GSP rat
Forward hPSGL-1/AflII
Forward b, pPSGL-1/AflII
Forward rPSGL-1/AflII
Forward ePSGL-1/AflII
Reverse h, b, p, r, ePSGL-1/AgeI/ClaI
Forward h, b, p, rFucT-VII
Reverse h, b, p, rFucT-VII
Forward h, b, p, rβ-actin
Reverse h, b, p, rβ-actin
5'-CGACTGGAGCACGAGGACACTGA-3'
5'-CAGACCATCTCGGTGGGGGAGTA-3'
5'-GGACACTGACATGGACTGAAGGAGTA-3'
5'-CACAGTGCACACGAAGAAGATAGTG-3'
5'GCTGTCAACGATACGCTACGTAACG-3'
5'-ACTCCACTGGCAGCCACAGAGG-3'
5'CGCTACGTAACGGCATGACAGTG-3'
5'-CCCTTCCTGTGGCCTCTGATACTC-3'
5'-ACCAGCACCCACGGAGGCACAGACC-3'
5'-CCCTGCCAGGGAGTTCAGATCTC-3'
5'-AGCCTTAAGCCACCATGCCTCTGCAACTCC-3'
5'-TATCTTAAGCCACCATGTTTCTGCAACTCC-3'
5'-CGCCTTAAGCCACCATGTTCCCACACT-3'
5'-AGCCTTAAGCCACCATGCCTCTGCCGCTC-3'
5'-TGGACCGGTATCGATAGGGAGGAAGCTGTG-3'
5'-TCCTTGTCTGGCACTGG-3'
5'-GCGGTGCTGGGAGTTCT-3'
5'-GAGACCTTCAACACCCC-3'
5'-GTGGTGGTGAAGCTGTAGCC-3'
64°C
55°C
55°C
56°C
54°C
50°C
50°C
Ta, annealing temperature; GSP, gene specific primer; h, human; b, bovine; p, pig; r, rat; e, equine; FucT-VII, α1–3 fucosyltranferase-VII.
Immunophenotypic analysis
Cell staining with mAbs or L-, P-, or E-selectin/IgM heavy
chain (μ) chimera was performed and analyzed with a
Cytomics™ FC 500 cytofluorimeter (Beckman Instruments, Nyon, Switzerland), as described [19].
Flow adhesion assays
Cells (106/mL) were perfused in a parallel plate flow
chamber (GlycoTech Corp., Rockville, MD) mounted on
a glass coverslip covered with a confluent monolayer of
CHO cells or coated with L-selectin/μ (2.0 μg in 100 μL
0.1 M borate buffer, pH 8.5, surface: 75 mm2) or P-selectin/μ (0.1 μg in 100 μL borate buffer) chimera or recombinant P-selectin (0.5 μg in 100 μL borate buffer) (R&D
Systems, Minneapolis, MN) adsorbed on goat antihuman IgM antibody (2.0 μg in 100 μL 0.1 M borate
buffer, pH 8.5; Caltag Laboratories, Burlingame, USA;
[16,19,69]. CHO-PSGL-1 cell and neutrophil interactions
were recorded for 5 min by videomicroscopy [16,19,69].
Rolling velocities illustrated in Fig. 6 were measured by
tracking individual cells every 0.1 s, for 1–20 s, using a
digital image analysis system (Mikado software, GPIL SA,
Martigny, Switzerland; [16,19,69]. 183–755 independent
determinations of cell rolling velocities were measured to
analyze the velocities of transfectants and 25–381 determinations for the analysis of neutrophil velocities. Frameby-frame velocities (Fig. 7) were measured by tracking
cells every 0.1 s for 6 s, within 0.28 mm2 microscopic
fields. The mean velocity of frame-by-frame tracked cells
was included between percentiles 40–60 of the velocity of
each cell population illustrated in Fig. 6. L- and P-selectindependent rolling was inhibited (>95%) by 10 mM EDTA
or LAM1–3 or WAPS12.2 mAbs (data not shown). Mocktransfected CHO cells did not roll on L- or P-selectin.
CHO transfectants used in adhesion assays expressed similar levels of cell surface PSGL-1 and sLex [19].
Sequences
PSGL-1 amino-acid sequences were either retrieved from
the Uniprot database [70], or deduced from our own
cDNA sequences (bovine, equine, pig and rat respective
accession numbers [EMBL: AM778464, AM778465,
AM778466, AM778467]), or inferred from their gene
sequences identified through homology searches (chimpanzee, rhesus monkey, dog, bat, northern tree shrew,
and bushbaby respective EMBL/Genbank/DDBJ accession
numbers:
AADA01122192,
AANU01210210,
AAEX02034222, AAPE01064070, AAPY01200400, and
AAQR01577322).
Most selectin sequences were retrieved from Uniprot.
Accession numbers of human, chimpanzee, rhesus monkey, rat, mouse and bovine L-selectins are P14151,
Q95237, Q95198, Q63762, P18337 and P98131, respectively. Those of human, rat, mouse, bovine, dog, pig,
equine and sheep P-selectin are P16109, P98106,
Q01102, P42201, Q28290, Q29097, Q5J3Q6 and
P98109, respectively. The chimpanzee and rhesus monkey P-selectin sequences were retrieved from the Refseq
database49 (IDs: XM_001137240 and XM_001094728).
Dog, pig, northern tree shrew, bushbaby, cat L-selectins,
and bat, northern tree shrew and cat P-selectins were predicted from their DNA sequences (EMBL/Genbank/DDBJ
respective
accession
numbers:
AAEX02026138,
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BMC Evolutionary Biology 2007, 7:166
BW973806,
AAPY01338510,
AANG01023786,
AAPE01015496,
AANG01023773).
http://www.biomedcentral.com/1471-2148/7/166
AAQR01653637,
AAPY01338519,
3.
4.
Sequence analysis
Multiple alignments were obtained by analyzing local and
global similarities between PSGL-1 (Fig. 1) or selectin
sequences (Fig. 4) using Clustal-W, T-Coffee and MEME
programs [71-73]. Alignment was edited and colored
using the Jalview program [74]. Signal peptides, propeptides and transmembrane domains were predicted with
the SignalP, ProP, and TMHMM programs [75-77].
5.
6.
7.
8.
9.
Statistical Analysis
Analysis of variance and the Bonferroni multiple comparison test or the Kruskal-Wallis non-parametric ANOVA
test were used to assess statistical significance of differences between groups. The Mann-Whitney test was used
to compare the medians of two unpaired groups. P values
< 0.05 were considered as significant.
Abbreviations
CHO cells - Chinese Hamster Ovary cells;
DR - Decameric repeats; FucT, fucosyltransferases;
10.
11.
12.
13.
PACE - Paired basic amino acid converting enzymes;
PSGL-1 - P-selectin glycoprotein ligand-1;
sLex - Sialyl Lewis-x;
TM - Transmembrane domain;
14.
15.
16.
mrv - Median rolling velocity.
Authors' contributions
OS, BB and FG designed research, analyzed data and
wrote the paper. BB and SG performed experiments. FG
performed multiple alignment. MS critically reviewed the
manuscript and contributed to writing. All authors read
and approved the final manuscript
17.
18.
19.
Acknowledgements
We would like to thank Dr Marco Burki, Dr Giuseppina Milano and Dr Urs
Muester for providing mammalian blood samples, and Dr Angela Ciuffi for
critical review of our manuscript. This work was supported by the grant
n°3200BO-105593 from the Swiss National Foundation for Scientific
Research.
20.
21.
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