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Regular Article |
From the Division of Molecular Pathology,
Graduate School of Medical Sciences, and the Departments of Laboratory
Medicine*
and Orthopaedic
Surgery,
School of Medicine, Kumamoto
University, Kumamoto; and the Department of Applied
Chemistry,
Kyushu Institute of Technology,
Kitakyushu, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We also found that the RP S19 dimer exhibits the chemotactic function by means of binding to C5a receptor on monocytes; the apparent chemotactic capacity of the RP S19 dimer as well as of C5a (the complement C5-derived leukocyte chemotactic factor) was strikingly reduced when the target monocytes were pretreated either with an anti-C5a receptor monoclonal antibody or with a synthetic C5a receptor antagonist, and the RP S19 dimer and C5a competed with each other displaying a similar affinity in binding to leukocytes.5 Namely, the RP S19 dimer and C5a share the chemotactic receptor known as C5a receptor, although a calculated homology in the amino acid sequence between RP S19 and C5a is only 4%.
C5a receptor is a member of the
G-protein-coupled receptor family and has the structural motif of seven
hydrophobic transmembrane 
-helices linked by extra- and
intracellular hydrophilic loops.6,7
Recent advance of the
study on the interaction between C5a and C5a receptor has been provided
by means of the nuclear magnetic resonance spectroscopic analysis of
C5a,8
the site-directed mutagenic analyses of C5a and C5a
receptor,9-13
and the peptide synthesis of C5a receptor
agonists and antagonists.14-16
By joining the accumulated
information, a two-step receptor ligand-binding model has been proposed
for the interaction between C5a and C5a receptor.17
The
first binding would occur between the
NH2-terminal acidic portion of the receptor
(possibly the second extracellular loop is also involved) and a basic
cluster at the core of C5a. The basic cluster is three dimensionally
formed by His15, Arg46, and Lys49 residues.8
The
high-affinity first binding does not activate the receptor, but
effectively raises the local concentration of C5a and thereby promotes
second binding. The second binding would occur between the
COOH-terminal portion of C5a, -Leu72-Gly73-Arg74-COOH,9
and transmembranous interhelical regions of the receptor. The second
binding triggers the G protein-coupled receptor signaling.
Based on the amino acid sequences of the receptor-binding sites
of C5a, we predicted the receptor-binding sites of the RP S19 dimer
(Figure 1)
. The first binding site should
be one of the basic clusters on the RP S19 molecule. We have previously
reported the presence of two basic clusters such as
-Lys23-Lys24-Ser25-Gly26-Lys27-Leu28-Lys29- and
-Lys38-Leu39-Ala40-Lys41-His42-Lys43- regions in terms of
heparin-binding characteristics.2
Using the alanine survey
of the basic residues at these sites in the site-directed mutagenesis,
and using a competition analysis with synthetic peptides mimicking the
basic cluster regions, we have currently determined the first binding
site.
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In the sequence of second ligand moiety of C5a, the side chains of
Leu72 and Arg74 are important. A Leu72Asp mutant of C5a reduced the
neutrophil chemotactic capacity to <1% compared to that of the native
C5a.9
An Arg74Ala mutant of C5a exhibited a 1000-fold less
activity than the wild type in the intracellular calcium mobilization
assay using neutrophils.10
The importance of the
-carboxyl group of Arg74 of C5a was also demonstrated. A C5a mutant
bearing one Gly residue elongated at position 75 possessed only 12% of
the neutrophil chemotactic potency of the wild form.9
According to the information, we found a sequence of
-Leu131-Asp132-Arg133- in RP S19. In this sequence, the side chains of
Leu and Arg are present with the same orientation as the COOH-terminal
of C5a. If the ß-carboxyl group of Asp132 of RP S19 functions
equivalently to the -carboxyl group of Arg74 of C5a, the
-Leu131-Asp132-Arg133- sequence of RP S19 would satisfy the conditions
required for the second ligand for the induction of the receptor
activation as the -Leu72-Gly73-Arg74 of C5a does. Using the
site-directed mutagenesis of recombinant RP S19, and using the peptide
synthesis, we currently examined our working hypothesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium was purchased from Nissui Pharm. Co. (Tokyo, Japan). Fetal bovine serum was a product of Gibco BRC (Paisley, Scotland). Mono-Poly Resolving Medium was a product of Flow Laboratory (Herts, UK). Bovine serum albumin (BSA), formyl-Met-Leu-Phe (f-MLF), and zymosan A were purchased from Sigma Chemical (St. Louis, MO). A multiwell chamber for chemotaxis assay was obtained from Neuro Probe (Bethesda, MD). Nuclepore filters were purchased from Nuclepore (Pleasant, CA). Restriction endonucleases (NdeI, BamHI), Plasmid Pure Prep, TaKaRa RNA PCR kit, DNA ligation kit, Microcon & Micropure, and Escherichia coli strain JM109-competent cells were purchased from TaKaRa Biomedicals (Otsu, Japan). Native Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). Jetsorb gel extraction kit was purchased from Genomed GmbH (Bad Oeynhausen, Germany). SeaKem GTG agarose and NuSieve GTG agarose were purchased from FMC (Rockland, ME). E. coli strain BL21(DE3)-competent cells and plasmid pET11a were obtained from Novagen Inc. (Madison, WI). Type II transglutaminase purified from the guinea pig liver was purchased from Oriental Yeast Co. (Osaka, Japan). Factor XIIIa was supplied by the Institute of the Chemo Sero Therapy (Kumamoto, Japan). NMePhe-Lys-Pro-dCha-dCha-dArg, which had been synthesized as described previously,18 was a kind gift from Dr. M. Mizuno of the Third Department of Internal Medicine, Nagoya University, Nagoya, Japan. All other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) or from Wako Pure Chemicals (Osaka, Japan) unless otherwise specified. Zymosan-activated plasma was prepared according to the method of Fernandez and colleagues19 with a modification as described previously.20
Oligonucleotides
Primer nucleotides were synthesized based on the cDNA sequence of
RP S19 previously reported.21
A list of the synthesized
primers named P1 to P12 is shown in Table 1
. The primers with odd and even numbers
are sense and antisense primers, respectively. P1 and P2 primers were
used for the preparation of a wild-type rRP S19, and primers from P3 to
P12 were used for the mutants of RP S19. P1 primer consisting of
nucleotides from number 13 to number 45, in which 20A, 21C, and 22G
were changed to C, A, and T, respectively, to construct the
NdeI restriction site, and P2 primer consisting of
nucleotides from number 472 to number 443, in which 466T, 464T, and
463G were changed to A, C, and C, respectively, to construct the
BamHI restriction site were synthesized by a DNA synthesizer
model 381A (Perkin Elmer-Applied Biosystems, Foster City, CA). P3 to
P12 primers that contain mutations were made to order by TaKaRa
Biomedicals. Vector pET11a primers (T7 promoter primer and T7
terminator primer) were also obtained from TaKaRa Biomedicals.
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Six types of recombinant RP S19, the wild type and five mutants (Lys23Ala, Lys24Ala, Lys27Ala, and Lys29Ala RP S19, Lys38Ala, Lys41Ala, His42Ala, and Lys43Ala RP S19, Leu131Asp RP S19, Asp132Gly RP S19, and Arg133Ala RP S19) were prepared. The cDNA for the wild-type RP S19 was prepared as described previously.5 The mutant RP S19 were prepared by the polymerase chain reaction-mediated site-directed mutagenesis according to the method of Brendan22 as described previously.2 pET11a bearing an insert RP S19 cDNA (recombinant plasmid) was purified from a positive colony culture, and analyzed for the DNA sequence using the Taq DyeDeoxy Terminator Cycle Sequencing kit for performing fluorescence-based dideoxysequencing reactions according to the method of Prober and colleagues23 to confirm the site-directed mutagenesis. These constructs of the wild-type and mutant RP S19 recombinant plasmids were transformed to the expression host E. coli BL21(DE3) competent cells.
The wild-type and mutant RP S19 molecules extracted from the periplasmic fraction of the E. coli were purified by high-performance liquid chromatography (HPLC) using a SP-5PW column (Tosoh, Tokyo, Japan) and a HiTrap heparin column (Pharmacia), in this order, as described previously.4 The preparations of the wild-type and mutant recombinant RP S19 thus obtained demonstrated single bands with an apparent molecular size of 15.5 kd in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), respectively (data not shown). Judging from the SDS-PAGE patterns, their purity was always >95%.
Preparation of Dimers of Recombinant RP S19
Factor XIIIa and type II transglutaminase catalyze the production of the chemotactically active RP S19 dimer, and the catalysis with the former enzyme requires heparin as a cofactor.2,3 Some types of the recombinant RP S19 were treated with factor XIIIa (final concentration, 1 U/ml) in the presence of heparin (1 U/ml), 1 mmol/L dithiothreitol, and 5 mmol/L CaCl2 for 60 minutes at 37°C as described previously.2 The remaining types of the recombinant RP S19 were treated with type II transglutaminase (final concentration, 0.5 U/ml) in the same conditions except for the absence of heparin as described previously.3 The cross-linked dimers of the recombinant proteins were then purified by immunoaffinity column chromatography with an anti-isopeptide bond monoclonal antibody column (Coval Ab, Lyon, France), and by HPLC with a Hitachi 5C4–300 column (Nacalai Tesque, Kyoto, Japan) as described previously.2 Each cross-linked dimer enriched in a HPLC fraction was evaporated with a vacuum centrifuge concentrator (Savant). When the quality of the preparations was examined by SDS-PAGE, it was always >90% (the minor contaminant in each preparation was the monomer, data not shown).
The protein concentration of these dimers thus prepared was determined by the absorbance at 280 nm under the assumption that absorbance unit 1.0 was equivalent to 1 mg/ml. The lyophilized samples were dissolved in sterilized phosphate-buffered saline (PBS, pH7.4) containing 0.1 mg/ml BSA, and used in the chemotaxis assay.
Preparation of Analogue Peptides
The peptides were synthesized by the solid-phase fluorenylmethoxycarbonyl (Fmoc) method on alkoxybenzyl alcohol resin. The synthesized peptides were separated from the resin with reagent K (TFA/phenol/H2O/thioanisole/ethanedithiol = 82.5/5/5/5/2.5), purified by HPLC with a YMC C18 preparation column (Yamamura, Kyoto, Japan) and freeze-dried. Their structures were confirmed by fast atom bombardment mass spectrometry. The lyophilized peptides were dissolved into PBS containing 0.1 to 2.0 mg/ml BSA and used in the chemotaxis assay.
Monocyte Chemotaxis Assay
Monocytes were isolated from heparinized human venous blood of healthy donors according to the method of Fernandez and colleagues19 as described previously.1 The monocytes were suspended at a density of 1 x 106 cells/ml in RPMI 1640 medium containing 10% fetal bovine serum for the multiwell chamber assay. In some experiments, the indicator monocytes were pretreated with analogue peptides for 10 minutes at 37°C. The multiwell chamber was handled according to the method of Falk and colleagues24 using a nuclepore filter with a pore size of 5 µm. When the synthetic peptides were used as the chemoattractant, we added BSA in the solvent at a concentration between 0.1 mg/ml and 2 mg/ml to prevent an apparent loss of the peptides. After incubation for 90 minutes, each membrane was separated, fixed with methanol, and stained with Giemsa solution. The total number of monocytes that had migrated beyond the lower surface of the membrane was counted in five high-power fields. The results were expressed by the number of migrated monocytes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In terms of heparin-binding capacity, RP S19 has two apparent basic clusters, which are -Lys23-Lys24-Ser25-Gly26-Lys27-Leu28-Lys29- and -Lys38-Leu39-Ala40-Lys41-His42-Lys43- regions.2 We hypothesized that one of these clusters would be the first binding site.
Monocyte Chemotactic Activity of Dimers of RP S19 Mutated at One of Basic Cluster Regions
We prepared two recombinant RP S19 mutants in which all of the basic amino acid residues in each basic cluster were substituted by Ala residues. The Lys23Ala, Lys24Ala, Lys27Ala, and Lys29Ala RP S19 and the Lys38Ala, Lys41Ala, His42Ala, and Lys43Ala RP S19 thus prepared, and the wild-type recombinant RP S19 were, respectively, treated with type II transglutaminase. The reason why type II transglutaminase was used instead of factor XIIIa (plasma transglutaminase) is that the former cross-links RP S19 independently to heparin3 different from the latter enzyme. The Lys23Ala, Lys24Ala, Lys27Ala, and Lys29Ala RP S19 is not effectively dimerized by factor XIIIa even in the presence of heparin, because of the loss of the heparin-binding capacity of the mutant RP S19.2 Judging from the SDS-PAGE patterns, there was no significant difference among the dimerization ratios of these RP S19 molecules when catalyzed by type II transglutaminase (data not shown).
The monocyte chemotactic capacities of these mutant and wild-type
dimers in the chemotaxis chamber assay are shown in Figure 2
. As the positive control, the wild-type
RP S19 dimerized with factor XIIIa in the presence of
heparin2
was used. Whereas, the dimers of the Lys23Ala,
Lys24Ala, Lys27Ala, and Lys29Ala RP S19 and the wild-type RP S19
exhibited comparable chemotactic activities to the positive control,
the dimer of the Lys38Ala, Lys41Ala, His42Ala, and Lys43Ala RP S19
demonstrated a significantly reduced activity.
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Inhibition of Chemotactic Activity of RP S19 Dimer by Basic Cluster Analogue Peptide
Basic cluster analogue peptides with 14 and 15 amino acids
corresponding to Leu22 to Asp35 and to Val37 to Asn51 were,
respectively, examined for the inhibitory capacities against the
monocyte chemotaxis attracted by the RP S19 dimer. In the chamber
assay, the indicator monocytes were pretreated with one of the peptides
at various concentrations for 10 minutes at 37°C and used. At
concentrations up to 10-3 mol/L, these peptides
did not influence the viability of the monocytes. A representative
experiment using these peptides at a concentration of
10-4 mol/L is shown in Figure 3
. The pretreatment with the peptide
(Ac-VKLAKHKELAPYDE) mimicking the sequence from Val37 to Asn51
prevented the chemotactic response to the RP S19 dimer. In contrast to
this, the peptide (Ac-LKKSGKLKVPEWVD), mimicking the sequence from
Leu22 to Asp35, did not prevent the response. Similar results were
obtained at the concentration of 10-3 mol/L. The
prevention by Ac-VKLAKHKELAPYDE was not observed when the monocytes
were attracted with the bacterial chemotactic peptide, f-MLF, instead
of the RP S19 dimer. This indicated the specific interaction of
Ac-VKLAKHKELAPYDE to the C5a receptor on the monocytes, because
monocytes recognize f-MLF with the formyl-peptide receptors but not
with the C5a receptor.
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. Although the experiment
at the concentration of 10-3 mol/L could not be
done because of a low solubility of this peptide in PBS containing BSA,
the experimental results at concentrations up to
10-4 mol/L indicate that the Lys41-His42-Lys43
moiety is essential for the first ligand function. Inhibition of Chemotactic Activity of C5a by First Ligand Analogue Peptide of RP S19 Dimer
The first ligand portion of C5a is also a basic cluster, which is three dimensionally formed by His15, Arg46, and Lys49 residues.8 The next question was whether the tandem basic sequence of the RP S19 dimer, the Lys41-His42-Lys43 moiety, was equivalent to the basic cluster of C5a in the C5a receptor binding. To answer this question, we examined the inhibitory capacity of the first ligand analogue peptide of the RP S19 dimer to the monocyte chemotaxis induced by C5a in the experimental system described above. In this experiment, ZAP was used as the source of C5a.
As shown in Figure 4
, the first ligand
analogue peptide of the RP S19 dimer (Ac-VKLAKHKELAPYDE), but not the
other basic cluster analogue peptide (Ac-LKKSGKLKVPEWVD), inhibited the
C5a-induced monocyte chemotaxis in a dose-dependent manner.
Furthermore, the first ligand analogue peptide where the tandem basic
residues were substituted by Ala residues (Ac-VKLAAAAELAPYDE) did not
possess the inhibitory capacity even at a concentration of
10-4 mol/L.
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C5a receptor activation capacities of these peptides were also
examined using monocytes without the pretreatment. None of these
peptides themselves attracted the monocytes at a wide range
concentration from 10-7 mol/L to
10-3 mol/L. A representative experiment at a
concentration of 10-4 mol/L is shown in Figure 5
.
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Identification of Second Binding Site of RP S19 Dimer to C5a Receptor
Our working hypothesis for the second binding site with the
activating capacity to C5a receptor is the moiety with
-Leu131-Asp132-Arg133- of the RP S19 dimer. This hypothesis bases on an
assumption that the ß-carboxyl group of Asp132 of RP S19 is
equivalent to the -carboxyl group of Arg74 of C5a as schematically
shown in Figure 6
.
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To examine the working hypothesis three mutants with either Leu131Asp, or Asp132Gly, or Arg133Ala of recombinant RP S19 were synthesized, and their cross-linked homodimers were prepared. In terms of the efficacy of the factor XIIIa-catalyzed dimer formation, no significant difference was present among the mutants and the wild-type (data not shown).
The monocyte chemotactic capacities of the dimers of the wild-type and
the mutants at the final concentration of 10-9
mol/L are comparatively shown in Figure 7
. All of the mutant protein dimers
demonstrated significantly lower chemotactic activities when compared
to that of the wild-type RP S19 dimer. These reduced activities of the
dimerized mutants were observed in a wide concentration range from
10-13 mol/L to 10-7 mol/L
(data not shown). Therefore, the side chains of Leu131, Asp132, and
Arg133 are all equally important for the chemoattractant capacity of
the RP S19 dimer.
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-carboxyl group of Arg74 of C5a. Monocyte Chemotactic Activity of Analogue Peptides
To confirm the above data, we prepared an analogue peptide
of RP S19 that possessed 18 amino acid residues from Gly127 to Lys144
(Ac-GQRDLDRIAGQVAAANKK). The COOH-terminal His145 was omitted in this
peptide to clear-cut that the COOH-terminal did not participate in the
monocyte chemoattraction. This peptide possessed the monocyte
chemotactic activity. The dose-activity relationship of this peptide is
shown in Figure 8
. The pattern is
bell-shaped, which is typical for the leukocyte chemotactic factors.
The apparent peak concentration of this peptide is
10-7 mol/L, that is, 100-fold higher than that
of the RP S19 dimer.
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. These peptides, as well as the chemotactic peptide, did
not exhibit any apparent toxic effect to monocytes at concentrations of
up to 10-3 mol/L. Competition between Chemotactic Analogue Peptide and RP S19 Dimer
The monocytes were pretreated with either the functional analogue
peptide (10-4 mol/L) or the Asp-Gly-substituted
peptide (10-4 mol/L) for 10 minutes at 37°C,
and used for the chemotaxis assay attracted by the RP S19 dimer
(10-9 mol/L). As shown in Figure 9
, the pretreatment with the functional
peptide, but not with the Asp-Gly-substituted peptide, prevented the
chemotactic response to the RP S19 dimer. The specificity of this
competition was further confirmed with f-MLF as the chemoattractant.
The pretreatment with the functional analogue peptide
(Ac-GQRDLDRIAGQVAAANKK) did not affect the f-MLF-induced monocyte
chemotaxis.
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Inhibition with Authentic C5a Receptor Antagonist of Receptor Activation by Second Ligand Analogue Peptide of RP S19 Dimer
The antagonist effect of NMePhe-Lys-Pro-dCha-dCha-dArg peptide to
C5a receptor has been established demonstrating the inhibition of
C5a18
and of the RP S19 dimer.5
The effect of
the C5a receptor antagonist on the monocyte chemoattraction induced by
the second ligand analogue peptide of the RP S19 dimer was examined. As
shown in Figure 10
, the C5a receptor
antagonist inhibited the monocyte chemotactic response to the RP S19
analogue peptide in a dose-dependent manner. By this result, it was
confirmed that the RP S19 analogue peptide attracts monocytes via the
C5a receptor. This result also indicates that the second ligand
interaction site of the C5a receptor is common in the C5a and the RP
S19 dimer bindings.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in inducing the maximum chemotactic response of monocytes.
The pretreatment of monocytes with the first ligand analogue peptide of
the RP S19 dimer also prevented the C5a-induced monocyte chemotaxis
(Figure 4)
. As the first ligand function, the Lys41-His42-Lys43
sequence of the RP S19 dimer seems equivalent to the basic cluster of
C5a formed by His15, Arg46, and Lys48 residues. This would indicate
that C5a and the RP S19 dimer share the first ligand acceptor site of
C5a receptor molecule. The first ligand acceptor site of the receptor
to C5a was indicated to be the NH2-terminal
acidic portion of the receptor.25
However, as for the role
of Asp residues forming the acidic portion, it has not yet been
determined whether they directly interact with the basic residues of
the first ligand of C5a via a salt bridge or they involve in folding a
high-affinity-binding pocket to the first ligand.25
The
present results, showing that the first ligand moiety of the RP S19
dimer is also composed of the basic residues, seem to support the
former possibility.
The present study also indicated that the moiety with
-Leu131-Asp132-Arg133- of the RP S19 dimer functions in the same way as
the moiety with -Leu72-Gly73-Arg74-COOH of C5a in the C5a receptor
activation. A good way to confirm this from the opposite side might be
to perform an experiment using a double mutant of RP S19 in which
Asp132 is changed to Gly and the COOH-terminal portion after Arg133 is
truncated. However, this experiment cannot be done theoretically,
because the RP S19 mutant having the truncated COOH-terminal sequence
of -Leu131-Gly132-Arg133-COOH lacks the Gln137 residue that is
essential to form the cross-linked dimer. Even in the absence of this
experiment, the present data strongly suggest that an identical region
of C5a receptor interacts with these moieties of the RP S19 dimer and
C5a, respectively, and that the receptor activation mechanism is,
therefore, the same between the RP S19 dimer and C5a. A mechanism model
in the C5a receptor activation by C5a was proposed.12
In
this model, the side chain guanidino group of Arg206 of C5a receptor is
initially recognized by the -carboxyl group of C5a by a positive
electrostatic interaction, but subsequently, the side chain of Arg206
relocates by a negative electrostatic interaction with the guanidino
group side chain of Arg74 of C5a. The conformational change with the
relocation of Arg206 of the C5a receptor is a positive signal for
triggering the G-protein-coupled transduction mechanism.12
In this molecular mechanism, the requirement of the orientation between
the guanidino group and carboxyl group of Arg74 of C5a does not seen
very restricted, because the Arg74 of C5a could be substituted by
D-Arg, preserving the agonistic capacity, at least when C5a
mimicking peptides were used.12
In the case of the RP S19
dimer, the ß-carboxyl group of Asp132 and the guanidino group of
Arg133 would drive the same molecular machinery. The molecular
mechanism without the requirement of a restricted orientation between
the guanidino and carboxyl groups would provide the same functional
capacity between the RP S19 dimer with the -Leu131-Asp132-Arg133-
moiety and C5a with the -Leu72-Gly73-Arg74-COOH moiety.
The RP S19 analogue peptides mimicking the first binding site and the second binding site, respectively, function in their monomer forms. In contrast to this, the cross-linked dimerization at Gln137 is essential for RP S19 to gain the chemotactic function in either case, catalyzed by factor XIIIa2, or by type II transglutaminase.3 The monomer form of RP S19 did not compete with the radiolabeled C5a in the C5a receptor binding.5 It seems reasonable to speculate that both of the moieties with the functions in the first binding and the second binding are hidden in the conformation of the monomer of RP S19.
As described previously, the RP S19 dimer functions as a receptor antagonist to the C5a receptor of polymorphonuclear leukocytes.5 How the RP S19 dimer behaves oppositely between monocytes and polymorphonuclear leukocytes in terms of the C5a receptor interaction is a very interesting issue to be solved in the near future.
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Footnotes |
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Supported by a Grant-in-Aid for Scientific Research B (to T. Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Y. S. and M. S. contributed equally to this work.
Accepted for publication August 23, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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