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*
Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104; and
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093
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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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Complement activation is essential for the development of normal inflammatory responses against foreign pathogens; however, its inappropriate activation has been a cause of tissue injury in many disease states. This tissue injury is directly mediated by the membrane attack complex, C5b-C9, and indirectly by the generation of anaphylatoxic peptides C3a, C4a, and C5a. Complement-mediated tissue injury has been reported in a variety of disease states, including autoimmune diseases (1), acute respiratory distress syndrome (2), Alzheimer’s disease (3, 4), stroke (5), heart attack (6), burn injuries (7), and reperfusion injuries (8, 9). Complement-mediated tissue injury has also been found to occur as a consequence of bioincompatibility situations, such as those encountered during dialysis and cardiopulmonary bypass (10, 11) and xenotransplantation (12, 13). Thus, there is a clear need for specific complement inhibitors.
To identify specific small-m.w. complement inhibitors that block all three pathways of complement activation, we previously screened a phage-displayed random peptide library with C3b, a proteolytically activated form of complement C3. This approach led to the identification of a 13-residue cyclic peptide (14), later named Compstatin. Studies of the mechanism of complement inhibition by Compstatin revealed that unlike natural inhibitors of complement that act on C3b, Compstatin binds to native C3 and inhibits its cleavage by C3 convertase (14); this inhibition was not caused by sterically hindered access to the C3a/C3b cleavage site (14). Three-dimensional (3D) structural analysis of a major conformer of Compstatin in solution, using two-dimensional (2D) nuclear magnetic resonance (NMR) and a hybrid distance geometry-restrained simulated annealing methodology, revealed that the backbone forms a type I ß-turn comprising residues Gln5-Asp6-Trp7-Gly8 (15). It is noteworthy that analyses of Ala substitution analogs indicated that in addition to Val3, type I ß-turn residues (Gln5-Gly8) contribute significantly to the inhibitory activity of the peptide (15).
Thus far, Compstatin has been tested in three different clinically relevant models. Hyperacute rejection in discordant kidney xenotransplantation has been studied ex vivo using a porcine-to-human perfusion model. In this model, Compstatin significantly prolonged the survival of the kidneys (12, 13). Its effect has also been tested in models for extracorporeal circulation (16), where it effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/C3 fragments to a polymer surface. As a result of the inhibition of complement activation, the activation of polymorphonuclear leukocytes (assessed by the expression of CD11b) and the binding of these cells (CD16+) to the polymer surface were almost completely lost (16). Most recently, Compstatin has been tested in vivo in primates to examine its effect on complement activation induced by a heparin-protamine complex; here it effectively inhibited complement activation (17).
This study was designed to characterize the interaction of Compstatin with C3. Here, using surface plasmon resonance (SPR), we have compared the interaction of Compstatin with native C3, C3(H2O) (C3 with a hydrolyzed thioester bond), and with the C3 fragments C3b, C3c, and C3d. The data we obtained point to differences in the mechanism of binding among these interactions. In addition, data from our structure-function analysis of Compstatin suggest that residues involved in type I ß-turn formation are also important for binding to C3. We have also performed biotransformation studies of this peptide to evaluate its stability in human blood; these findings indicate that Compstatin is stable in human blood and that N-acetylation of this peptide enhances its stability against plasma proteases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemicals and reagents used for peptide synthesis were purchased from Applied Biosystems (Foster City, CA), with the exception of F-moc amino acids, which were obtained from Nova Biochem (San Diego, CA). Veronal-buffered saline (VBS), pH 7.4, contained 5 mM barbital and 145 mM NaCl. Gelatin VBS (GVB) was VBS containing 0.1% gelatin; GVB2+ was GVB containing 0.5 mM MgCl2 and 0.15 mM CaCl2; and GVBE was GVB with 10 mM EDTA. MgEGTA contained 0.1 M MgCl2 and 0.1 M EGTA. PBS contained 10 mM phosphate, 145 mM NaCl, pH 7.4. PBS-T was PBS containing 0.05% Tween 20.
Purified proteins
Human complement protein C3 was purified from normal human plasma as described (18). To separate native C3 from C3(H2O), the C3 sample was incubated at 37°C for 2 h, loaded onto a Mono S column (Pharmacia, Piscataway, NJ), and eluted with a linear salt gradient (19). C3b was generated by limited trypsin cleavage of C3 and purified on a Mono Q column (Pharmacia) (20). C3c and C3d were generated by elastase treatment of C3 and purified on a Mono Q column (Pharmacia) (20).
Peptide synthesis, purification, and characterization
Compstatin and its analogs were synthesized in an Applied Biosystems peptide synthesizer (model 431A) using F-moc amide resin (4-(2',4'-dimethoxyphenyl-F-moc-aminomethyl)-phenoxy resin). The side-chain protecting groups were Cys (Trt), Asp (otBu), Arg (Pmc), Thr (tBu), Gln (Trt), Trp (Boc), and His (Trt). All peptides were cleaved from the resin by incubation for 3 h at 22°C with a solvent mixture containing 5% phenol, 5% thioanisole, 5% water, 2.5% ethanedithiol, and 82.5% trifluoroacetic acid (TFA). The reaction mixture was filtered through a fritted funnel, precipitated with cold ether, dissolved in 50% acetonitrile containing 0.1% TFA, and lyophilized. The crude peptides obtained after cleavage were dissolved in 10% acetonitrile containing 0.1% TFA and purified using a reversed-phase C-18 column (Waters, Milford, MA). Disulfide oxidation of peptides VII-XV was performed by stirring a 0.15-mM solution of the peptide in 0.1 M ammonium bicarbonate, pH 8.0, and bubbling with oxygen at 22°C for 48 h. Disulfide oxidation of all the other peptides was done on-resin as described elsewhere (13). Purified peptide II was reduced and alkylated with 10 mM DTT and 40 mM iodoacetamide. N-Acetylation of the peptides was achieved by treating them with a solution of 0.5 M acetic anhydride, 0.125 M diisopropylethylamine, and 0.015 M 1-hydroxybenzotriazide for 5 min (Applied Biosystems User Bulletin no. 35). On-resin biotylation of the peptides was performed as follows. The Dde group on the Lys side chain was removed by treating the peptide-resin with three short treatments (3 min each) of 2% hydrazine in dimethylformamide (21). It was then placed on the synthesizer and washed with N-methylpyrrolidone. Biotin (1 mmol) was dissolved in equal volumes (2.9) of DMSO and N-methylpyrrolidone, placed in an amino acid cartridge, and activated according to Applied Biosystems User Bulletin no. 35. The purity and identity of Compstatin and its analogs were critically monitored by analytical chromatography on a reversed-phase C-18 column and by laser desorption mass spectrometry (22). Formation of a disulfide bond in each cyclic peptide was confirmed by mass spectrometry using a mass shift assay that involves reaction of thiols with p-hydroxy mercuribenzoic acid (23).
SPR measurements
The kinetics of Compstatin binding to C3 and its fragments were
determined on SPR-based biosensors Biacore X and Biacore 3000 (Biacore,
Piscataway, NJ) at 25°C. Binding experiments were performed in
physiologic ionic strength buffer (PBS-T) using a streptavidin chip
(Sensor Chip SA; Biacore). Nonspecific adsorption of analytes to the
sensor chip was blocked by adding 0.05% Tween 20 to the buffer. To
obtain a homogeneous ligand surface, Compstatin analogs were oriented
on the sensor chip. Approximately 100–120 response units (RUs) of
biotinylated Compstatin (I*CVVQDWGHHRC*TAGHMANLTSHASAK-biotin
or biotin-KYSSI*CVVQDWGHHRC*T-NH2)
was immobilized on the test flow cell, and the control flow cell was
immobilized with equivalent RUs of the linear analog
(IAVVQDWGHHRATAGHMANLTSHASAK-biotin). Addition
of a spacer (extra residues) at the N or C terminus was based on the
sequence of the parent phage peptide. Binding was measured at 30
µl/min by injecting various concentrations of analyte for 120 s;
dissociation was followed for an additional 120 s. The sensor chip
was regenerated by brief pulses of 0.2 M sodium carbonate, pH 9.5.
Biosensor data for the control analog were subtracted from those
obtained for biotinylated Compstatin and globally fit to a 1:1 Langmuir
binding model:
 | (1) |
), the association and
dissociation processes are described, respectively, as:
 | (2) |
 | (3) |
), a plot of
ln(RU0/RU) vs time will
yield a straight line with a slope of
koff. The equilibrium dissociation
constant (KD) is calculated from the
equation KD =
koff/kon. Complement-mediated hemolytic assays
Inhibition of complement activity by Compstatin and its peptide analogs was studied by measuring their effect on the alternative pathway of the complement. Inhibition of complement activation was determined by measuring the lysis of rabbit erythrocytes (Er) in normal human serum (NHS) as previously described (26). Various concentrations of peptides were mixed with 5 µl of NHS, 5 µl of 0.1 M MgEGTA, and 10 µl of Er (1 x 109/ml), and the final volume was adjusted to 100 µl with GVB. The reaction mixture was incubated at 37°C for 20 min and stopped by adding 200 µl of GVB with 10 mM EDTA. After centrifugation, lysis of Er was determined at 405 nm. The percentage of lysis obtained was normalized by considering 100% lysis to be equal to the lysis occurring in the absence of the peptide. The concentration of the peptide causing 50% inhibition of hemolytic activity was taken as the IC50.
In vitro biotransformation
One milliliter of fresh heparinized human blood or NHS was mixed with 0.3 mg of Compstatin or one of its analogs (dissolved in 1 ml of PBS) and incubated at 22°C. Samples (200 µl) were removed at various time intervals and immediately centrifuged at 4°C to separate plasma from cells. One hundred microliters of plasma was removed, mixed with an equal volume of 0.1% TFA in H2O, and centrifuge-filtered through Ultrafree-MC filters (Millipore, Bedford, MA) with a molecular mass cut-off of 5000 Da. Half of the filtrate was injected onto HPLC (Waters LC 600) connected to a reverse-phase C-18 column (Vydac, Heperia, CA), and the other half was analyzed by matrix-assisted laser desorption mass spectrometry using a time-of-flight mass spectrometer (MicroMass TofSpec, Beverly, MA). Each peak recovered from HPLC was also individually characterized by matrix-assisted laser desorption mass spectrometry (22) and Edman degradation. The first sample, removed immediately after starting the incubation, was considered the zero time point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To measure the affinity of Compstatin for C3, and to better
understand the nature of the Compstatin-C3 interaction, we used SPR
technology. In general, immobilization of the ligand is achieved by
amine-coupling chemistry in this technology; however, this approach
produces heterogeneous surface ligands and precludes the measurement of
homogeneous binding constants. Thus, we synthesized Compstatin analogs
with biotin added to its N or C terminus and oriented these analogs
onto a chip surface. Both of these analogs (peptides XXII and XXIII)
were as active as Compstatin in inhibiting complement activity (Table I
). A linear analog of Compstatin
(peptide XXIV) was used as a control, because it did not bind to C3.
Compstatin analog oriented via its C terminus (peptide XXIII) bound to
C3 (Fig. 1), whereas the analog oriented
via its N terminus (peptide XXII) failed to bind to C3 (data not
shown), suggesting that a free N terminus is important for peptide
binding to C3. It should be pointed out that Compstatin, when
originally isolated from a phage-displayed random peptide library, was
oriented in the M13 phage via its C terminus.
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and 3,
respectively, showed nonlinear plots. Nonlinearity in the binding data
could have been due to the presence of C3(H2O) in
the sample, because freeze-thawing (27) and spontaneous
hydrolysis of the thioester bond convert C3 into
C3(H2O) (28). To test this
possibility, we separated native C3 from C3(H2O)
on a Mono S column (Pharmacia) (19).
C3(H2O) was generated in bulk by repeated cycles
of freezing and thawing and then purification over a Mono S column
(19). When purified native C3 and
C3(H2O) were tested for binding to Compstatin,
both bound in a nonlinear fashion (Fig. 2), indicating that binding follows
complex models. Thus, the dR/dt plots were
divided into fast and slow components. Such approximations have been
used previously (24, 25). The
ks values for the fast and slow
components obtained from the slopes of the dR/dt
plots (Equation 2) were plotted against C3 concentrations to calculate
kon values (Fig. 2, insets,
and Table II).
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). Dissociation is typically measured
using the highest concentration, because doing so yields the most
reproducible data. The dissociation rate constant was calculated from
the slope of the line defined by the initial data points of this plot.
Analysis of the data indicated that native C3 and
C3(H2O) bound to Compstatin with nearly identical
affinity (Table II).
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and Table II). The kinetics of binding of C3b and
C3c were distinctly different from those of native C3 and
C3(H2O). The sensograms for C3b and C3c, except
during the later part of the dissociation, showed a close fit to a 1:1
binding model. We reasoned that the poor fit during the later part of
the dissociation might be due to rebinding of the analyte molecules. To
verify this possibility, we coinjected 110 µM Compstatin during the
dissociation phase. As shown in Fig. 4, injection of the free peptide during the dissociation of C3b decreased
the residual signal during the later part of the dissociation and
yielded a close fit to a 1:1 binding model; similar results were
obtained when the free peptide was injected during the dissociation of
C3c (data not shown). Both C3b and C3c showed a decrease in their
on-rates when compared with C3; little change was observed in the
off-rates. This decrease in on-rates resulted in 22- and 74-fold
decreases in affinity for C3b and C3c, respectively.
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In an earlier study (14) we showed that Compstatin,
the cyclic 13-aa N-terminal region of the parent phage-displayed
peptide, effectively inhibits human complement activity. In this study,
we have tested the activity of Compstatin and its several analogs by
measuring their ability to inhibit the alternative pathway-mediated
lysis of Er (Fig. 5). We interpreted the
loss of activity in some of these analogs to their inability to bind to
C3. Hemolytic assays use serum as a source of complement; therefore, a
loss or reduction in activity could result from proteolytic cleavage of
the Compstatin peptide in serum. To conclusively rule out this
possibility, we checked for peptide cleavage under the same conditions.
None of the analogs tested, with the exception of peptides I and III,
were cleaved under these conditions (Table I). Peptide I showed limited
loss of Ile1, and peptide III showed limited
cleavage between Ile1-Ala2,
Ala2-Val3, and
Val3-Val4.
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). To further reduce the size of
the peptide, we generated short constrained peptides by changing the
ring size; one to six residues inside the 11-member ring were deleted
(peptides VIII-XV, Table I). However, we found that we were unable to
further reduce the size of the Compstatin peptide and still retain
activity. Identification of the residues of Compstatin that are
involved in the interaction with C3 and elucidation of the 3D structure
of these analogs, if amenable to NMR analysis, are needed to interpret
these results. Nevertheless, these findings indicate that the 11-member
peptide between disulfide-linked Cys2 and
Cys12 constitutes a minimum structure required
for optimal activity.
Protease-resistant peptide mimetics synthesized entirely from
D-amino acids in the reverse order of their
all-L-homologs (retro-inverso) are known to retain
biological activity (29). In such peptides, the side-chain
configurations correspond to the natural all-L-form
derivative, but the carbonyl and amine groups forming the backbone
amide bonds are reversed (30). Therefore, retro-inverso
peptides are considered as useful probes for dissecting the relative
contribution of side-chain and main-chain interactions to a molecular
recognition process. Therefore, we synthesized the retro-inverso
mimetic (peptide XVI, Table I) of Compstatin and tested its activity.
It was found to be inactive, suggesting that main-chain atoms of
Compstatin contribute to C3 recognition.
Our previous data on Ala scan analogs indicated that
Val3, Gln5,
Asp6, Trp7, and
Gly8 are important for maintaining the inhibitory
activity of Compstatin (15). From our 2D NMR studies, it
was clear that Val3 participates in side-chain
hydrophobic interactions and that
Gln5-Gly8 form a type I
ß-turn, which is important for the structural stability of Compstatin
and probably for specific side-chain orientation. However, it was not
clear whether type I ß-turn residues are also involved in the
interaction with C3. To dissect the role of these residues in the
structural stability of Compstatin, as contrasted with its interaction
with C3, we synthesized ß-turn analogs and analyzed their activity.
The residues
Gln5-Asp6-Trp7-Gly8,
which form the type I ß-turn in Compstatin, were substituted either
with Gly-Pro-Phe-Gly (peptide XVII) or with Gly-Pro-DPhe-Gly (peptide
XXI), which have the propensity to form type I and type II ß-turns,
respectively (31). Both analogs were inactive (Table I),
suggesting that in addition to providing structural stability, some of
the ß-turn residues must also interact with C3. Substitution of
individual turn residues with residues typical of those positions in a
type I ß-turn (i.e., Gln5-Gly,
Asp6-Pro, and Trp7-Phe
substitutions, peptides XVIII-XX) resulted in a substantial decrease in
the inhibitory activity of the peptide. The maximal loss (>100-fold)
in activity was observed with the Gln5-Gly and
Trp7-Phe substitutions, suggesting that these
residues might be involved in intermolecular recognition. Analysis of
complete sets of 2D NMR experiments for a number of analogs with
substituted ß-turn residues is underway to dissect the role of the
turn in binding and inhibitory activity of Compstatin.
In vitro biotransformation of Compstatin in human blood
The properties of Compstatin (13, 16) suggest that it
has therapeutic usefulness as an anticomplement drug; thus, it is
important to determine its biochemical and functional stability in
human blood. Compstatin was incubated in human blood, and its
biotransformation products were separated by HPLC on a reversed-phase
C-18 column and characterized by matrix-assisted laser
desorption-ionization mass spectrometry and Edman degradation. The
membrane filtration method used in this study recovered about 70% of
the peptide added to the blood. Incubation of Compstatin in human blood
resulted in its rapid cleavage at the N terminus and generation of
Compstatin (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) (Fig. 6). The second
slow cleavage occurred between Arg11 and
Cys12. No cleavage was seen at the C terminus,
because it was protected with amide. To determine whether the cyclic
nature of the peptide blocks further processing, we incubated a linear
analog (peptide III, Table I, in which Cys2 and
Cys12 were replaced with Ala) with human blood.
It is clear from the data (Fig. 6, B2) that the linear
analog was efficiently processed from the N-terminal end.
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-amino group by acetylation. We reasoned that blocking of the
amino terminus might protect the molecule from cleavage at
Ile1. As shown in Fig. 6, acetylation of the
amino terminus blocked this cleavage. Further analysis of the
acetylated Compstatin (Ac-Compstatin) showed that it was stable in
human blood (half-life of 24 h at 37°C). These experiments were
also repeated using normal human plasma and serum; the results were
essentially similar to those obtained with human blood. Functional activity of the biotransformation products of Compstatin
As described above, biotransformation of Compstatin in human blood
resulted in its cleavage into a major product, Compstatin (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) and a
minor linearized product, Compstatin (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) cleaved between
Arg11 and Cys12. Large
quantities of these products were generated and purified by HPLC over a
C-18 reversed-phase column (Vydac) and tested for their functional
activity in a complement-mediated hemolytic assay. Compstatin (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
was 2-fold less active than Compstatin, and its further cleavage
between Arg11 and Cys12,
which linearized the peptide, resulted in the formation of an inactive
species (Table I). These data emphasize that the cyclic nature of the
peptide is important for its functional activity. Acetylation of the
amino terminus blocked the cleavage of Ile1;
therefore, Ac-Compstatin was expected to have increased complement
inhibitory activity in serum. Indeed, when it was tested in the
hemolytic assay, Ac-Compstatin was three times more active than the
unprotected Compstatin (Fig. 5 and Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Binding of Compstatin to C3
Analysis of the binding of Compstatin to C3 and its fragments C3b,
C3c, and C3d (Fig. 2) indicated that the binding site is located in the
C3c region. These results are in good agreement with previously
published binding data obtained by ELISA (14). It is
interesting that in our earlier study we observed that Compstatin
displayed on the phage surface would bind to C3c when C3c was
immobilized on a microtiter plate, but not when Compstatin was adsorbed
to the plate and C3c was presented in solution (14). Based
on this finding, we speculated that the binding site is not exposed in
C3c unless the conformation of C3c is altered, e.g., by adsorption to a
solid support (14). The SPR data presented here (Fig. 2, Table II) show that Compstatin binds to C3c, though with a 72-fold
lower affinity than that of native C3. These data indicate that the
binding site is clearly exposed in C3 as well as in its physiologically
generated fragments C3b and C3c. We suggest that the lack of binding of
C3c to immobilized Compstatin in the ELISA could have been due to its
lower affinity for C3c, whereas the binding of phage-displayed peptide
to immobilized C3c could be attributed to the presence of multiple
copies of the peptide expressed on the phages (five copies per
phage).
SPR analyses make it possible to probe the mechanism of protein-protein
interaction in this system. Our real-time kinetic binding data from SPR
measurements indicate that binding of Compstatin to C3 does not follow
a simple 1:1 binding model (
2 = 15.5). Linear
transformation of the data showed that the reaction is multistep (Fig. 2). Such deviations could simply result from heterogeneity in the
surface and/or analyte being tested. However, we have ruled out both of
these causes. First, homogeneity of the ligand surface was assured by
orienting Compstatin in one direction. Further support came from the
fact that the binding to C3b and C3c followed a 1:1 binding model (Fig. 2). Second, spontaneous hydrolysis of the thioester of C3 converts
native C3 into C3(H2O) (0.005% per min at
37°C) (28); thus, C3 preparations are mixtures of native
C3 and C3(H2O). For SPR analyses, native C3 was
separated from C3(H2O) before the experiment and
kept at 0°C until assay, and the protein sample was reanalyzed at the
end of the experiment to detect the presence of
C3(H2O); <2% of the C3 was hydrolyzed to
C3(H2O). Therefore, the nonlinear
dR/dt plot shown in Fig. 2 could mean genuine
multistep binding. Such plots could reflect the presence of multiple
binding sites with different affinities, a conformational change, or a
more complex model. Experiments designed to localize the Compstatin
binding site on C3 indicated that the binding site is located within
the 40-kDa C-terminal half of the ß-chain of C3 (38).
Thus, it is unlikely that the nonlinear dR/dt
plot we obtained is related to the presence of multiple binding sites.
Moreover, the data did not fit well to a bivalent analyte model
(A + B 
AB, AB +
B AB2,
2 = 13). The possibility that Compstatin
undergoes a structural reorientation upon binding remains open. The
occurrence of conformational changes in peptides upon binding has been
reported for cyclosporine complexed to cyclophilin (39, 40) and for the Bak peptide complexed to the antiapoptotic
protein Bcl-xL (41). To determine
whether our data supported this possibility, we tried to fit our data
to a two-state conformational change model (A + B
AB AB*). The data fit well with this
model (see fit, Fig. 2; 2 = 1.57). Currently,
studies of the bound peptide are underway to verify this premise.
Cleavage of native C3 by C3 convertases results in generation of C3b
(185 kDa) and C3a (9 kDa). C3b possesses the ability to bind to a large
number of serum and membrane proteins (42). The binding
sites for these proteins are buried in native C3 and become available
only after a conformational change that occurs upon cleavage of C3 to
C3b. Like C3b, C3(H2O) binds to these serum and
membrane proteins. In addition, both C3b and
C3(H2O) show enhanced binding to the fluorophore
8-anilino-1-nephthalensulfonate and a similar positive shift in near-UV
circular dichorism spectrum when compared to C3 (27).
Based on these results, it is largely considered that C3b and
C3(H2O) have similar conformations. The data
presented in Fig. 2 shows that binding of Compstatin to
C3(H2O) is biphasic, whereas that to C3b follows
a 1:1 binding model. Thus, our results indicate that at least the
conformation of the Compstatin binding site in
C3(H2O) is different from that of C3b.
Structure-activity relationships in Compstatin
Earlier analysis of the functional activity of Compstatin showed that reduction and alkylation of Compstatin destroys its inhibitory activity in serum, a finding that indicated that oxidation of cysteine residues is required for its functional activity (14). However, the reason for this loss of activity was not clear. By using a direct binding SPR assay we have now shown that linearization of Compstatin results in the loss of its ability to bind to C3, a result that suggests that oxidation of cysteine maintains the structure of Compstatin that is optimal for binding and thus preserves its functional activity.
The interaction between a peptide and its receptor/binding protein can
involve an interaction between side-chain and main-chain atoms.
Retro-inverso peptidomimetic analogs are attractive tools for probing
the relative contributions of both sets of atoms to a bimolecular
recognition process. In such analogs, activity is preserved if the
orientation of the side chain is the most important factor in the
specific ligand interaction. For example, recent studies have
demonstrated the preservation of the antigenicity of a retro-inverso
peptide mimetic derivative of the C terminus of the histone H3 sequence
IRGERA (43) and the transport function of a 16-mer
retro-inverso form of a homeobox domain (44). In contrast,
if main-chain atoms play a significant role in the ligand interaction,
the analogs are inactive, as has been inferred from the analysis of a
hormone-binding domain of a vasopressin receptor (45). In
our current study, the retro-inverso mimetic of Compstatin was inactive
(Table I); thus, it appears that binding to C3 is not solely dependent
on the side chain interactions and that correctly oriented main-chain
atoms also contribute to binding.
Turn structures, which have been recognized for >30 years
(46), are intrinsically polar in nature, with the backbone
groups packed together and the side chains projected outward. Turns are
often considered potential sites for molecular recognition. In small
peptides, they are known to provide conformational stability by
optimizing backbone-chain compactness and to facilitate intermolecular
recognition through side-chain clustering (31). Compstatin
contains a type I ß-turn encompassing residues
Gln5-Gly8. Alanine scan
studies of Compstatin analogs have suggested that these residues are
important for the inhibitory activity of the peptide (15).
However, from these results it was not clear whether the ß-turn is
critical only for preservation of the structural stability or whether
it is also important for intermolecular recognition. We have now
addressed this question by synthesizing ß-turn substitution analogs.
Peptide analogs in which the ß-turn segment was replaced with
residues having the tendency to form a type I turn
(Gly5-Pro6-Phe7-Gly8,
peptide XVII) (31) or type II turn
(Gly5-Pro6-DPhe7-Gly8,
peptide XXI) (31), and analogs in which individual turn
residues were substituted (Gln5-Gly and
Trp7-Phe) were functionally inactive (Table I).
Therefore, it appears that side-chain interactions exist between some
of the turn residues and C3.
Biotransformation of Compstatin
The proteolytic susceptibility of a biologically active peptide is
as important to its biological effect as its affinity for the target
protein and its in vivo half-life. To determine the susceptibility of
Compstatin to proteolytic cleavage by serum proteases, we incubated
this peptide in human blood. Under these conditions, Compstatin was
processed rapidly at the N terminus, resulting in the removal of
Ile1. However, the N-terminal processing
apparently stopped after the first amino acid residue. One explanation,
although unlikely, is that cleaved products were produced but were not
detected because of selective loss during the sample preparation.
However, under the same condition, the linear analog of Compstatin was
successively cleaved at the N terminus, and all of the transformed
products were detected (Fig. 6). Therefore, it is very unlikely that
Compstatin products were not detected because of a selective loss on
the filter membranes. Thus, our data demonstrate that the cyclic nature
of Compstatin is important not only in stabilizing its structure but
also in protecting it from enzymatic processing. To our knowledge,
Compstatin is the first example of a peptide whose proteolytic
processing in blood has been shown to be blocked by cyclization.
In the past, many attempts have been made to develop synthetic peptides
as complement inhibitors. These peptides were based on the C3
convertase cleavage site in C3 (47), the factor D cleavage
site in factor B (48), sites of length polymorphism in C3
or C5 (49), or the C terminus of the serine protease
inhibitor (48). None of these approaches has yielded a
potent peptide that can inhibit complement activation. One possibility
that must be considered to explain the failure of these approaches is
the biotransformation of the peptides in serum. Fig. 6 demonstrates
that linear peptides are very susceptible to protease processing. In
the above-mentioned studies, the inhibitory activities of the peptides
were tested in complement-mediated hemolytic assays that use serum as a
source of complement. Therefore, it is possible that failure of these
approaches could be explained at least in part by the processing of the
peptides by serum proteases.
In this study we have shown that acetylation can prevent the N-terminal
processing of Compstatin (removal of Ile1) and
thereby enhance the inhibitory activity of the peptide. The acetylated
analog had an inactivation rate of 0.01%/min at 22°C (Fig. 6) and
0.03%/min at 37°C (data not shown) in serum. These data indicate
that Ac-Compstatin is stable against proteolytic cleavage in human
blood and has the potential to be developed as an in vivo complement
inhibitor.
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Acknowledgments |
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Footnotes |
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2 Current address: National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India.
3 Address correspondence and reprint requests to Dr. John D. Lambris, Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, 401 Stellar-Chance Laboratories, University of Pennsylvania, Philadelphia, PA 19104.
4 Abbreviations used in this paper: C3, third component of complement; C3(H2O), C3 with a hydrolyzed thioester bond; C3b, the proteolytically activated form of C3; C3c, the 135,300 Mr fragment of C3 generated using elastase; C3d, the 35,000 Mr fragment of C3 generated using elastase. Er, rabbit erythrocytes; NMR, nuclear magnetic resonance; VBS, Veronal-buffered saline; GVB, VBS containing 0.1% gelatin; SPR, surface plasmon resonance; RU, response unit; NHS, normal human serum; 3D, three-dimensional; 2D, two-dimensional; TFA, trifluoroacetic acid; Ac-Compstatin, acetylated Compstatin.
Received for publication May 3, 2000. Accepted for publication June 7, 2000.
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