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Department of Biochemistry, University of Texas Health Science Center, Tyler, TX 75708
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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28-fold (5.2–0.18 µM)
when the density of C3b was increased from 18,000 to 400,000
C3b/cell. Very-high-affinity C5 convertases were generated when
preformed C3 convertases were allowed to self amplify by giving them
excess C3. These convertases exhibited Km
from 0.016 to 0.074 µM, well below the normal plasma concentration of
C5 in blood (0.37 µM). The results suggest that in serum convertases
formed with monomeric C3b will be relatively inefficient in capturing
C5 but will continue to cleave C3 opsonizing the cell surface for
phagocytosis, whereas convertases formed with C3b-C3b complexes in
areas of high C3b density will primarily cleave C5. The catalytic rate
of these convertases approaches maximum velocity, thereby switching the
enzyme from cleavage of C3 to cleavage of C5, and production of the
cytolytic C5b-9 complex. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C5 convertases of the alternative and classical pathways of complement are complex serine proteases that are made up of two or more subunits (1, 6, 7, 8, 9, 10, 11). The study of C5 convertases has been difficult because the enzyme complex that cleaves C5 is the same serine protease (C3 convertase) that cleaves C3 (1). Cleavage of C3 produces the proteolytically activated form of C3 (C3b),3 which forms the nonenzymatic subunit of the enzyme complex responsible for C3 and C5 cleavage (1, 3). C3 convertases of the alternative pathway (monomeric C3/C5 convertase assembled with monomeric C3b (C3b,Bb)) and the classical pathway (C4b,C2a) are bimolecular complexes (1, 3). The alternative pathway C3 convertase (C3b,Bb) has C3b as the noncatalytic subunit attached in a Mg2+-dependent fashion to the catalytic subunit (Bb) of the enzyme complex. In the classical pathway C3 convertase C4b,C2a has a structure similar to the alternative pathway C3 convertase in having C2a as the catalytic subunit noncovalently bound to a C4b molecule. The soluble serine protease C3 convertase (C3b,Bb) assembled with monomeric C3b has been shown to be the simplest form of C5 convertase (12). The natural surface-bound C5 convertases are the more complex forms of the enzyme and are made up of a C3 convertase and an additional C3b molecule (6, 7, 8, 9, 10, 11, 13, 14). Attachment of the additional C3b molecules near the C3 convertase was originally thought to be required for generating C5 convertase activity (6, 8, 15). Vogt et al. (13) revealed that the role of the additional C3b was to bind the substrate, C5. Later a more specific structure was described. High C5 convertase activity was associated with the formation of C3b-C3b or C3b-C4b dimers in which the additional C3b molecule was shown to be covalently attached to a specific site on the first C3b or C4b molecule (10, 16). The dimers were suggested to form complexes C3b-C4b,C2a and C3b2,Bb, which are currently considered to be functional C5 convertases of the classical and the alternative pathways, respectively (9, 10, 17, 18).
To enhance our understanding of the structure/function of natural surface-bound C5 convertases we examined the effect of C3b density on the functional role of the noncatalytic subunit of surface-bound C5 convertases. In this study we show that, whereas the density of C3b/cell influences the Km and not the catalytic rate constant (kcat) of C5 convertases, the type of surfaces do not influence either the Km or the kcat of the enzyme. The data suggest that the initial monomeric C3/C5 convertases formed on the surface because of their weak affinity for C5 will function as C3 convertases amplifying C3b around them. As C3b deposition occurs, C3b-C3b complexes are formed. These complexes form convertases that have higher affinities for C5 and will primarily cleave C5 leading to a higher production of the cytolytic C5b-9 complex. Based on the present kinetic analysis of the natural surface-bound C5 convertases together with results published by other groups on the structure of C5 convertase, we propose a model for the formation of high-affinity C5 convertases during complement activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chicken erythrocytes (EC) were isolated from chicken blood purchased from Colorado Serum (Denver, CO). Nonidet P-40, a nonionic detergent, and EDTA were purchased from Sigma (St. Louis, MO). Veronal-buffered saline (VBS) contained 5 mM barbital and 145 mM NaCl (pH 7.4). Gelatin VBS (GVB) was VBS containing 0.1% gelatin, while GVB containing 10 mM EDTA (GVBE) was also used.
Purified proteins
Complement proteins, C3 (19, 20), factor B (21), factor D (22), and C6 (23) were all purified from normal human plasma as described in the references cited. C5 was also isolated from normal human plasma as described (24) except that ceramic hydroxylapatite (Bio-Rad, Richmond, CA) was used instead of hydroxylapatite. C3b was prepared from C3 by cleavage with factors B and D in the presence of Ni2+ at 37°C as described previously (12). Purified C5b,6 was obtained from Advanced Research Technologies (San Diego, CA). All proteins were homogenous by polyacrylamide gel electrophoresis. Protein concentrations of C3b, C5, C6, factor B, factor D, and C5b,6 were determined spectrophotometrically using 11.0, 11.0, 10.8, 12.7, 11.0, and 10.3, as the values for E280 nm1%, respectively. All purified proteins were stored at -76°C. Mr values employed in the calculations were 176,000 for C3b, 190,000 for C5, 179,000 for C5b, 120,000 for C6, 299,000 for C5b,6, 93,000 for factor B, 63,000 for Bb, and 24,000 for factor D.
Preparation of ZymC3b, ERC3b, and ESC3b
ZymC3b was prepared as described previously (12). C3b was deposited on zymosan by resuspending 1 x 1010 zymosan particles in 0.2 ml of 10 mg/ml C3 and adding 5 µg of trypsin, followed by a 10-min incubation at 22°C. The deposition of C3b by trypsin was repeated and the cells were washed six times with 5 ml GVB. The zymosan particles were resuspended in 100 µl GVB and mixed with 50 µl GVB containing factors B (35 µg) and D (0.5 µg) and 50 µl of 10 mM NiCl2. After 5 min of incubation at 22°C, 5 µl of 0.2 M EDTA was added. The bound C3b was amplified by adding 50 µl C3 (500 µg) and incubating the cells for 30 min at 22°C. The zymosan particles bearing C3b were washed and the amplification procedure was repeated until the desired numbers of C3b/zymosan were obtained (25). The total number of C3b molecules bound to zymosan was measured by two independent methods using 125I-labeled C3 (26) or radiolabeled factor B (12) as described previously. The number of C3b deposited on the zymosan particles was adjusted so as to obtain different densities of C3b/cell ranging from 18,000 to 405,000 C3b/cell. ESC3b and ERC3b bearing different amounts of C3b were prepared similarly.
Formation and assay of surface-bound C5 convertases
Because formation of C5 convertase took less than 1 min, enzyme
was formed in the same reaction mixture in which the assays were
performed. Enzyme velocities were determined under saturating
concentrations of factors B and D and C6 in 0.5 ml siliconized
microfuge tubes as described previously (12). Assay
mixtures contained varying concentrations of C5 (preincubated for 20
min at 37°C to eliminate freeze/thaw-generated background C5b,6-like
activity), factor B (1.2 µg, 516 nM), factor D (0.1 µg, 167 nM), C6
(2.5 µg, 833 nM), and 0.5 mM NiCl2. The
reaction was started by the addition of ZymC3b,
ESC3b, or ERC3b. Depending
on the density of C3b per cell, the concentration of cells was adjusted
so as to have 9–35 ng of bound C3b in a final volume of 25 µl GVB
resulting in 2–8 nM enzyme concentration. After 15 min of incubation
at 37°C, further cleavage of C5 was prevented by transferring the
assay tubes to an ice bath and adding ice-cold GVBE. Appropriately
diluted assay mixtures were immediately titrated for C5b,6 formation by
hemolytic assays using EC. C5b,6 was quantitated
using standard curves generated with purified C5b,6. Controls
established that the cold temperature and the dilution were sufficient
to reduce the cleavage of C5 during subsequent steps to undetectable
levels. Lysis of rabbit erythrocytes (ER) or
sheep erythrocytes (ES) was shown to contribute
<2% to C5b,6 titers using lysis of EC as the
endpoint.
Quantitation of reaction products
C5b,6 was measured hemolytically using the sensitivity of EC to hemolytic lysis by human C5b-9 as described (12). To an aliquot (25 µl) of the diluted sample from C5 convertase assays was added a mixture of 1.2 x 107 EC and 5 µl of pooled normal human serum (NHS) as a source of complement proteins C7-C9 in a final volume of 225 µl GVBE. The reaction mixtures were incubated for 10 min at 37°C after which the unlysed cells were removed by centrifugation for 1 min at 10,000 x g. The amount of hemoglobin released was quantitated spectrophotometrically at 414 nm. One-hundred percent lysis was measured as EC lysed in 2% Nonidet P-40. Controls containing C5 and C6 but no C5 convertase, were subtracted as the background. Controls containing C5 convertase but no purified C5 or C6 demonstrated that no significant amount of C5b,6 was formed from NHS used as a source of C7–9 during the lysis of EC.
Data analysis
The reaction velocity data was analyzed according to the Michaelis-Menten equation: v = (Vmax) [S]/(Km + [S]). The results were fit to this equation using nonlinear regression analysis and the kinetic parameters, Km, Vmax, and kcat were determined using Grafit version 4.12 software (Erithacus Software, London, U.K.).
Preparation of 125I-labeled factor B and C3
Factor B (100 µg) was radiolabeled with 125I for 30 min at 0°C in a glass tube coated with Iodogen (Pierce, Rockford, IL). Free 125I was removed by centrifugal desalting (27). Specific activities of radiolabeled factor B ranged from 0.12 to 0.95 µCi/µg. C3 was labeled as described for factor B. The radiolabeled C3 was diluted with cold C3 to give a specific activity of 0.08 µCi/µg.
Determination of the number of C3b molecules bound per cell
Cells were amplified with C3b using radiolabeled C3 at 9.8 mg/ml
(specific activity = 0.08 µCi/µg) instead of unlabeled C3 as
described above. The amplification procedure was repeated to obtain
cells bearing different densities of radiolabeled C3b ranging from
18,000 to 700,000 C3b/cell. ER bearing
radiolabeled C3b were incubated for 5 min at 37°C in 25 µl GVB
after which the reaction mixture was diluted with 75 µl GVB
containing 0.5 mM NiCl2. Bound and free
radiolabel was separated by layering 75 µl of the mixture on 250 µl
of 20% sucrose in GVB containing 0.5 mM NiCl2
followed by centrifugation for 1 min at 10,000 x g at
22°C. The amount of radiolabeled C3b bound to cells was determined by
cutting the tube and counting the amount of radioactivity in the pellet
(26). The number of C3b bound to ER
calculated from the radioactivity (cpm) bound to
ER, is reported after subtracting nonspecific
binding to control ER.
Determination of the number of Bb binding sites (C5 convertase sites)
The number of C5 convertase sites formed on ZymC3b, ERC3b, or ESC3b was determined by measuring only those C3b molecules that were capable of forming an enzyme with radiolabel factor B under saturating assay conditions (12). ZymC3b, ERC3b, or ESC3b employed in the binding assays were washed three times with GVB to remove any free C3b. Various amounts of 125I-labeled factor B were added to assay mixtures containing factor D (0.1 µg, 167 nM), C5 (5 µg, 1053 nM), C6 (2.5 µg, 833 nM), and 0.5 mM NiCl2. Formation of enzyme was started with the addition of ZymC3b, ERC3b, or ESC3b to give a final volume of 25 µl GVB. After 5 min of incubation at 37°C, assays were diluted with 75 µl GVB containing 0.5 mM NiCl2. Bound and free radiolabel were separated by layering 75 µl of the mixture on 250 µl of 20% sucrose in GVB containing 0.5 mM NiCl2 followed by centrifugation for 1 min at 10,000 x g at 22°C. The amount of factor B bound to ZymC3b as Bb was determined by cutting the tube and counting the amount of radioactivity in the pellet (26).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To count the number of C3b/cell, ER cells
bearing different amounts of C3b ranging from 18,000 to 700,000
C3b/cell were prepared using radiolabeled C3. The number of C5
convertase sites that could be generated on these
ER cells bearing different densities of
radiolabeled C3b was determined as the number of Bb binding sites. Only
those C3b molecules capable of binding
125I-labeled factor B as Bb under saturating
concentrations of factor D, Ni2+, and
radiolabeled factor B were quantitated. The specific activity of
125I-labeled C3 (0.079 µCi/µg) was adjusted
such that it was 9-fold less than that of
125I-labeled factor B (0.70 µCi/µg). This was
necessary so as to have a 3-fold increase in the radioactivity over the
background from C3b if all the bound C3b molecules could be saturated
with Bb (12). The assays employed
Ni2+ to minimize the decay of the Bb subunit of
the enzyme during the 1-min spin through cold 20% sucrose.
Quantitation of the number of C3b/cell and the number of Bb binding
sites was done at the same time to minimize the possible effect of C3b
decay from the cell surface. As seen in Fig. 1
, the ratio of the number of Bb binding
sites to the number of C3b/cell did not vary significantly with a
35-fold variation in density of C3b. The average ratio of Bb per C3b
was determined to be 0.83 ± 0.13, (i.e., one Bb per 1.2 molecules
of C3b) suggesting that most of the C3b can bind Bb even when the C3b
density is greater than 400,000 C3b/cell. Given the errors associated
with determining radioactive and specific activities of the two
proteins, these results suggest that equivalent numbers of C3b and Bb
bind at all levels of C3b/cell. Therefore, throughout this study we
have determined the number of C5 convertase sites formed by measuring
Bb binding under saturating levels of factor B and used it also as a
measure of the number of C3b bound per cell.
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Zymosan particles bearing C3b ranging from 18,600 to 405,000
C3b/Zym were employed for assembling C5 convertase. Enzyme activity was
measured as the amount of C5 cleaved by quantitating the amount of
C5b,6 formed during a 15-min assay under saturating concentrations of
factors B and D and C6. Initial velocities were determined at various
concentrations of C5. Data for ZymC3b,Bb made with 31,000 and 300,000
C3b/Zym are shown in Fig. 2, whereas data
obtained with ZymC3b,Bb formed with other densities of C3b/cell are
summarized in Fig. 3A and in
Table I. The velocity data obtained were
found to fit well to the theoretical curve based on the
Michaelis-Menten equation: v =
(Vmax)
[S]/(Km + [S]) (Fig. 2A). The data were also observed to fit well to the
linearized form of the Michaelis-Menten equation, shown as the
Eadie-Hofstee plot (Fig. 2B). C5 convertase formed with
300,000 C3b/cell had a 10-fold lower
Km (0.3 µM) than the enzyme formed
with 31,000 C3b/cell (2.8 µM). These results suggest that the density
of C3b/cell influences the Km of the
enzyme. The effect of the density of C3b/cell on the
Km of C5 convertase is apparent in the
results shown in Fig. 3A, which illustrates a spectrum of
different Km values depending on the
number of C3b/cell employed to assemble the enzyme. The average
Km of the enzyme decreased from 5.15
µM at 18,600 C3b/cell to 0.177 µM at 405,000 C3b/cell (Table I).
These results suggest that stronger binding interactions between
the enzyme complex and the substrate C5 occurred with increasing C3b
density. In contrast to the Km, which
varied widely, the catalytic rate
(kcat) of C5 convertases varied
comparitively little at different densities of C3b/cell (Fig. 3B and Table I).
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A and Table I,
the Km of
ERC3b,Bb decreased 33-fold from 3.14 µM at
18,600 C3b/cell to 0.095 µM at 434,000 C3b/cell while the catalytic
rate of the enzyme decreased slightly by 3-fold. These results
suggest that the C3b density influences the
Km of C5 convertase but the type of
surface employed for assembling the enzyme does not influence the
Km or the
kcat of C5 convertase. Measurement of kinetic parameters of C5 convertases formed on ES, a nonactivator of the alternative pathway of complement
Although the data obtained with zymosan and
ER, both activators of the alternative pathway of
complement, indicated that the surface does not influence the
Km of the enzyme we thought it
important to examine the properties of C5 convertase formed on a
nonactivating surface (ES). The enzyme assembled
on ES exhibited kinetic properties similar to
those obtained on the two activators of the alternative pathway of
complement (Fig. 3 and Table I). The velocity vs substrate plots for
ESC3b,Bb assembled with ES
bearing 18,000 and 115,000 C3b/cell (Fig. 4, A and B,
respectively) showed an excellent fit to the theoretical curve based on
the Michaelis-Menten equation as well as to the linearized form of the
equation (Eadie-Hofstee plot) shown as insets in Fig. 4. Thus, while
the Km of
ESC3b,Bb varied significantly (100-fold)
depending on the density of C3b/cell, the
kcat of the enzyme varied slightly,
decreasing by 4-fold when the C3b density was increased from 18,000
to 479,000 C3b/ES (Table I).
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The conversion of low affinity to high-affinity C5 convertases was
studied by examining the enzymatic properties of self-amplified C5
convertases. These were the highest affinity C5 convertases that were
generated in this study. They were prepared by allowing preformed C3
convertases to amplify C3b around themselves in the presence of excess
C3. After amplification, the cells were washed and the convertases that
had not yet decayed were assayed for C5 cleavage. The velocity data
obtained were found to fit well to the theoretical curve based on the
Michaelis-Menten equation (Fig. 5). The
data were also observed to fit well to the linearized form of the
Michaelis-Menten equation, shown as the Eadie-Hofstee plot (Fig. 5, inset). The Km of
self-amplified C5 convertases examined on all three surfaces exhibited
values in the range of 0.074–0.016 µM (Table I). Due to the low
Km, care was taken to ensure that
substrate consumption was kept low during the assays by using low
concentrations of enzyme. In most assays, cleavage of C5 was <1%,
whereas in those at substrate concentrations below 100 nM cleavage of
C5 was less than 5% at the end of the 15-min assay. The very low
Km of these C5 convertases indicates a
stronger interaction with C5 that is greater by three orders of
magnitude than that observed with monomeric C3/C5 convertase, C3b,Bb
(Km = 24 µM).
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). These results imply that C5
convertases with different affinities for C5 exist and that the
observed Km is an average
Km. If the observed
Km was an average of a high-affinity
C5 convertase (Km = 0.016 to 0.074
µM, Table I)) and a low-affinity C5 convertase
(Km = 5.0 or 24 µM, Table I), then
because the two C5 convertases differ 100-fold or more in their
affinity for C5 the Eadie-Hofstee plots would be biphasic. However,
this was not observed to be the case in most of the experiments that
employed different densities of C3b/cell to assemble the enzyme as
observed in Figs. 2B, 4A, 4B, 5, and
6 indicating that the observed
Km is not an average of just a high
and a low-affinity C5 convertase but instead is an average of different
affinities. To investigate this further we determined the kinetic
properties of C5 convertases formed on zymosan bearing 84,000 C3b/cell.
Because these zymosan particles do not have a very high density of
C3b/cell we would expect a larger percent of C5 convertases formed at
this density of C3b/cell to have a Km
(Km = 5.0 or 24 µM, Table I) that is
above the average Km of 1.39 µM
obtained when all the C3b molecules are converted to enzymes under
saturating levels of factor B (Table I). Therefore, initial velocities
for C5 cleavage were determined up to 53 µM of C5 (10.0 mg/ml), a
concentration that is 38-fold above the average
Km (1.39 µM) of the enzyme (Fig. 6).
Errors involved in measuring velocities at very high substrate
concentrations are large. Nevertheless, within experimental error, the
data obtained appeared to fit well to the theoretical curve for a
homogeneous population of enzymes based on the Michaelis-Menten
equation and the Eadie-Hofstee plot had no curvature to it (Fig. 6, inset). Modeling the data indicated that subpopulations of
C5 convertases that have Km values
10-fold or less apart would within experimental errors give linear
Eadie-Hofstee plots indicating a homogeneous population of enzymes.
Considered together, these results suggest that the observed
Km of C5 convertases is an average
Km of C5 convertases with varying
affinities for C5.
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The activities of surface-bound C5 convertases assembled with
different densities of C3b/cell have been compared at the normal plasma
concentration of C5. Because all the three surfaces exhibited similar
enzymatic properties, only data for zymosan is shown in Fig. 7. Fig. 7 shows that because C5
convertases formed with few C3b/cell have a
Km above the normal physiologic
concentrations of C5 in plasma (0.37 µM; Fig. 7, vertical dashed
line) these convertases will be relatively inefficient in binding C5
and, therefore, inefficient in cleaving C5. And because C5 convertases
formed with a high density of C3b/cell have a
Km well below the normal plasma
concentration of C5 in blood, these convertases will be occupied with
C5 most of the time and the enzyme will cleave C5 at a catalytic rate
close to Vmax.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The average
Km of the enzyme was observed to
decrease with increasing C3b density (Fig. 3A) to levels
lower than the normal plasma concentration of C5 (0.37 µM; vertical
dashed line in Fig. 7). These results imply that as amplification
continues depositing C3b molecules on the cell surface, C5 convertases
that have high affinities for C5 are generated.
The study of self-amplified C5 convertases in particular has provided
new insights into the mechanism of transformation of low-affinity
monomeric C3/C5 convertases to high-affinity C5 convertases. These were
the highest affinity C5 convertases that were observed on cell
surfaces. Preformed C3 convertases were allowed to amplify C3b around
themselves by providing excess C3. After amplification, the cells were
washed and the remaining initial C3 convertases were assayed for C5
cleaving properties. The Km of these
convertases for C5 (0.016–0.074 µM) (Table I) was observed to be
well below the normal plasma concentration of C5 in blood (0.37 µM)
suggesting that they will cleave C5 at velocities approaching
Vmax (Fig. 7) under physiological
conditions. In contrast, monomeric C3/C5 convertase (C3b,Bb) because of
its weak affinity for C5 (Km = 24
µM) has been suggested to cleave 9,000 C3 for every C5 cleaved under
physiological conditions (12, 28). Considered together,
these results suggest that because C5 convertases formed with few C3b
per cell have Km values well above the
normal concentration of C5 in blood, these convertases will be
relatively inefficient in binding C5 like the monomeric C3/C5
convertase. Instead they will spend most of their time cleaving C3,
thereby opsonizing the cell surface with C3b for phagocytosis. As
amplification continues, C3b density increases and C3b complexes form
convertases that have high affinities for C5. These convertases will
predominantly cleave C5 at catalytic rates close to
Vmax (Fig. 7) thus switching the C3/C5
convertase to production of the cytolytic C5b-9 complex.
Several studies have examined the binding interaction between the
substrate C5 and the noncatalytic subunit of C5 convertase and binding
constants that vary by about three orders of magnitude have been
reported (9, 10, 14, 28, 29, 30). The binding of C5 to C3b
molecules deposited in clusters on ES has been
reported as the highest binding affinity
(Kd = 0.005 µM) (9, 10), whereas that between C5 and soluble monomeric C3b has been
shown to be the weakest (Kd = 37 µM)
(28). Although the dissociation binding constant,
Kd, and the
Km of an enzyme are not always
similar, the binding affinity measured between C5 and the noncatalytic
subunit of the soluble forms of C3/C5 convertases (C3b,Bb; cobra venom
factor from Naja naja kaouthia-dependent C5 convertase; and
cobra venom factor from Naja haje haje-dependent C5
convertase) has been shown to be nearly identical with the
Km of the respective enzyme
(28). In the present study, the lowest
Km measured was for the self-amplified
C5 convertase assembled on ES (0.016 ±
0.007 µM) (Table I). The Km is
observed to be within 3-fold of the Kd
(0.005 µM) reported for the binding of C5 to C3b molecules deposited
in clusters on ES (9, 10). These
observations suggest that surface-bound C5 convertases like soluble C5
convertases may have similar Kd and
Km. The studies of Hong et al.
(16) and Kinoshita et al. (10) have suggested
the formation of C4b-C3b and C3b-C3b dimers as high-affinity C5 binding
sites within the C3b clusters deposited on ES.
The additional C3b has been shown to be covalently bound to the C3b
(10) or C4b (9, 18) subunit of the C3
convertase resulting in structures C3b,C4b,C2a and
C3b2,Bb, considered to be C5 convertases of the
classical and the alternative pathway, respectively (9, 10, 17, 18). The covalent C3b-binding site has been identified as a
serine residue at position 1217 on the 
'-chain of C4b
(9). Although no evidence is provided in the present study
for the structure of high-affinity C5 convertases, we attribute the low
Km of the enzyme to the formation of
C3b-C3b complexes.
The functional role of C3b dimers has been suggested to be able to hold the substrate C5 in a rigid conformation so as to enable cleavage by Bb. Hong et al. (16) reconstituted C5 convertase activity with purified C3b dimers or oligomers in the presence of Ni2+ and factors B and D. Their studies showed higher C5 convertase activity with C3b oligomers and dimers than with monomeric C3b. Studies by Jelezarova et al. (31) have suggested C3b-C3b dimers in complex with IgG molecules to form better precursors of convertases than C3b. Recent studies by Sandoval et al. (32) involving indels have identified at least two binding sites on C5 for the classical pathway C5 convertase in addition to the convertase cleavage site. Based on all these studies it appears that C5 binds with high affinity to multimeric C3b complexes through interactions of at least two distinct sites on C5 and at least two sites on C3b.
In contrast to the Km of surface-bound
C5 convertases, which varied by approximately three orders of magnitude
(Fig. 3A and Table I), the
kcat of the enzyme varied very little
(Fig. 3B). C5 convertases assembled on zymosan particles
exhibited similar catalytic rates at all densities of C3b/cell. Those
formed on ER and ES at low
density of C3b/cell exhibited kcat
that was similar to that of the soluble monomeric C3b,Bb but showed a
3- to 4-fold decrease with increasing density of C3b/cell (Table I).
Although this decrease in kcat was
consistently observed with increasing density of C3b/cell, its origin
is not apparent. High densities of C3b,Bb, or bound C5 could interfere
with efficient C5 cleavage or restrict access to neighboring active
sites. The kcat of the self-amplified
C5 convertases might be revealing in this regard, but the
kcat of these enzymes was impossible
to measure accurately due to the low numbers of enzymes per cell and to
enzyme decay before and during the assays (these enzymes were premade
and no factor B was available for enzyme reformation as in other
kinetic assays). Nevertheless, the average
kcat for cell-bound alternative
pathway C5 convertases was 0.0056 s-1 indicating
that the rate of C5 cleavage is one of the slowest enzymatic reactions
known with one C5 cleaved per 3 min at
Vmax. Apparently this level of
activity is sufficient to generate biologically effective levels of
cytolytic C5b-9 and the chemotactic fragment C5a.
The proposed mechanism underlying the conversion of low affinity to
high-affinity C5 convertases is schematically represented in Fig. 8. The first C3b molecule deposited on
the surface, forms a monomeric C3/C5 convertase with factor B. This
C3/C5 convertase, because of its weak affinity for C5, will function
primarily as a C3 convertase cleaving thousands of C3 for each C5
cleaved (12), thereby amplifying C3b around the first
C3/C5 convertase. As C3b deposition occurs, C3b complexes are formed
that have higher affinities for C5. This results in the conversion of
low-affinity C5 convertase to high-affinity C5 convertase. At
physiological C5 concentrations these enzymes exceed 90% saturation
with C5, which prevents C3 cleavage and allows C5 cleavage at a
velocity approaching Vmax. The C3b
molecules at the edges of the expanding amplification ring will be
mostly monomers and will form monomeric C3/C5 convertase. The monomeric
C3/C5 convertases will spend most of their time cleaving C3 because of
their weak affinity for C5. As amplification continues outward,
deposition of C3b converts this region into high-affinity C5
convertases and produces another ring of monomers. This process
continues until the surface is completely coated with C3b and most
C3/C5 convertases have been converted to high-affinity C5 convertases.
At this point, net C3b deposition ceases and a plateau in C3b per cell
is reached (33).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nenoo Rawal, Department of Biochemistry, University of Texas Health Center, 11937 U.S. Highway 271, Tyler, TX 75708-3154.
3 Abbreviations used in this paper: C3b, proteolytically activated form of C3; EC, chicken erythrocytes; ER, rabbit erythrocytes; ES, sheep erythrocytes; VBS, Veronal-buffered saline; GVB, gelatin VBS; GVBE, GVB containing 10 mM EDTA; NHS, normal human serum; ZymC3b,Bb, surface-bound C5 convertase assembled on zymosan particles; ERC3b,Bb, surface-bound C5 convertase assembled on ER; ESC3b,Bb, surface-bound C5 convertase assembled on ES; C3b,Bb, monomeric C3/C5 convertase assembled with monomeric C3b; Vmax, maximum velocity; kcat, catalytic rate constant.
Received for publication November 1, 2000. Accepted for publication December 1, 2000.
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),
ERC3b,Bb (
), and ESC3b,Bb (
) formed with
cells bearing varying amounts of C3b/cell as indicated. The number of
C3b/cell was measured as the number of Bb binding sites as described in
Fig. 1
