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INTRODUCTION |
Cyclophilins are highly conserved proteins first identified as the
main binding proteins for cyclosporin A
(CsA),1 an immunosuppressive
drug widely used in the prevention of graft rejection (1, 2). They were
later identified as peptidyl-prolyl cis/trans isomerases
(PPIase) (3, 4). Such an activity consists of the acceleration of the
cis/trans isomerization of Xaa-Pro peptide bonds and has
been proposed to be involved in protein folding (5). The enzymatic
activity of cyclophilins is strongly inhibited by CsA because of the
binding of the drug over the catalytic site of these proteins.
Different members of the cyclophilin family have been described. They
all contain a conserved core domain, carrying both the CsA binding and
isomerase sites, flanked by distinct N and C termini accounting for
their specificities (6). The prototype of this family is the abundant
cytosolic 18-kDa form now named cyclophilin A (CyPA) (1). Cyclophilin B
(CyPB) (7) and cyclophilin C (CyPC) (8) are closely related, but their
mRNA encodes a signal peptide that directs them to the secretory pathway. A mitochondrial form called cyclophilin D (9) and a second
larger cytosolic form named cyclophilin 40 (10) have also been
described. Alignment of amino acid sequences reveals 65% identity
between CyPA and CyPB (6, 7) and more than 70% between CyPB and CyPC
(8). In the central core of the three forms the conservation in amino
acid sequence is over 80%, implying that the regions bearing the CsA
binding and isomerase activity are very similar in the different
cyclophilins. The three-dimensional structures of CyPA (11), CyPB (12),
and CyPC (13) have been solved and as expected the central core is
similarly shaped. The structure includes eight antiparallel
strands
forming a right-handed
-barrel overlaid by connecting loops and
helices (11). Both active sites are closely localized in a large
hydrophobic pocket formed by four
strands and their connecting
loops, whereas the N and C termini are located on opposite sides of the molecule.
In the case of CyPB, the C-terminal VEKPFAIAKE sequence has been
described as a signal for retention in intracellular vesicles (14). The
protein however was reported to follow the secretory pathway (15). Our
finding that CyPB is present in human milk and blood plasma provided
the first evidence that it is effectively secreted into biological
fluids (7, 15, 16). Mariller et al. (17) furthermore
demonstrated that CyPB isolated from milk is truncated because of the
absence of the C-terminal AIAKE sequence. The presence of CyPB in
plasma spurred us on to find out whether specific binding sites for
this protein existed on blood cells. Indeed we were able to detect
surface binding sites for CyPB on human peripheral blood T lymphocytes
(18). The binding parameters were estimated to be 10-20 nM
for the dissociation constant (Kd) and
30,000-120,000 for the number of binding sites/cell. We also found
that the surface-bound ligand was specifically internalized into T
cells and that CsA-complexed CyPB retained its cellular binding
properties while promoting increased uptake of the drug and enhanced
immunosuppressive activity (19-21). More recently, we were able to
distinguish two classes of CyPB binding sites on the surface of
peripheral blood T lymphocytes (22). Although both types of sites
exhibit similar binding affinities, they are easily discriminated by
their sensitivity to 0.6 M NaCl. The type I class of
binding sites is insensitive to ionic strength and corresponds to
functional lymphocyte receptors for CyPB, because endocytosis of the
ligand follows the binding (22). Interaction with type I sites is
reduced in the presence of CsA, suggesting that the conserved CsA
binding region of CyPB is involved. CyPC also interacts with the
lymphocyte receptors, however with a 5-fold lower affinity, whereas
CyPA does not, indicating that fine differences in the
three-dimensional structure and/or a few specific amino acids may be
responsible for variations in binding affinity. The type II class of
binding sites represents at least 70% of the total lymphocyte binding
capacity and corresponds to sulfated glycosaminoglycans (GAG) (22).
Interestingly, the interaction of CyPB with GAG is strongly inhibited
by a peptide corresponding to the 24 N-terminal amino acid residues of
CyPB, but remains unchanged in the presence of CsA (22). On the other
hand, the binding to lymphocyte type II sites is found exclusively for
CyPB, because neither CyPA nor CyPC is able to reduce the ligand
binding (22). These results suggest that two distinct regions of CyPB may be involved in the interactions with lymphocyte GAG and specific membrane receptors.
In this study, which aims to understand the functional implications of
CyPB association with the two classes of binding sites on T cells, we
have tried to identify the GAG binding region and the functional
receptor binding region in the CyPB molecule. Using either fragments of
CyPB obtained by proteolysis or mutant forms modified by genetic
engineering, we localized two amino acid clusters necessary for binding
to sulfated GAG. In addition, we provide evidence that the region
interacting with the type I binding sites is close to the CsA binding
site of CyPB and that the amino acids of the enzymatic site of the
protein are not required for the interactions with the receptor.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human CyPA and CyPB were purified as
described previously (7, 23). Recombinant human CyPC was a generous gift from Novartis (Basel, Switzerland). For site-directed mutagenesis, the previously described bacterial expression construct, which contains
the coding sequence of mature human CyPB placed between the
NcoI and HindIII sites of the pKK233-2 plasmid
(Amersham Pharmacia Biotech, Uppsala, Sweden) was used as template.
Construction of the CyPBL17A/R18A mutant has already been
outlined (24). For the CyPBR76A/G77A/D78A mutant
(CyPBRGD-), DNA fragments covering the 5' moiety from the
NcoI site up to the mutated residues flanked by a
KpnI restriction site (introduced simultaneously as a silent mutation) and the 3' moiety from the same site to the
HindIII site following the stop codon were generated by
polymerase chain reaction. After gel purification and appropriate
digestion, both fragments were introduced into the pKK233-2 plasmid in
a three-way ligation. The CyPBK3A/K4A/K5A
(CyPBKKK-), CyPB
14YFD16
(CyPB
YFD), CyPBR62A, CyPBF67A,
and CyPBW128A mutants were generated using the Quickchange
kit (Stratagene, La Jolla, CA) with minor modifications. Briefly,
complementary primers covering the region to be modified were used to
generate mutated unmethylated strands. Upon treatment with the
DpnI endonuclease twice for 3 h at 37 °C, the
methylated template DNA was digested allowing the selective rescue of
the mutated strand after bacterial transformation. The DNA sequence of
all the mutant plasmids was verified using the dideoxy chain termination method (25) with dATP[
S] and the Sequenase kit (both
from Amersham Pharmacia Biotech, Amersham, UK), and vector- and
insert-specific primers. Production and purification of recombinant wild-type and mutated CyPB were performed using the procedure described
by Spik et al. (7). Protein separation was performed at pH
6.0 using a UnoS-12 cation exchange column (Bio-Rad).
Partial Enzymatic Proteolysis of Recombinant Human CyPB and
Purification of Peptides--
Before hydrolysis, clostripaïn
(E.C. 3.4.22.8) (Pharmacia) was activated by incubation in 0.1 M phosphate buffer, pH 7.4, containing 2.5 mM
dithiothreitol and 1 mM CaCl2 for 2 h.
Hydrolysis was then performed at 37 °C for 24 h with a molar
ratio enzyme/substrate equal to 1/20. Clostripaïn mainly
hydrolyzes peptide bonds located C-terminal of Arg residues. At the end
of the incubation, the sample was loaded on a QAE-Sephadex A-50 column
(Pharmacia). The acidic isoelectric point (pI) of clostripaïn
allowed its retention on the column while peptides were collected in
the elution fraction. The peptides obtained were then separated by
reverse phase high performance liquid chromatography (HPLC) using an
Ultrapore C8 column (Beckman). Elution was achieved by a continuous
H2O/acetonitrile gradient in the presence of 0.08%
trifluoroacetic acid, and monitored at 215 nm using a SP8450 detector
(Spectra-physics, San José, CA). First analysis of peptides of
interest was done by mass spectrometry using a matrix-assisted laser
desorption ionization technique coupled with an analyzer of time of
flight (MALDI-TOF). This preliminary identification was confirmed by
partial amino acid sequence determination.
Enzymatic Activity Assay and CsA Binding Analysis--
The
peptidyl-prolyl cis/trans isomerase activity was assessed
according to the method of Fischer et al. (26) with the
difference that the reaction was allowed to proceed for 90 s at
10 °C. For CsA binding analysis, an automated LH-20 column
(Pharmacia) binding assay was used, according to the procedure
described by Hanschumacher et al. (1) and Spik et
al. (7). The CsA binding capacity of each mutant was calculated by
comparing the initially applied amount of radioactivity to that
recovered in void volume fractions.
Interactions with Heparin--
The capacity of wild-type and
mutant CyPB to interact with heparin was analyzed on prepacked Hi-trap
heparin-Sepharose columns (Pharmacia) previously equilibrated in a 20 mM phosphate buffer, pH 7.4. Purified proteins (1 mg) were
loaded onto the column and elution was performed with a NaCl gradient
from 0 to 1 M in phosphate buffer at a flow rate of 0.5 ml/min. The elution profile was monitored by following the absorbance
at 280 nm.
Cellular Binding Experiments--
To provide a reference model
for the study of CyPB binding to T lymphocytes, the human lymphoblastic
Jurkat cell line (E6-1 clone, ATCC TIB-152) was used for binding
experiments according to the method described by Allain et
al. (18). To discriminate between type I and type II binding
sites, cells were washed three times with 3 ml of cold Dulbecco's
phosphate-buffered saline and once with phosphate buffer containing 0.6 M NaCl, as described by Denys et al. (22). The
binding curves for each mutant were fitted using the wild-type
dissociation constants determined for each type of sites as fixed parameters.
Molecular Modeling--
Molecular modeling was carried out on a
personal computer using the WinMGM software (27). The structures for
human CyPA and CyPB were obtained from the Brookhaven National
Laboratory protein data bank, as files 1CWA and 1CYN, respectively. The
crystallographic coordinates of N-terminal area
1Asp-Glu-Lys-Lys-Lys (5) of CyPB are not available;
therefore the determination of the putative three-dimensional structure of this area was performed on the basis of the partial known
coordinates of the molecule by energy minimization on a Silicon
Graphics Optane workstation using the SYBYL program.
Statistical Analysis--
Results are expressed as the mean
values ± S.D. from at least three separate experiments performed
in triplicates. Statistical analysis was performed using a Student's
t test and a P value less than 0.05 was
considered significant.
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RESULTS |
Discrimination of CyPB Binding Sites on Jurkat cells--
As a
prerequisite for our binding experiments, we examined whether,
similarly to those found on T lymphocytes, the surface binding sites on
Jurkat cells can be divided in two classes. The total surface binding
of [125I]-CyPB to Jurkat cells was characterized by a
Kd estimated to be 9.2 ± 2.0 nM
(Table I) and a number of sites of
80,000/cell. After washing with a buffer containing 0.6 M
NaCl, the binding capacity was strongly reduced to less than
20,000/cell, indicating that about 75% of CyPB binding sites were
sensitive to NaCl treatment and corresponded to the type II sites
previously identified as sulfated GAG. As seen in Table I, the ligand
binding affinity for type I sites (Kd = 20 ± 10 nM) was in the same range as that of type II sites
(Kd = 9 ± 5 nM). These
dissociation constants were used as fixed parameters for fitting the
binding curves for each competitor. To verify the specificity of the
interactions, we analyzed the binding of CyPA and CyPC to Jurkat cells.
CyPA did not interact significantly, whereas CyPC exhibited binding only to the NaCl-resistant sites that correspond to the functional CyPB
receptor (not shown). The binding affinity of CyPC to these type I
sites was however 6-fold lower (Kd = 117 ± 65 nM) than that of CyPB. Taken together, our results indicate
that the type I and type II binding sites previously described on
peripheral blood T lymphocytes (22) are also expressed on the membrane of Jurkat cells.
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Table I
Binding parameters studies of CyPB mutated in its N-terminal part
The Kd values for the total binding sites were
determined by the Scatchard linearization method. Jurkat cells were
incubated for 1 h at 4 °C with increasing concentrations of
radiolabeled ligands. To discriminate between the type I and type II
binding sites, cells were washed with Dulbecco's phosphate-buffered
saline containing 0.6 M NaCl and the indicated ligands were
used to compete with wild-type [125I]-CyPB for binding to
Jurkat cells. Radioactivity was then measured in the supernatant
(ligand released from the type II sites) and in cellular pellets
(ligand remaining bound to the type I sites). The binding curves for
each mutant were fitted using the wild-type dissociation constants
(Kd) as fixed parameters.
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Strategy of Identification of Binding Regions of CyPB to
GAG--
To delineate specific regions of CyPB possibly involved in
the interactions with the type II binding sites on Jurkat cells, we
compared the amino acid sequence of CyPB with those of CyPA and of CyPC
(Fig. 1). CyPB and CyPC are highly
homologous and the main difference occurs in the N-terminal region.
Indeed the interactions of CyPB with T lymphocyte binding sites have
earlier been shown to involve, at least in part, the N-terminal
extension of the protein (22, 24). This region contains an Arg residue at position 18 which, when derivatized with
p-hydroxyphenylglyoxal inhibits the binding of CyPB by 70%.
Mutation of this residue does not however affect ligand binding,
suggesting that other residues located nearby are involved in the
interaction with GAG (24). It is known that such interactions are
largely electrostatic and require basic amino acids. Indeed, CyPB
possesses a highly accessible and basic
3Lys-Lys-Lys5 cluster in its N-terminal part,
which might establish ionic interactions with the sulfated GAG.
However, CyPC, though possessing a basic RKR cluster at a similar
position (Fig. 1), does not interact with the lymphocyte GAG. Another
region possibly implicated in the establishment of interactions with
GAG is the 14Tyr-Phe-Asp16 sequence, which is
specific of CyPB and near enough to the Arg18 residue to be
affected by the chemical modification (22). The residues
Glu22 and Asp23 are other residues specific of
CyPB and close to Arg18. Importantly, three-dimensional
structure analysis (12) has previously revealed these two amino acids
to be localized in a loop structure (loop 19-24) not found in other
cyclophilins. To study the importance of all these residues of
the N-terminal region in the interactions with type II sites, we
engineered a series of CyPB mutants. In CyPBKKK- the
sequence 3Lys-Lys-Lys5 was replaced by AAA. In
the case of CyPB
YFD the
14Tyr-Phe-Asp16 sequence was deleted.
CyPBE22K and CyPBD23P were engineered by substituting Glu22 and Asp23 with the
corresponding amino acid residues of CyPC and CyPA. In addition, we
used a second method to identify the receptor binding regions of CyPB.
It consisted of generating peptides by partial proteolytic cleavage of
CyPB and searching which ones inhibited the binding of wild-type
CyPB.

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Fig. 1.
Amino acid sequence alignment of CyPB with
CyPA and CyPC. The MKVLLAAALIAGSVFFLLLPGPSAA peptide is
boxed and corresponds to a signal peptide that does not
exist in mature CyPB. Gray areas indicate amino acid
residues conserved among CyPA, CyPB, and CyPC. The residues that were
mutated in CyPB are underlined. The
3Lys-Lys-Lys5 and
14Tyr-Phe-Asp16 sequences were substituted by
an AAA sequence and deleted, respectively. The residues
Glu22 and Asp23 were respectively substituted
by Lys (as in CyPC) and Pro (as in CyPA). The
76Arg-Gly-Asp78 sequence was replaced by AAA.
The Arg62, Phe67, and Trp128
residues, which correspond to the active site residues
Arg55, Phe60, and Trp121 of CyPA,
were substituted by Ala.
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Analysis of CsA Binding and Enzymatic Activities of CyPB Mutated in
Its N-terminal Part--
Before the analysis of their cellular binding
activity, all the CyPB mutants were tested for PPIase activity and
efficiency to form a complex with CsA. All four mutants,
i.e. CyPB
YFD, CyPBKKK-, CyPBE22K, and CyPBD23P retained both activities
with values comparable to those found for wild-type CyPB (data not
shown). These results are not surprising because the mutations made are
confined to the N-terminal region, which is spatially localized on one
side of the molecule (12), far away from the regions involved in PPIase
or CsA binding activities.
Interactions of CyPB
YFD and CyPBKKK-
Mutants with Heparin--
To support the hypothesis that
14Tyr-Phe-Asp16 and
3Lys-Lys-Lys5 are required for the interactions
of CyPB with GAG, the mutants CyPB
YFD and
CyPBKKK- were analyzed for their ability to interact with
heparin (Fig. 2). Wild-type CyPB and
mutants were applied onto a heparin-Sepharose column in a phosphate
buffer, pH 7.4. After extensive washing with the same buffer, elution
of the proteins was performed by increasing concentrations of NaCl from
0 to 1 M. As previously reported, CyPB was eluted at 0.6 M NaCl only, indicating that it was strongly retained on
the column. In contrast, CyPBKKK- and
CyPB
YFD were more rapidly eluted from the column, at 0.1 and 0.3 M NaCl, respectively. These results indicate that both tripeptides are likely to be involved in the interactions with
sulfated GAG. The 3Lys-Lys-Lys5 tripeptide
seems to be of more crucial importance, because its replacement
dramatically reduced the avidity of CyPB for heparin. CyPBKKK- was eluted at an ionic strength lower than 0.15 M, indicating that cyclophilin lacking the
3Lys-Lys-Lys5 sequence was unable to
interact with sulfated GAG under physiological conditions. In
contrast, the elution of CyPB
YFD required a 0.3 M NaCl concentration, showing that interactions with
heparin may occur in an isotonic medium and supporting our hypothesis
for a role of this sequence in stabilizing the interactions of CyPB with sulfated GAG. Molecular modeling was used to examine the relative
positions of the two potential GAG binding sites in the folded
N-terminal extension of CyPB. The model shown in Fig.
3 clearly illustrates that the two
clusters are juxtaposed in the three-dimensional structure and could
act synergistically to interact tightly with GAG chains.

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Fig. 2.
Comparison between the binding of CyPB and of
N-terminal CyPB mutants to heparin-Sepharose. 1 mg of
CyPBKKK- (dotted line), CyPB YFD
(dashed line), and CyPB (solid line) were applied
onto a heparin-Sepharose column (1-ml HiTrap column) equilibrated with
a 20 mM phosphate buffer at pH 7.4. Elution was performed
by incremental increases of the NaCl concentration and monitored at 280 nm.
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Fig. 3.
Ribbon diagram of the 1-31 N-terminal part
of CyPB. The model was visualized with the WinMGM program using
the coordinate file obtained after energy minimization. The unknown
structure of the 1Asp-Glu-Lys-Lys-Lys5
extremity was determined using the SYBYL program and extended to the
partial known structure of CyPB (1CYN from the Brookhaven National
Laboratory protein data bank). Side chains with hydrogen atoms of
residues Lys3, Lys4, Lys5,
Tyr14, Phe15, Asp16, and
Arg18 are indicated.
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Cellular Binding Properties of CyPB
YFD and
CyPBKKK- Mutants--
To find out whether the interactions
of CyPB with heparin reflect those with the surface binding to the type
II sites, we tested the ability of CyPB
YFD and
CyPBKKK- to compete with wild-type CyPB for binding to
Jurkat cells (Table I). Cells were incubated in the presence of 50 nM [125I]-CyPB and increasing concentrations
of unlabeled wild-type or mutated proteins. After washing, cells were
treated with 0.6 M NaCl, and the distribution of remaining
surface-bound (type I sites) and released (type II sites) ligand was
analyzed. As expected, CyPB
YFD and CyPBKKK-
were ineffective in reducing the binding of [125I]-CyPB
to the type II sites. In contrast, interactions with the type I sites
were reduced to an extent similar to what was measured with the
wild-type unlabeled CyPB, implying that both mutants could still
interact with the type I binding sites but had lost their ability to
recognize the type II binding sites. To support the hypothesis that
both mutants only interacted with the type I binding sites, we
performed direct binding assays. Cells were incubated in the presence
of increasing concentrations of either [125I]-CyPBKKK- or
[125I]-CyPB
YFD. The nonspecific
interactions obtained in the presence of a 200-fold molar excess of the
corresponding unlabeled mutant were subtracted from total counts. The
binding parameters were determined and compared with those obtained
with the wild-type radioiodinated ligand (Table I). The apparent
dissociation constants (Kd) of
[125I]-CyPBKKK- and
[125I]-CyPB
YFD binding were estimated to
be 10.0 ± 3.0 nM and 12.5 ± 1.5 nM,
respectively. These values are close to the Kd value
of the wild-type [125I]-CyPB binding (9.2 ± 2.0 nM). In contrast, the number of binding sites/cell was
estimated to be 17,000 ± 2,000 and 15,000 ± 2,000 respectively, less than that found for the wild-type ligand
(80,000 ± 5,000). Thus, the substitution of the
3Lys-Lys-Lys5 cluster by AAA and the deletion
of the 14Tyr-Phe-Asp16 cluster led to a similar
loss of about 80% of the whole binding capacity of CyPB, without
affecting the affinity of the ligand for its binding sites. Indeed, the
20% remaining binding capacity probably corresponds to the
NaCl-resistant fraction referred to as type I sites, which explains why
both CyPB
YFD and CyPBKKK- were only
efficient to displace radiolabeled ligand from these sites. Taken
together, these results demonstrate the involvement of the
3Lys-Lys-Lys5 and
14Tyr-Phe-Asp16 clusters in the establishment
of tight interactions with the type II binding sites present on Jurkat
cells. In addition, the fact that both CyPBKKK- and
CyPB
YFD interact with the type I binding sites similarly
to wild-type CyPB implies that a second region, not located in the
N-terminal extension of the protein, is required here.
Cellular Binding Properties of CyPBE22K and
CyPBD23P Mutants--
To find out whether other
CyPB-specific residues of the N-terminal extension are implicated in
the interactions with type II sites, we examined the cellular binding
properties of CyPBE22K and CyPBD23P mutants.
The dissociation constants of these modified proteins were estimated to
be 23 ± 9 nM and 25 ± 14 nM for the type I sites and 11 ± 6 nM and 12 ± 7 nM for the type II sites, respectively (Table I). These
values differed little from those measured for wild-type CyPB,
indicating that all three ligands exhibited a similar affinity for the
two types of binding sites. These results therefore demonstrate that
the Glu22 and Asp23 residues, and by extension
the 19-24 loop of CyPB, are not involved in the interactions with GAG
sites and further substantiate that the region close to
Arg18 and required for cellular binding under physiological
conditions is most probably the 14Tyr-Phe-Asp16 cluster.
Identification of CyPB Binding Regions after Proteolytic
Cleavage--
Additionally, a proteolytic approach was used to
identify regions of CyPB implicated in the interactions with Jurkat
cells. Several proteases were tested but only the results obtained
after clostripaïn treatment are presented here because they
were the most conclusive. After clostripaïn cleavage, the
fragments obtained were purified by reverse phase HPLC and the ability
of the 16 fractions eluted to compete with [125I]-CyPB
binding to Jurkat cells was analyzed (Fig.
4). Only two fractions, referred to as
fractions A and B, were found to contain fragments able to reduce the
binding of [125I]-CyPB to Jurkat cells. Fraction A,
eluted with 30% of acetonitrile in HPLC, inhibited the binding of
radiolabeled CyPB to T cells by 55%, whereas an equivalent amount of
unlabeled CyPB inhibited ligand binding by 75%. Thus, fraction A
probably contains a large part of the CyPB areas involved in the
interactions with T cells. The fraction B, eluted with 50% of
acetonitrile, inhibited the binding of [125I]-CyPB to T
cells by 25% only. Because it represented the last fraction and was
eluted together with uncleaved CyPB, it probably corresponded to a
small residual portion of uncleaved protein. To identify the contents
of fraction A, we performed mass spectrometry in the MALDI-TOF mode
(data not shown). Fraction A contained three fragments, a major one
characterized by its low molecular mass (812.4 Da) and two minor
fragments, representing less than 35% of the contents, with respective
molecular masses of 1287.2 Da and 1474.9 Da. The precision of the
method allowed us to identify these three fragments, based solely on
their molecular masses (7, 24). They corresponded to the
13Val-Tyr-Phe-Asp-Leu-Arg18,
66Asp-Phe-Met-Ile- ... -Phe-Thr-Arg76 and
99His-Tyr-Gly-Pro ... -Ala-Gly-Lys112
regions of CyPB, which have calculated molecular masses of 811.92, 1287.42, and 1475.66 Da, respectively. The identity of the peptides was
confirmed by partial N-terminal amino acid sequence analysis (data not
shown). Our results imply that clostripaïn has, in one
instance, cleaved a peptide bond at the C-terminal side of a Lys
residue. Such a secondary site cleavage of Lys-containing peptide bond
has already been reported (29). Because of their localization in a
highly conserved region of CyPB and presence in only small amounts in
fraction A, the 66Asp-Phe-Met-Ile- ...
-Phe-Thr-Arg76 and 99His-Tyr-Gly-Pro- ...
-Ala-Gly-Lys112 peptides are unlikely to play a role in the
inhibition of [125I]-CyPB binding. In contrast, the major
fragment identified corresponds to a region from the N-terminal part of
CyPB that contains the 14Tyr-Phe-Asp16 sequence
we previously singled out. This result further confirms the involvement
of this sequence in the binding of CyPB to the GAG sites on T
cells.

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Fig. 4.
Purification of proteolytic peptides obtained
after cleavage of CyPB by clostripaïn and determination of
their ability to compete to [125I]-CyPB for binding to
Jurkat cells. A, elution profile of CyPB peptides
separated by reverse phase HPLC on a C8 column. Elution was performed
by an acetonitrile gradient and monitored at 215 nm. B,
inhibition of [125I]-CyPB binding to T cells in the
presence of the previously obtained fractions. The control value
(100%) was determined in the absence of competitor. The fractions,
named A and B, are discussed in the text. The CyPB bar corresponds to
the inhibition of binding observed in the presence of CyPB.
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Strategy to Study a Possible Interaction with the Integrin
Receptor--
Comparison with the amino acid sequence of other
cyclophilins shows that only CyPB possesses an RGD sequence at
positions 76-78 (Fig. 1). This sequence is located in an outward
extending loop of the protein close to the central core of CyPB,
allowing connection between two
sheets that support in part the
spatial structure of the conserved central domain of CyPB (Fig.
5). The tripeptide RGD is a consensus
binding sequence for integrins (30), suggesting that interactions
between CyPB and receptors for RGD-bearing proteins may occur. To check
the possible involvement of the RGD sequence in the interactions with
the functional receptor, we generated a mutant, termed
CyPBRGD-, where the 76Arg-Gly-Asp78
tripeptide was replaced by AAA.

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Fig. 5.
Three-dimensional representation of the
72-126 region of CyPB. The model was visualized with the WinMGM
program using the coordinate file 1CYN from the Brookhaven National
Laboratory protein data bank. The CsA molecule is represented in
sticks. Side chains of residues Arg76, Gly77,
and Asp78 are indicated.
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Analysis of CsA Binding and Enzymatic Activities of
CyPBRGD---
The
76Arg-Gly-Asp78 tripeptide is not directly
located in the central core of CyPB and the corresponding amino acid
residues of CyPA, 69Arg-His-Asn71, have not
been reported to be essential for activities. Surprisingly the
CyPBRGD- mutant exhibited strongly reduced PPIase activity and no CsA binding (Table II). We assume
that the substitution of the 76Arg-Gly-Asp78
tripeptide led to a wrong folding of the central core of the protein so
that the enzymatic activity of the protein was lost. This mutant was
retained for our next experiments as an inactive form of CyPB.
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Table II
PPIase activity and CsA binding efficiency of central core mutant forms
of CyPB compared with wild-type CyPB
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Binding Properties of CyPBRGD---
The binding
properties of CyPBRGD- to Jurkat cells were examined (Table
III). Increasing concentrations of
CyPBRGD- reduced the amount of radioactivity released in
the 0.6 M NaCl wash comparably with the wild-type CyPB,
indicating that the mutant has conserved its binding properties for the
type II sites. In contrast, a 5-fold higher concentration of
CyPBRGD- than of CyPB was required to reduce by 50% the
amount of [125I]-CyPB associated with the NaCl-resistant
fraction. These last results reflect a marked decrease in the affinity
of this mutant for the type I sites. When using a synthetic RGDS
peptide to compete with the cellular binding of
[125I]-CyPB to Jurkat cells no interference was observed,
however, which seems to rule out the possibility that the type I sites correspond to proteins of the integrin family (data not shown). We
rather surmise that substitution of the
76Arg-Gly-Asp78 tripeptide most probably led to
substantial conformational modifications of CyPB, which perturbed the
interactions with type I surface binding sites.
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Table III
Binding competition studies with central core mutant forms of CyPB
The indicated ligands were used to compete with wild-type
[125I]-CyPB for binding to Jurkat cells. To discriminate
between both type I and type II binding sites, cells were washed with
Dulbecco's phosphate-buffered saline containing 0.6 M
NaCl. Radioactivity was then measured in the supernatant (ligand
released from the type II sites) and in cellular pellets (ligand
remaining bound to the type I sites). The binding curves for each
mutant were fitted using the wild-type dissociation constants
(Kd) as fixed parameters.
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Strategy of Identification of Binding Regions of CyPB to the
Functional Receptor--
Denys et al. (22) have
demonstrated that large excesses of CsA inhibit the binding of CyPB to
the type I sites, without affecting the interactions with GAG. These
results indicate that the drug probably overlays the domain of CyPB
involved in binding to type I and consequently that this domain might
be part of the conserved central part of the protein. Zydowski et
al. (31) have shown that the areas of CyPA supporting PPIase and
CsA binding activities are distinct. In particular, they identified
Arg55 and Phe60 as being essential for the
PPIase catalytic activity, whereas Trp121 and to a lesser
extent Phe60 are required for the CsA binding activity.
Even though similar studies have not been performed yet for CyPB, an
analysis of the sequence alignment of CyPA and CyPB and of the
three-dimensional structures of both proteins allowed us to pinpoint
the three corresponding amino acid residues in CyPB, i.e.
Arg62, Phe67 and Trp128. Indeed,
they are positioned similarly to the Arg55,
Phe60, and Trp121 residues of CyPA, and are
located in a comparable spatial environment, suggesting that they could
participate in either PPIase or CsA binding activities (Fig.
6). We therefore engineered three
mutants, termed CyPBR62A, CyPBF67A, and
CyPBW128A, where Arg62, Phe67, and
Trp128 were replaced by an Ala residue.

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Fig. 6.
Three-dimensional comparison of CyPA
(A) and CyPB (B). The models
were visualized with the WinMGM program using the coordinate files 1CWA
(A) and 1CYN (B) from the Brookhaven National
Laboratory protein data bank. CsA is represented with violet balls.
A, The amino acid residues indicated, Arg55,
Phe60, and Trp121, are in the peptidyl-prolyl
isomerase activity site and the CsA binding site of CyPA. B,
The corresponding residues of CyPB that were mutated,
Arg62, Phe67, and Trp128, are
indicated.
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Analysis of CsA Binding and Enzymatic Activities of CyPB Active
Site Mutants--
The mutants of the conserved central domain were
tested for their enzymatic and CsA binding properties (Table II). As
expected, substitution of either Arg62 or Phe67
residues led to a dramatic loss of PPIase activity, down to 8 and 12%
of wild-type CyPB isomerase activity. Moreover, the ability of
CyPBF67A to interact with CsA was reduced by 80%, whereas
CyPBR62A was as efficient as wild-type CyPB in complexing
the drug. These results are in agreement with the published work of
Zydowski et al. (31) concerning CyPA and demonstrate the
conservation of the enzymatic site and CsA binding domain between CyPA
and CyPB. Moreover, we found the CyPBW128A mutant unable to
interact with CsA, demonstrating that the requirement of this amino
acid residue in the interactions with the drug is also a common feature
of CyPA and CyPB. Surprisingly, the CyPBW128A mutant
retained 60% of the capacity to accelerate the cis-trans
isomerization of a Pro-containing substrate, indicating that the
Trp128 residue in CyPB was not essential for PPIase
activity. This is not the case for CyPA where mutation of the
corresponding Trp121 residue was reported to strongly
reduce the enzymatic activity. Fine differences in the spatial
conformation of the central domains of CyPA and CyPB exist, explaining
why such variations in the CsA binding capacity might occur. In support
of this observation, it was found that the half-inhibitory
concentration of CsA required to inhibit PPIase activity is 3-fold
higher for CyPA when compared with CyPB, indicating that indeed
differences in the three-dimensional structure of the central core may
be related to variations in the activities of each cyclophilin (12). In
conclusion, we produced three different mutants of the conserved
central domain of CyPB, namely CyPBR62A,
CyPBW128A, and CyPBF67A, lacking either PPIase activity, CsA binding capacity, or both, respectively.
Cellular Binding Properties of CyPBR62A,
CyPBF67A, and CyPBW128A--
Competitive
binding experiments conducted with CyPBR62A,
CyPBF67A, and CyPBW128A demonstrated that the
three mutants were as efficient as wild-type protein in inhibiting the
binding of [125I]-CyPB to the type II binding sites
(Table III), in line with our previous results. Moreover,
CyPBR62A and CyPBF67A were as efficient as
wild-type CyPB to reduce the amount of radiolabeled ligand associated
with the NaCl-resistant fraction. Because both these mutants have lost
their PPIase activity, the results strongly suggest that the amino
acids of the catalytic site of CyPB are not directly involved in the
interactions with the type I sites. In contrast, a 7-fold higher
concentration of CyPBW128A was required to displace 50% of
the radiolabeled ligand bound to the type I sites. Interestingly, this
last mutation completely abolished the CsA binding capacity of CyPB but
was less critical for enzymatic activity. It may therefore be
postulated that the areas of CyPB involved in the interaction with the
type I sites are close to the Trp128 residue and probably
overlay the CsA binding site of this protein.
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DISCUSSION |
Before mapping the CyPB regions involved in functional receptor
and GAG binding, we checked that the two types of binding sites
described by Denys et al. (22) on the membrane of peripheral blood T lymphocytes were also expressed on the membrane of Jurkat cells. Indeed we found that 75% of the total Jurkat cell binding capacity was due to interactions with type II sites and that the remainder corresponded to type I functional receptors previously shown
to be involved in ligand endocytosis. Binding to type I receptors was
also found for CyPC and was strongly reduced in the presence of CsA,
suggesting that part of the conserved CsA binding domains of CyPB and
CyPC corresponds to the binding region. The type II binding sites
represent about 75% of the total binding capacity and probably involve
interactions with sulfated GAG present on cell surface. This binding is
highly specific for CyPB and can be effectively reduced in the presence
of a synthetic peptide corresponding to the N-terminal part of the
protein (22). The role of this N-terminal extension of CyPB in the
cellular binding has first been emphasized by Mariller et
al. (24) who have also suggested that another region of CyPB may
be involved in the interactions.
The results presented here identify two regions of CyPB involved in the
interactions with both types of CyPB binding sites present on the T
cell membrane. We clearly demonstrate that CyPB possesses two receptor
binding regions: one, located in the specific N-terminal extension of
the protein, required for the interactions with sulfated GAG and the
second one, corresponding to a part of the central conserved core of
CyPB, involved in the recognition of a specific functional receptor.
Both binding regions are spatially located on opposite sides of the
CyPB molecule (12), raising the possibility that
interactions with both types of binding sites could occur simultaneously.
The use of CyPB mutants corresponding to the protein modified within
its N-terminal extension provided evidence for the role of the
14Tyr-Phe-Asp16 cluster in the interactions
with heparin and the type II binding sites constituted by sulfated GAG.
Proteolytic cleavage confirmed the requirement of the
13VYFDLR18 peptide of CyPB in the interactions
with these binding sites, indicating that this region covers at least
in part the actual binding region present in the N-terminal end of the
protein. Most probably the 13VYFDLR18 peptide
and more specifically the 14Tyr-Phe-Asp16
sequence are necessary but not sufficient for interactions with the
type II binding sites. Conceivably the
14Tyr-Phe-Asp16 sequence is required to
stabilize the primary interactions of a highly exposed basic sequence
with the type II sites, explaining why a large excess of free
13VYFDLR18 peptide inhibits the binding of
CyPB. In this respect, we found that another region which contains the
3Lys-Lys-Lys5 basic cluster was also required
for tight interactions, in line with the involvement of two regions
from the N-terminal part of CyPB in the binding to GAG chains.
CyPBKKK- and CyPB
YFD were eluted from a
heparin-Sepharose column, respectively, with 0.1 and 0.3 M
NaCl whereas CyPA, which lacks a KKK cluster, and CyPC, which lacks a
YFD cluster, were eluted from 0.2 and 0.25 M NaCl (22). The
three-dimensional model of the N-terminal extension of CyPB showed that
both the 3Lys-Lys-Lys5 and
14Tyr-Phe-Asp16 clusters can be spatially
juxtaposed and may act synergistically to form a cradle-like binding
site for the sulfated polysaccharide chain. Unfortunately, the
published crystallographic data on CyPB (12) do not give information on
the three-dimensional conformation of the
1Asp-Glu-Lys-Lys-Lys5 extremity, which would
help confirm this model. This is because of the absence in the crystal
structure of the five N-terminal amino acid residues, which were
probably cleaved off by proteases during the isolation procedure. Using
the molecular modeling, we showed that the
3Lys-Lys-Lys5 and the
14Tyr-Phe-Asp16 clusters form a structural
arrangement that could permit the interactions with the GAG. In
addition, a YFDLR peptide was described in type IV collagen as a
heparin binding domain (32, 33). Indeed, this YFDLR peptide is located
in a discontinuity of the triple helix of collagen and was reported to
be directly involved in promoting keratinocyte adhesion to heparin
sulfate proteoglycans. Taken together, these data clearly document the
role of such regions in the interactions with GAG and strongly support
our hypothesis that the binding region of CyPB involved in the
interactions with type II sites and heparin is probably restricted to
the 3Lys-Lys-Lys5 and
14Tyr-Phe-Asp16 clusters.
A role of sulfated GAG was suggested to be related to localization or
local presentation of HBP. Because the biological functions of secreted
cyclophilins are as yet largely unclear, it is difficult to speculate
on the implications of the interaction with GAG. Some cyclophilins were
reported to exhibit chemotactic activity for eosinophils, neutrophils,
and monocytes (34, 35). Also, CyPB levels measured in blood plasma from
CsA-treated graft recipients (21) and from patients suffering from HIV
infection (36) or sepsis (37) are increased for reasons not yet
understood. Taken together, these observations suggest however that
secreted cyclophilins might act as pro-inflammatory mediators. Another
family of small chemoattractant proteins are the chemokines that are
implicated in the attraction and activation of a variety of leukocytes
(38). Like CyPB, they bind to functional receptors on target cells and interact with sulfated GAG. The importance of the interactions with
sulfated GAG present on cell membrane has also been shown for many
growth factors and cytokines. Indeed these primary interactions are a
prerequisite for the binding to specific receptors and the enhancement
of cellular responses. For instance, soluble heparin was reported to
support binding of interleukin-8 to its neutrophil receptor and to
increase the intracellular free calcium concentration (39). On the
other hand, it has been suggested that interactions with GAG, either at
the surface of endothelial cells or in the extracellular matrix, could
be responsible for the establishment of an immobilized chemokine
gradient and the presentation of these molecules to leukocytes (40,
41). In support of this idea, it was demonstrated that MIP-1
and
RANTES (regulated on activation normal T cell expressed) interact with
solid phase GAG or activated endothelium and that these immobilized
chemokines are then capable of stimulating leukocyte adhesion (42, 43).
It is therefore conceivable that the type II sites contribute to the
binding of CyPB to its type I lymphocyte receptor and regulate its
activity. This possibility may only be further clarified by elucidating the biological role of CyPB and identifying the functional receptor.
Finally, we examined in detail the implication of the CsA binding and
PPIase domains of CyPB in type I receptor binding, because the
interaction between CyPB and the drug leads to impaired receptor binding. Upon analysis of the homologies between the sequences of CyPA
and CyPB, three amino acids residues, namely Arg62,
Phe67, Trp128, were identified in CyPB as being
possibly required for either PPIase, CsA binding, or both activities.
We found that substitution of the Trp128 residue in CyPB
prevented the interaction with CsA and markedly reduced the affinity of
the protein for the type I sites. These results agree with our previous
finding that addition of CsA inhibits the interactions of CyPB with the
type I sites present on blood T lymphocytes (22). Surprisingly, PPIase
activity was not directly related to the cellular binding properties of
CyPB. The substitution of Arg62 and Phe67
effectively resulted in the loss of enzymatic activity but did not
affect the interactions with the type I sites. Taken together, our
results indicate that the second binding region of CyPB is probably
conformational and emphasize the crucial role of the Trp128
residue in the interactions with either CsA or type I sites. The
presence of a Trp residue in the CsA binding domain is a common feature
of all cyclophilins and may explain in part why CyPC exhibits binding
activity for the type I sites. The binding affinity of CyPC was however
found to be 6-fold lower than that of CyPB, whereas CyPA was unable to
interact with any of the binding sites present on T cells. Such a
discrimination of the cyclophilins for the recognition of a receptor
has been already reported for CyPC with which a 77-kDa protein, termed
CyCAP for CyPC-associated protein, was found to specifically interact
(44, 45). CsA inhibits this interaction although neither CyPA nor CyPB
is able to bind to this membrane protein. The areas of CyPC that
interact with CyCAP are thought to be localized in a loop close to the
catalytic site but without any sequence homology with the other
cyclophilins, explaining the specificity of recognition. Inhibition of
CyCAP binding by CsA is probably because of steric hindrance (13). Such
fine divergences in the spatial conformation of the central domain of
the different cyclophilins might therefore well explain the variations
seen in the binding affinities of CyPB and CyPC for their specific
receptors. In addition, Sherry et al. (35) have very
recently characterized a signaling receptor for CyPA on T lymphocytes
and suggested that here also the CsA binding domain is involved in the
interaction. Possibly, the type I binding sites for CyPB, CyCAP and the
lymphocyte CyPA receptor might all be members of a family of related
cyclophilin-binding proteins. The possibility that the different
cyclophilins interact to some extent with the various receptor family
members cannot be ruled out presently. Resolution of this question will
require purification and structure elucidation of the different
cyclophilin-binding proteins expressed on the membrane of T cells.
Our studies clearly localize two distinct binding regions in the CyPB
molecule that are separately involved in the recognition of GAG and of
a functional receptor on T cells. This should allow the design of new
experiments to test the importance of the cellular binding of CyPB and
to provide new insights into the biological activity of this protein.