Generation of Multiple Fluid-Phase :Plasma Complexes during Complement Activation: Possible Implications in C3 Glomerulopathies This information is current as of September 26, 2021. Mahalakshmi Ramadass, Berhane Ghebrehiwet, Richard J. Smith and Richard R. Kew J Immunol 2014; 192:1220-1230; Prepublished online 23 December 2013; doi: 10.4049/jimmunol.1302288 Downloaded from http://www.jimmunol.org/content/192/3/1220

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes during Complement Activation: Possible Implications in C3 Glomerulopathies

Mahalakshmi Ramadass,* Berhane Ghebrehiwet,*,† Richard J. Smith,‡ and Richard R. Kew*

The is tightly regulated to safeguard against tissue damage that results from unwanted activation. The key step of C3 cleavage to C3b is regulated by multiple mechanisms that control the initiation and extent of activation. This study dem- onstrated that C3b:plasma protein complexes form in the fluid-phase during complement activation. Several different plasma pro- teins displayed a discrete high molecular SDS-resistant band when any of the three complement activating pathways were triggered

in normal human serum or plasma. Serum depleted of individual complement revealed that C3 and factors B and D were Downloaded from essential for complex formation. Inactivation of the thioester bond in C3 also prevented complex formation. In vitro, complexes could be generated using four purified proteins—C3, factor B, , and target protein—and Mg2+ to allow C3 convertase formation. These studies showed that the complexes consisted of a plasma protein covalently bound to C3b in a 1:1 molar ratio; the C3b portion was rapidly degraded by factors H and I. Analysis of plasma samples from patients with dense deposit disease and C3 glomerulonephritis demonstrated that C3b:protein complexes form spontaneously in the blood of patients with dense deposit disease and, to a lesser extent, in C3 glomerulonephritis patients, but not in healthy controls. This finding supports the underlying http://www.jimmunol.org/ hypothesis that these C3 glomerulopathies are diseases of fluid-phase complement dysregulation. These complexes could normally function as a passive mechanism to intercept C3b from depositing on host cells. However, excessive generation and/or defective clearance of fluid-phase C3b:protein complexes may have pathological consequences. The Journal of Immunology, 2014, 192: 1220–1230.

he complement system is the primary humoral component but regulated on host cells, and is controlled in both the fluid- of innate immunity, and activation of the cascade by phase and on host cell surfaces by regulators of complement ac-

T recognition pathways (classical, lectin, and alternative) tivation (RCA), a class of proteins that regulate C3 cleavage (4, 5). by guest on September 26, 2021 converges at the crucial step of C3 cleavage (1). The alternative Because covalent attachment of C3b to targets is indiscriminate pathway is unique in that it is constitutively active owing to the and somewhat inefficient, only ∼ 10% of activated C3b attaches slow spontaneous hydrolysis of the labile thioester bond in the to intended targets (6, 7). The majority of C3b thioesters react thioester domain (TED) of C3 (2). This “tick-over” mechanism with water molecules in the fluid-phase, neutralizing the reactive forms an active “C3b-like” C3 (C3-H2O) that binds factor B, as- thioester and limiting host cell (and target cell) deposition (7). sembles a C3 convertase, and generates additional C3b to amplify Dysregulation of C3 and the ensuing spontaneous activation of the pathway. Proteolytic cleavage of C3 to C3b exposes the un- the alternative pathway have been associated with several ultra-rare stable thioester bond, which then reacts very quickly with avail- complement-mediated renal diseases. One such example comprises able amine or hydroxyl groups to covalently attach C3b on a target the C3 glomerulopathies (C3Gs), the two prototypical examples of surface (3). This initial “tagging” step is amplified on pathogens, which are C3 glomerulonephritis (C3GN) and dense deposit dis- ease (DDD) (8). Both C3GN and DDD are defined by their shared *Department of Pathology, Stony Brook University School of Medicine, Stony C3-dominant glomerular immunofluorescence and differentiated Brook, NY 11794; †Department of Medicine, Stony Brook University School of by the location and appearance of glomerular deposits that can be Medicine, Stony Brook, NY 11794; and ‡Molecular Otolaryngology and Renal Re- search Laboratories, Division of Nephrology, Department of Internal Medicine, resolved on electron microscopy. Genetic studies have identified Carver College of Medicine, University of Iowa, Iowa City, IA 52242 loss-of-function mutations in RCA proteins or gain-of-function mu- Received for publication August 27, 2013. Accepted for publication November 20, tations in C3 in these diseases (9). In addition, DDD (and, less often, 2013. C3GN) is often associated with the development of autoantibodies This work was supported by National Institutes of Health Grants GM063769 (to to the alternative pathway C3 convertase (C3bBb) that are known as R.R.K.), AI060866, and AI084178 (to B.G.). C3 nephritic factors (C3Nefs) (10–13). C3Nefs stabilize the C3bBb Address correspondence and reprint requests to Dr. Richard R. Kew, Department of complex, dramatically increasing its half-life (14). Persis- Pathology, Stony Brook University, Stony Brook, NY 11794-8691. E-mail address: [email protected] tence of the C3 convertase consumes C3, leading to the very low plasma C3 levels characteristic of this disease (15). Abbreviations used in this article: a1AG, a-1 acid ; aHUS, atypical hemolytic uremic syndrome; C3G, C3 glomerulopathy; C3GN, C3 glomerulonephri- The current study initially investigated the effect of complement tis; C3Nef, C3 nephritic factor; CVF, cobra factor; CVFAS, CVF-activated activation on the vitamin D binding protein (DBP) and made the serum; DBP, vitamin D binding protein; DDD, dense deposit disease; HK, high m.w. kininogen; IVIG, i.v. Ig; LK, low m.w. kininogen; NFDM, nonfat dry milk; NHS, unexpected observation that DBP forms covalent complexes with a a normal human serum; 1PI, -1 proteinase inhibitor; RCA, regulator of complement C3b. We hypothesized that this interaction would not be unique to activation; TED, thioester domain. DBP and explored the interaction of C3b with several other plasma Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 proteins of diverse size, charge, and abundance. All proteins formed www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302288 The Journal of Immunology 1221 complexes with C3b that could be degraded by the fluid-phase RCA microscopy) and the availability of sufficient plasma samples to complete protein factors H and I. Although it is intuitive that nascently gen- all assays multiple times. The human research institutional review board at erated C3b should be able to attach to proteins in close proximity, the University of Iowa approved all procedures, and all patients gave in- formed consent. there are very few descriptions of these complexes and the obser- vations that have been reported focus on C3b binding to other SDS-PAGE and immunoblotting complement proteins or activators (16–18). Thus, the existence of fluid-phase C3b:plasma protein complexes generally is not recog- All samples were separated using 8% polyacrylamide gels with SDS at 80 V for the stacking gel and 100 V for the resolving gel. Resolved gels then nized and their physiological significance has not been described. In were transferred onto an Immobilon polyvinylidene difluoride membrane this study we observed these complexes in plasma samples from (Millipore, Bedford, MA) at 100 V for 75 min. The polyvinylidene difluoride DDD patients and, to a lesser extent, in plasma samples from C3GN membrane was blocked with 5% nonfat dry milk (NFDM) in TBS with 0.1% patients. Their presence further supports the pathophysiological basis Tween 20 (TBST) for 30 min, followed by primary and HRP-labeled sec- ondary Ab incubations in 5% NFDM. Finally, blots were developed using of these two C3Gs as fluid-phase dysregulation of the C3 convertase. HyGLO Quick Spray Chemiluminescent Detection Reagent (Denville Sci- Our findings also suggest that in the normal state, covalent attach- entific, Denville, NJ) and x-ray film. ment of C3b to plasma proteins may be a passive mechanism to minimize host cell deposition at sites of complement activation. In vitro complex formation and breakdown Excessive generation or defective clearance of these circulating C3b: To evaluate the role of activated C3 in complex formation, the alternative plasma protein complexes in the C3Gs may contribute to disease but pathway was assembled in vitro, using the purified proteins C3 (1.3 mg/ml), may also offer therapeutic targets to limit the observed renal damage. factor B (200 mg/ml), factor D (1 mg/ml), and DBP (400 mg/ml) or a1AG 2+

(1 mg/ml) with 0.5 mM Mg and incubated at 37˚C for the specified Downloaded from amount of time. In control experiments, factor B was eliminated from the Materials and Methods mixture to prevent activation. The molar ratios of the various components Reagents were maintained at physiological levels even when the exact concen- trations could not be maintained owing to dilution effects. The roles of Purified cobra venom factor (CVF), preactivated zymosan A, and sheep regulatory proteins factors H and I in the breakdown of C3 complexes were erythrocytes were obtained from Complement Technology (Tyler, TX). determined by adding purified alone (340 mg/ml), factor I alone Human serum depleted of individual complement components (C3, C4, C5, (54 mg/ml), or both together along with C3 (1 mg/ml), factor B (177 mg/ factor B, factor D, , factor H, and factor I) and purified human http://www.jimmunol.org/ ml), factor D (0.76 mg/ml), DBP (307 mg/ml), and 0.5 mM Mg2+ at 37˚C complement proteins (C3, C3b, factor B, factor D, factor H, and factor I) along with CVF (416 U/ml) to activate the protein mixture. Complexes were all purchased from Complement Technology. The following purified were evaluated by SDS-PAGE and immunoblotting. human proteins were all obtained from Athens Research and Technology (Athens, GA): a-1 proteinase inhibitor (a1PI), a-1 acid glycoprotein (a1AG), IgG, and DBP. C5a sandwich ELISA The IgG fraction of goat polyclonal anti-human DBP was purchased from A sandwich ELISA to detect human C5a and C5a des-Arg was developed as DiaSorin (Stillwater, MN) and then affinity purified in our laboratory using previously described in detail (19). Briefly, MaxiSorp 96-well plates were immobilized DBP. Chicken polyclonal anti-a1PI Ab was obtained from a coated with 500 ng of mouse monoclonal anti-human C5a (clone 295003; ProSci (Poway, CA). Biotinylated rabbit polyclonal anti- 1AG, goat poly- R&D Systems, Minneapolis, MN) capture Ab at 4˚C overnight. The clonal anti-human albumin, and rabbit polyclonal anti-kininogen Abs were coating solution was removed and the wells were blocked with 300 ml by guest on September 26, 2021 all obtained from Abcam (Cambridge, MA). Mouse monoclonal anti-human blocking solution (3% NFDM in PBS with Tween) for 1 h at room tem- factor B (clone D33/3) was obtained from Santa Cruz Biotechnology (Santa perature. After three washes, 100 ml standards (78 pg/ml to 5 ng/ml purified Cruz, CA). Polyclonal anti-human C1 inhibitor Ab was developed in rabbits. natural human C5a; Complement Technology) or experimental samples were Chicken polyclonal anti-human C3 was obtained from Gallus Immunotech added and incubated at room temperature with shaking for 90 min. This was (Cary, NC). Rabbit IgG Ab developed against epitopes on human C3d followed by four washes and incubation with 100 ng detection Ab, bio- (#ab15981) was purchased from Abcam. Mouse monoclonal anti-human tinylated mouse monoclonal anti-human C5a (clone 295009, R&D Systems) factor I neutralization Ab (#A247) was obtained from Quidel (San Diego, for 60 min at room temperature with shaking. Finally, 40 ng of HRP- CA). Williams plasma (deficient in high and low m.w. kininogen) was a conjugated streptavidin (KPL, Gaithersburg, MD) was added to each well generous gift from Dr. Alvin Schmaier, Case Western Reserve University, and incubated at room temperature for 30 min. After five washes, 100 ml Cleveland, OH. HRP substrate solution (KPL) was added until color development, followed m Collection of human blood and in vitro activation of by addition of 100 l stop solution (KPL), and the absorbance was measured at 450 nm using a SpectraMax M2 plate reader (Molecular Devices, Sun- complement nyvale, CA). Blood was collected from healthy, medication-free human subjects who gave informed consent using a protocol approved by the Stony Brook Hydrolysis of the C3 thioester University Institutional Review Board. Vacutainer tubes (BD, Franklin m Lakes, NJ) containing either 3.2% sodium citrate (for plasma) or a silica clot The thioester bond in C3 was inactivated by treating 250 g C3 with 0.5 M hydroxylamine (NH2OH), pH 7.5, for 2 h at 21˚C followed by exhaustive activator (for serum) were used. Individual serum and plasma samples from 3 at least five subjects were pooled, aliquoted, and frozen at 280˚C. Pooled dialysis against PBS (1.8 L, 3 ) at 4˚C for a total of 18 h (20). Inactivation serum or citrated plasma (0.3 ml) was activated either with CVF (416 U/ of thioester was confirmed by hemolytic assays using both rabbit eryth- ml), zymosan A (10 mg/ml), or 50 ml heat-aggregated (heated at 63˚C for rocytes for the alternative pathway, and Ab-coated sheep erythrocytes for 20 min) human IgG (10 mg/ml) and incubated at 37˚C for the time indi- the classical pathway, as well as a C5a ELISA of the supernatant from the cated in each experiment. For activation of citrated plasma samples, 2 mM hemolysis assay with Ab-coated sheep erythrocytes. C3-depleted serum 2+ alone or C3-depleted serum reconstituted with either native C3 or C3- Mg was added to overcome the chelation effect of sodium citrate. Serum ++ depleted of the complement regulators factors H and I spontaneously NH2OH (at 0.5 mg/ml) was taken in a final dilution of 1:100 in GVB buffer (gelatin veronal buffer) along with 50 ml Ab-coated sheep eryth- activates the alternative pathway and hence was stored in 0.1 mM EDTA. 3 7 These samples were activated by adding just 0.5 mM Mg2+ and incubating rocytes (5 10 /ml) and incubated at 37˚C for 1 h, after which the cells at 37˚C. In certain experiments, depleted serum samples were reconstituted were centrifuged, supernatant was collected, and C5a levels were quantified. by adding back the purified protein to achieve its mean plasma concen- tration (factor B: 210 mg/ml; factor D: 1 mg/ml; C3: 1.3 mg/ml) along with Ab neutralization of factor I 0.4 mM Mg2+ and then incubating at 37˚C for 15 min. Mouse monoclonal anti-human factor I, which inhibits the Human subjects with C3G domain, was used to determine whether enzyme activity of factor I is re- quired to cleave the C3b:plasma protein complexes. Anti-factor I (100 mg/ A total of 12 patients with biopsy-proven C3G (6 DDD and 6 C3GN) were ml) was added to factor H–depleted serum and incubated on ice for 15 min selected from our C3G registry for inclusion in this study on the basis of to allow Ab binding, followed by incubation at 37˚C for a specified amount histopathological data (light microscopy, immunofluorescence, and electron of time. 1222 COMPLEMENT ACTIVATION GENERATES C3b:PLASMA PROTEIN COMPLEXES

a Results band is specific to DBP, two other plasma proteins, 1PI and Complement activation correlates with generation of high m.w. a1AG, both of similar size but greater abundance than DBP, were SDS-resistant forms of several plasma proteins examined in the CVF-activated serum (CVFAS) samples. Fig. 1 shows that activation of any complement pathway also induced Because DBP has been shown to function as a chemotactic formation of high m.w. SDS-resistant bands of a PI and a AG; these for C5a in vitro, the initial goal of this study was to determine whether 1 1 bands were not observed in untreated NHS or CVFAS treated with complement activation converts DBP into an active chemotactic EDTA. Factor B cleavage is shown in the bottom panel of Fig. 1 to cofactor (21). Possible structural changes in DBP were investigated verify complement activation. Thus, activation of serum complement in normal human serum (NHS) by treating with three distinct in vitro correlates with the appearance of high m.w. SDS-resistant pathway activators and then analyzing the samples by SDS-PAGE bands of DBP, a PI, and a AG. and immunoblotting. No diminutioninthe56-kDanativeDBPband 1 1 The temporal correlation between complement activation and was apparent, and neither were lower m.w. DBP bands detected generation of these high m.w. bands was investigated in serum and (Fig. 1 and data not shown). However, Fig. 1 shows that activation citrated plasma to determine whether the clotting process alters of any complement pathway did generate an SDS-resistant DBP band formation. Samples were blotted for several proteins with dif- band at ∼ 200 kDa that was not observed in untreated serum. ferent abundance, m.w., and isoelectric points. All proteins examined Moreover, simultaneous addition of EDTA with CVF to serum formed similar SDS-resistant bands upon complement activation in inhibited complement activation and prevented formation of this both serum and plasma (Fig. 2A), suggesting that this process is not high m.w. band. To determine whether formation of this high m.w. affected by blood clotting and is not restricted to a certain class of

proteins with a common structural motif. The temporal appearance Downloaded from of these SDS-resistant bands correlated with complement activation in both CVFAS and plasma (Fig. 2A). In serum, high m.w. bands begin appearing at the 5-min time point (Fig. 2A), which is the precise time that complement activation products begin to appear in the CVF-treated serum: Bb (Fig. 2A) and C5a generation

(Fig. 2B). Peak band formation occurs at ∼ 15 min and then http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Complement activation induces formation of high m.w. SDS- FIGURE 2. Formation of SDS-resistant bands correlates temporally with resistant complexes with plasma proteins. Pooled NHS was sham treated complement activation in vitro. Pooled NHS or citrated plasma was activated with PBS (lane 1), or complement was activated by incubating serum at at 37˚C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 37˚C, using 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), or 0.5 mM Mg2+ was added to plasma to overcome the chelation effects of sodium mg/300 ml heat-aggregated human IgG (lane 4). As a control to inhibit citrate. At each time point, activation was stopped by placing the sample on complement activation, 10 mM EDTA was added to serum prior to the ice. (A) Aliquots were separated on an 8% SDS-PAGE and then immuno- addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS- blotted for the indicated proteins. Gels were cropped to focus only on the

PAGE and then immunoblotted for DBP, a1PI, or a1AG. Arrows indicate high m.w. SDS-resistant bands. Samples also were blotted for factor B to the position of high m.w. SDS-resistant bands formed during complement verify cleavage as an indicator of complement activation. The 75-kDa b-chain activation. The same samples also were blotted for factor B (fB) to verify of C3 was used as a loading control. (B) C5a ELISA of complement acti- cleavage as an indicator of complement activation (bottom panel). vation in serum. (C) C5a ELISA of complement activation in citrated plasma. The Journal of Immunology 1223 gradually decreases in intensity (Fig. 2A). In CVF-treated citrated plasma (Fig. 2A), complement activation is delayed owing to chelation of divalent cations by sodium citrate, but bands do con- sistently appear at the 30-min time point (Fig. 2A), their appearance correlating with factor B cleavage (Fig. 2A) and C5a generation (Fig. 2C). Of interest, complement activation generates a single kininogen band in serum but a doublet in plasma, reflecting the presence of two forms of kininogen in blood, high m.w. kininogen (HK) and low m.w. kininogen (LK). In serum, in contrast, HK is cleaved during the clotting process and only the LK band appears (Fig. 2A). Thus, the formation of high m.w. SDS-resistant bands of several plasma proteins is not unique to specific blood proteins and correlates temporally with in vitro activation of complement. The role of erythrocytes in the generation of these high m.w. bands was investigated using citrated whole blood. Fig. 3 demon- strates that SDS-resistant bands of a1PI and a1AG form in whole blood during complement activation, indicating that surface ex- FIGURE 4. High m.w. SDS-resistant bands of the different plasma pression of RCA proteins on erythrocytes does not prevent high proteins can form independently of each other. Normal human citrated m.w. band formation. Using Williams plasma (deficient in both plasma or citrated Williams plasma (deficient in both HK and LK) were Downloaded from 2+ HK and LK), we also found that these bands form independently of activated with CVF (416 U/ml) and 2 mM Mg for 60 min at 37˚C. each other. As seen in Fig. 4, although no kininogen bands formed Aliquots were separated using 8% SDS-PAGE and immunoblotted for kininogen, DBP, a PI, a AG, and factor B. in C-activated Williams plasma, the generation of SDS-resistant 1 1 bands of DBP, a1PI, and a1AG was not altered. ponent was performed in samples where depletion abolished band Activation of the C3 thioester during C-activation causes

formation. C3-, factor B–, and factor D–depleted sera were unable http://www.jimmunol.org/ formation of covalent complexes of C3b with plasma proteins to generate high m.w. bands upon addition of CVF (Fig. 5A), but The evidence presented above indicates a strong correlation be- formation was restored with addition of the deficient proteins. In tween complement activation and formation of high m.w. SDS- contrast, generation was not altered in serum depleted of proper- resistant bands of several plasma proteins. To identify the steps din, C4, or C5, clearly demonstrating that C3 cleavage is essential involved in the generation of these complexes, sera depleted of to this process. different complement components were used. Fig. 5A shows the The role of the C3 cleavage product C3b was examined next formation of high m.w. SDS-resistant bands of DBP, a1PI, and (Fig. 5B). Purified C3b added to factor B-depleted serum showed a1AG in various depleted serum samples activated with CVF; no complex formation. Addition of C3b to C3-depleted serum reconstitution of depleted serum with the purified missing com- generated a C3 convertase but was not able to induce formation of by guest on September 26, 2021 high m.w. forms of DBP, a1PI, and a1AG, even though robust factor B cleavage could be demonstrated, indicating the require- ment for both native C3 and the components needed to form C3 convertase (factors B and D). Because native C3 is essential for this process, the role of the thioester bond was investigated by treating C3 with hydroxylamine (NH2OH) to inactivate this bond and then using this chemically modified C3 to reconstitute C3- depleted serum (Fig. 6). Hydroxylamine-treated C3, like C3b, could not attach to an activating cell surface and generate C5a, indicating effective thioester inactivation (Fig. 6A). This inactivated C3 also failed to induce formation of high m.w. complexes of DBP, a1PI, or a1AG upon addition of CVF (Fig. 6B), indicating that an intact thioester bond is essential for this process. Thus the SDS-resistant high m.w. bands of plasma proteins formed during complement activation are covalent complexes with nascently generated C3b (note high m.w. bands in the C3 blot of Fig. 6B). Consistent with this mechanism, multiple high m.w. C3 bands were observed when the samples described in Figs. 1 and 2 were blotted for C3. Fig. 6C shows immunoreactive C3 bands above the 110-kDa C3 a-chain in the samples from Fig. 1. Furthermore, the temporal appearance and intensity of these multiple C3 bands correlate precisely with the individual proteins detected in CVFAS and plasma in Fig. 2 (shown in Fig. 6D). Moreover, Fig. 6C and 6D show that the appearance of FIGURE 3. High m.w. SDS-resistant bands form in whole blood with high m.w. C3 bands correlate with a reduction in the intensity of the complement activation. Citrated whole blood or citrated plasma from the native C3 a-chain (110 kDa) that contains the thioester bond. same blood donor was treated with 2 mM Mg2+ and CVF (416 U/ml) for 30 min at 37˚C. PBS was added to plasma to compensate for blood cell Factors H and I degrade the C3b:plasma protein complexes volume and to equalize the protein concentrations between the plasma and whole-blood samples. After CVF activation, whole blood was centrifuged The time course of complex formation (Fig. 2A) shows that band to pellet the cells, and aliquots of both plasma samples were separated intensity decreases over time, consistent with the breakdown of using an 8% SDS-PAGE and immunoblotted for a1PI, a1AG, and factor B. these complexes. Because C3b is cleaved by factors H and I, their 1224 COMPLEMENT ACTIVATION GENERATES C3b:PLASMA PROTEIN COMPLEXES Downloaded from http://www.jimmunol.org/

FIGURE 5. Generation of C3 convertases and C3 are both essential for formation of high m.w. SDS-resistant bands. (A) Serum samples depleted of individual complement components (fB, fD, fP, C3, C5, and C4) were activated with CVF (416 U/ml) for 15 min at 37˚C. Samples in which by guest on September 26, 2021 complex formation was abolished were reconstituted with physiological levels of missing purified protein (fB, fD, or C3) followed by activation with CVF. Aliquots were separated using 8% SDS-PAGE and immuno- blotted for DBP, a1PI, and a1AG. Samples also were immunoblotted for C3 b-chain band as a loading control and factor B to show complement activation. (B) Factor B–depleted and C3-depleted sera were reconstituted FIGURE 6. Reaction with the thioester bond in C3 during complement with purified C3b (1.3 mg/ml) and incubated at 37˚C for 1, 3.5, or 5 min, activation causes formation of covalent complexes with plasma proteins. (A) The amount of C5a generated in reconstituted C3-depleted serum separated using 8% SDS-PAGE and immunoblotted for DBP, a1PI, and samples measured by ELISA. Ab-coated sheep erythrocytes were treated a1AG. Samples also were blotted for factor B to show complement acti- vation in C3-depleted serum reconstituted with C3b. CVF-activated NHS with either C3-depleted serum alone or C3-depleted serum reconstituted was included as a positive control. with 1.3 mg/ml native C3, hydroxylamine-treated C3 (C3-NH2OH), or C3b and incubated at 37˚C for 1 h. The supernatant was removed, and C5a levels were quantified by C5a ELISA. (B) C3-depleted serum alone or possible role in this process was examined. Factor H–depleted and reconstituted with native C3 or hydroxylamine-treated C3 (C3-NH2OH), factor I–depleted sera were incubated at 37˚C for 15 min to allow both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for C3b:protein complex formation, following which breakdown for 15 min at 37˚C. CVF-activated NHS was included as a positive control. was monitored for 16 h, with or without the addition of purified Aliquots were separated using 8% SDS-PAGE and immunoblotted for a a C factor H or factor I (Fig. 7A). No breakdown was observed in the DBP, 1PI, 1AG, factor B, and C3. ( ) Pooled NHS was sham treated factor I–depleted serum, but addition of purified factor I induced with PBS (lane 1), or complement was activated by incubating serum at complete degradation of each complex, consistent with the known 37˚C, using 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), or 0.5 mg/300 ml heat-aggregated human IgG (lane 4). As a control to inhibit role of this protease (Fig. 7A). In factor H–depleted serum, there complement activation, 10 mM EDTA was added to serum prior to addition was significant breakdown of C3b:protein complexes that was of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and only marginally increased by the addition of purified factor H then immunoblotted for C3. (D) Pooled NHS or citrated plasma was ac- (Fig. 7A). Next, factor H–depleted serum was examined at very tivated at 37˚C with 416 U/ml CVF for the indicated times. In addition to early time points to assess its role in complex breakdown. DBP, CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects a1PI, and a1AG complexes spontaneously formed in factor H– of sodium citrate. At each time point, activation was stopped by placing depleted serum (without a complement activator) and the com- the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then plexes appeared as doublets within 1 min but were all cleaved to immunoblotted for C3. the lower m.w. form by 5 min (Fig. 7B). No doublets were ob- served in normal serum even 1 min after CVF was added (data not facilitates complex breakdown, other serum cofactors (factor H– shown), indicating that complex cleavage is delayed in the ab- like 1, factor H–related proteins) can substitute in the absence of sence of factor H, further confirming that, although factor H factor H (22–24). To confirm the role of factor I in the initial rapid The Journal of Immunology 1225 Downloaded from http://www.jimmunol.org/

FIGURE 7. C3b:protein complexes are cleaved in serum by factor I with the help of a cofactor. (A) Factor I–depleted serum and factor H–depleted serum were reconstituted with 0.5 mM Mg2+ and incubated at 37˚C for 15 min, to allow for complex formation, followed by addition of 10 mM EDTA to stop the reaction. The samples were then reconstituted with either PBS or the purified depleted component (factor I or factor H) and incubated for 16 h at 37˚C.

Samples were separated using 8% SDS-PAGE and immunoblotted for DBP, a1PI, and a1AG. (B) Factor H–depleted serum was reconstituted with 0.5 mM 2+ Mg and incubated at 37˚C for the indicated times. Serum aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, a1PI, a1AG, and factor B. (C) Factor I–depleted serum alone or reconstituted with 25% (0.253) or 100% (13) of the plasma concentration of factor I (70 mg/ml) was activated with CVF (416 U/ml) for 15 min at 37˚C. CVF-activated NHS was included as a positive control for complex formation. Serum aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, a1PI, and a1AG. (D) Factor H–depleted serum was treated with 100 mg/ml of either an by guest on September 26, 2021 irrelevant mouse IgG or an Ab that neutralizes the serine protease domain of factor I for 15 min on ice. Serum samples then were incubated with 0.5 mM 2+ Mg at 37˚C for the indicated times. Immunoblots for DBP and a1PI complex formation are shown. (E) Pooled NHS and C3-depleted serum were activated with CVF for 15 min, and factor I–depleted serum was incubated at 37˚C for 15 min. The samples were separated using 8% SDS-PAGE and immunoblotted for C3. cleavage reaction, purified factor I was added to factor I–depleted over, hydroxylamine-treated C3 was capable of activating the serum. As expected, reconstitution of factor I to the depleted se- alternative pathway, as evidenced by factor B cleavage, but, in rum restored the initial cleavage (Fig. 7C). Furthermore, an Ab contrast to native C3, it could not form a complex with DBP (Fig. that specifically blocks the serine protease domain in factor I also 8B). These results confirm that only four components are neces- inhibits complex cleavage (Fig. 7D). These results indicate that sary and sufficient for C3b:protein complex formation. factor I is essential for the initial cleavage and subsequent break- Because the use of purified proteins provides a well-defined down of these complexes, a process that can be facilitated by the in vitro model, the breakdown of C3b:protein complexes was in- cofactor activity of factor H. C3b:protein complexes are initially vestigated using purified factors H and I. Fig. 9A shows C3b:DBP generated as higher m.w. forms, and factor I along with cofactors complexes after 15 min, 2 h, or 16 h with no regulators, factor H mediates the cleavage of C3b to iC3b and smaller C3b cleavage alone, factor I alone, or both factors H and I. Only the combina- products. This degradation pattern is readily observed on a C3 blot tion of both factor H and factor I could initiate cleavage and of CVFAS versus factor I–depleted serum (Fig. 7E), where in breakdown of the C3b:DBP complex. These results using purified CVFAS there is a distinct 67-kDa iC3b product of the a-chain, proteins confirm that C3b:DBP (or other plasma proteins) are whereas in factor I–depleted serum the prominent C3 cleavage initially generated as higher m.w. complexes that are rapidly product is the 100-kDa a-prime chain of C3b. cleaved by factor I with the help of a cofactor to a lower m.w. form, and that this complex represents the relatively stable SDS- In vitro generation of C3b:DBP complexes using purified resistant complex observed in serum and plasma following com- proteins plement activation (Figs. 1–5). Although the iC3b:DBP band The preceding data demonstrate that formation of C3b:protein completely disappeared after a 16-h incubation, an intermediate complexes requires native C3, a C3 convertase, and a target plasma cleavage product, which could potentially be C3dg:DBP, appeared protein. To examine whether this system can be assembled in vitro, at 15 min and was not further degraded even after 16 h (Fig. 9B). purified C3, factor B, factor D, and a target protein of interest (DBP) These results were confirmed using purified a1AG as the target were mixed at physiological ratios in the presence of Mg2+.Fig.8A protein, where a prominent band at an approximate m.w. corre- shows that a C3b:DBP complex was formed upon incubation at 37˚C sponding to C3dg:a1AG also appeared at 15 min and was present for either 15 or 30 min. This complex was the same size as the at 16 h (Fig. 9C, center panel). Similar results were also observed uncleaved complex in factor I–depleted serum (Fig. 8A). More- using purified a1PI (data not shown). This putative C3dg:protein 1226 COMPLEMENT ACTIVATION GENERATES C3b:PLASMA PROTEIN COMPLEXES

of factors H and I. Defective regulation may allow excessive gen- eration of C3b:plasma protein complexes in the circulation. The C3Gs are a type of ultra-rare renal diseases characterized by fluid-phase dysregulation of the alternative pathway of complement. Aliquots of EDTA plasma from six C3GN and six DDD patients were analyzed by SDS-PAGE and immunoblotting for C3b:protein complexes, but no sample showed high m.w. SDS-resistant complexes when blotted for C3 and a1AG (Fig. 10A). To determine whether complexes could form in these patients’ plasmas, samples were incubated at 37˚C for 60 min in the presence of 7.5 mM Mg2+ to overcome the EDTA chelation, and in three samples (DDD patient samples 1, 4 and 12) complexes were then observed (Fig. 10B). No complex formation was observed in normal controls and C3GN samples, although multiple high m.w. bands that did not correspond to complexes on the a1PI and a1AG blots were present on the C3 blot in C3GN sample 5 (Fig. 10B). Because many of the samples from patients were significantly depleted of C3, purified C3 (1.3 mg/ml) and 7.5 mM Mg2+ were

added to all samples from patients, followed by 37˚C incubation Downloaded from for 30 min or 60 min. Five of six DDD samples (1, 4, 6, 11, 12) formed complexes (Fig. 10C). Sample 8, the only DDD sample that showed no complex formation, was also the only DDD sample negative for C3Nefs. C3GN sample 7 formed a clear complex with a1AG, whereas samples 2 and 9 formed weak complexes and

samples 3, 5, and 10 were essentially negative. Of interest, the http://www.jimmunol.org/ FIGURE 8. C3b:protein complexes can be formed in vitro using four bands formed in the plasma samples from patients corresponded to purified proteins. (A) Purified C3, fD, DBP, and 0.5 mM Mg2+ were mixed the approximate size of bands in factor H–depleted serum (Fig. in the presence or absence of fB at physiological concentrations and in- 10C). The presence of C3b:protein complexes in the blood of cubated at 37˚C for either 15 or 30 min. Factor I–depleted serum sample 2+ patients with DDD and, to a lesser extent, in C3GN patients, but incubated at 37˚C for 15 min in the presence of Mg was included as not in plasma samples from healthy controls, indicates that there is a positive control. Mixtures were immunoblotted for DBP (for complex dysregulation of the C3 convertase. formation) and factor B (for complement activation). (B) Purified fB, fD, DBP, and 0.5 mM Mg2+ were mixed with either native C3 or hydroxyl- Discussion amine-treated C3 (C3-NH2OH), at physiological ratios, and were incu- bated at 37˚C for 15 min. Factor I–depleted serum sample incubated at Excessive activation and/or poor regulation of the complement by guest on September 26, 2021 37˚C for 15 min in the presence of Mg2+ was used as a positive control. system can trigger significant host cell damage, the possibility of Samples were immunoblotted for DBP to assess complex formation and which is minimized by multiple levels of regulation that control factor B to verify complement activation. both the initiation and the extent of complement activation. Among the key steps in complement activation is the cleavage of C3 to C3b, band does not appear in factor I-depleted CVFAS reconstituted which exposes the labile thioester bond in the TED of C3b (27). with purified factor I (Fig. 9C, left panel), indicating that in serum The extremely rapid ability of the thioester to react with available a factor I–independent mechanism further cleaves the C3dg por- nucleophile acceptor groups (amino or hydroxyl) covalently attaches tion (25, 26). This factor I–independent process is consistent with C3b to the surface of cells or tissue debris. This essential step of C3b the known cleavage of C3dg by and trypsin-like proteases tagging marks foreign targets for destruction and/or removal by (25, 26). To verify that the C3d fragment (containing the TED phagocytes (28). However, the C3b thioester also reacts with H2Oin covalent linkage site) is indeed attached to a1AG, the same samples plasma, resulting in fluid-phase hydrolysis. In this article, we dem- were blotted using an Ab against C3d-specific epitopes (Fig. 9C, onstrate that upon complement activation in serum (or plasma), C3b right panel). Results show that the size and temporal appearance of also forms transient covalent complexes with $ 10 different plasma C3d correlated with iC3b:a1AG and C3dg:a1AG complexes. Be- proteins of various sizes, isoelectric points, and abundance, and that cause no other intermediate cleavage products could be detected in these C3b:protein complexes are subsequently degraded by factors H serum samples, even when using a broad-range gradient gel (data and I. Covalent binding of C3b to IgG (18), C4 (16), and properdin not shown), it is reasonable to surmise that following the initial (17) has been reported previously, but these studies were examining rapid factor I–mediated cleavage of C3b (to iC3b), a second, slower formation of C3 convertases or immune complex clearance, and not factor I–dependent cleavage removes most of the remaining C3 the role of plasma proteins in binding and neutralizing C3b. Given (C3c fragment), leaving C3dg attached to the plasma protein. In our current knowledge of the reactivity of the thioester in C3, this serum or plasma, this is then followed by a rapid factor I–inde- process of generating C3b:plasma protein complexes is predictable pendent cleavage that removes the remaining C3d and perhaps but, surprisingly, is neither well recognized nor documented. leaves a small C3 peptide tag attached to the plasma protein, which The amount of C3b:plasma protein complexes generated in would be indistinguishable from the native protein by immuno- complement activated normal serum, and particularly in factor I– blotting. depleted serum, is substantial, accounting for a large percentage of the total immunoreactive C3 bands (Figs. 6C, 6D, 7E). Complexes C3b:plasma protein complexes form spontaneously in patients form despite the fact that 91.5% of plasma is water (55 M) and only with DDD 7.5% is protein. Given that water reacts with the thioester bond The data presented above show that C3b:protein complexes are andisingreatmolarexcesscomparedwithplasmaproteins,itis generated by complement activation and then degraded by the actions noteworthy that C3b:protein complexes form and can be detected The Journal of Immunology 1227

FIGURE 9. The breakdown of C3b:plasma protein complexes requires cleavage by fac- tor I in the presence of factor H. (A) Purified C3, fB, fD, and DBP were mixed with factor H alone, factor I alone, or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immunoblotted for DBP to assess complex formation and factor B to verify complement activation. C3 b-chain was used as a loading control. (B) The un- cropped DBP immunoblot of the last six lanes of (A) shows intermediate cleavage products. (C) Purified C3, fB, fD, and a1AG were mixed with either factor I alone or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immu- Downloaded from noblotted for a1AG (center panel) or C3d (right panel) to assess complex formation and breakdown. The positions of C3b, iC3b, and C3dg complexes with a1AG are indi- cated. To compare complex degradation in serum with the purified protein system, factor http://www.jimmunol.org/ I–depleted serum alone, or factor I–depleted serum reconstituted with factor I and acti- vated with CVF for 15 min, was separated and immunoblotted for a1AG (left panel).

in serum or plasma after complement activation. Furthermore, the the plasma protein is followed by a rapid factor I–independent C3b:protein complex attachment must have some level of selec- cleavage reaction that leaves behind a small C3 peptide tag (a tivity because if the reaction were entirely nonspecific then C3b: portion of C3d) attached to the protein (25, 26), which would be by guest on September 26, 2021 albumin would be prevalent, because albumin is the most abun- indistinguishable from the native protein band by immunoblotting. dant plasma protein. However, formation of a high m.w. albumin Uncontrolled alternative pathway activation caused by deficiency band was considerably weaker than other much less abundant of factor H has been associated with several diseases, including plasma proteins (Fig. 2A). Thus, the binding reaction is not en- atypical hemolytic uremic syndrome (aHUS), C3Gs (C3GN and tirely indiscriminate, even though the C3b thioester has a wide DDD) and age-related (AMD) (29, 30). aHUS range of protein targets in plasma. The results suggest that it may and AMD are surface-phase diseases, with loss of tight complement prefer available nucleophile acceptor groups in certain proteins. regulation at the level of either the endothelial cell or Bruch’s The covalent attachment of C3b to multiple plasma proteins membrane, respectively. The C3Gs, in contrast, are fluid-phase dis- reflects the highly reactive nature of the thioester bond, but very few eases in which complement dysregulation occurs in the plasma. The descriptions of these complexes have been reported (16–18) and consequence of fluid-phase dysregulation is massive conversion of their breakdown has not been described. This collateral by-product C3 to C3b, which would lead to the formation of multiple different of complement activation could serve a passive regulatory role by C3b:protein complexes. However, complexes were detected only restraining unintended deposition of C3b on host cells and by when plasma was either incubated at 37˚C or spiked with native limiting complement-mediated tagging to the intended target at C3 in vitro (in patients’ samples in which C3 was depleted), indi- sites of activation. Moreover, we have noted that C3b binds with a cating that although dysregulation allows complexes to spontane- much greater propensity to chemically or thermally denatured plasma ously form, they must be rapidly degraded and/or cleared from the proteins, suggesting that C3 may be involved in clearing denatured circulation in vivo. Furthermore, these results are consistent with the proteins from the circulation (M.Ramadass,B.Ghebrehiwet, clinical finding of extremely low plasma C3 levels in these patients. and R.R. Kew, manuscript in preparation). The C3b:protein complexes It is intriguing to speculate whether these C3b:protein complexes we have described are first produced as higher m.w. forms at a 1:1 have a direct role in the pathogenesis of the C3Gs. Clearly, factor H molar ratio of C3b/plasma protein. Consistent with what is known deficiency has been associated with aHUS, AMD, and C3Gs, but about C3b sequential cleavage following complement activa- it is also relevant to consider the clinical phenotype associated tion, the C3b:protein complexes are then cleaved very rapidly by with factor I deficiency (30–34). This very rare primary immu- factor I and its cofactors to iC3b and then more slowly to C3dg, nodeficiency is inherited as an autosomal recessive trait and has which is followed by a rapid factor I–independent cleavage, possibly been reported in only ∼ 30 families. In the absence of factor I, C3 leaving a very small peptide tag attached to the plasma protein (25, is depleted, and any C3b:protein complexes that form cannot be 26). Factor I is absolutely essential to the process of complex deg- broken down (Fig. 7). Because iC3b and its smaller cleavage radation. That we could detect no further cleavage products, even product C3d are not generated, there is, consequently, a lack of using a broad-range gradient gel, suggests that a second slower recognition by the C3 receptors CR2 (binds C3d on B lymphocytes) factor I–dependent cleavage reaction resulting in C3dg bound to and CR3 (binds iC3b on phagocytic cells), which are needed to 1228 COMPLEMENT ACTIVATION GENERATES C3b:PLASMA PROTEIN COMPLEXES

FIGURE 10. C3b:plasma protein complexes form spontaneously in DDD patient samples. (A)EDTA–plasmasam- ples from DDD patients (n =6)and C3GN patients (n = 6) were separated using 8% SDS-PAGE and immunoblotted for C3, factor B, and a1AG. As a refer- ence for complex size, factor H– and factor I–depleted serum samples were included. (B) EDTA–plasma samples Downloaded from from DDD patients (n = 6) and C3GN patients (n = 6) or healthy controls (n = 2) were reconstituted with 7.5 mM Mg2+ and incubated at 37˚C for 60 min. The samples were then separated using 8% SDS-PAGE and immunoblotted for C3, http://www.jimmunol.org/ factor B, a1PI, and a1AG. (C) Next, 1.3 mg/ml of C3 and 7.5 mM Mg2+ were added to the EDTA–plasma from DDD patients (n = 6) and C3GN patients (n = 6) and incubated at 37˚C for either 30 min or 60 min. As a reference for com- plex size, factor H– and factor I–depleted serum samples were included. The sam- ples were separated using 8% SDS-PAGE

and immunoblotted for C3, factor B, by guest on September 26, 2021 and a1AG.

stimulate phagocytosis and link the innate and adaptive immune samples from factor I–deficient as well as H and I double knockout responses. Therefore, defective opsonization makes factor I–deficient mice, but not factor H–deficient mice, showed multiple high m.w. patients susceptible to recurrent pyogenic infections and aseptic SDS-resistant forms of C3 that correspond to the uncleaved C3b: meningitis (31–34). What is notable, however, is that although renal plasma protein complexes reported in this article. However, these disease has been described in these patients, it is very uncommon bands were noted but were thought to be C3b oligomers (37). These and is associated with deposition of immune complexes, suggesting in vivo findings are consistent with our results and support the activation of the classical pathway (35, 36). Thus the clinical picture premise that C3b:protein complexes can be detected in the cir- of factor I deficiency suggests that C3b:protein complexes alone do culation. Moreover, a follow-up study analyzed the glomerular C3 not promote C3G and that functional factor I is required for the deposits (obtained by laser capture microdissection) in factor I– disease to develop, perhaps by generating iC3b and C3dg fragments deficient mouse kidneys (38). Analysis by SDS-PAGE showed no of the C3b:plasma protein complexes. C3 a-chain band (which contains the thioester bond), but a prom- Of note, factor I is also required for the development of C3G-like inent C3 b-chain, in factor I–deficient mouse kidneys. However, disease in mice. Factor H–deficient mice develop kidney disease a higher SDS-resistant band was observed, indicating that the C3 that is similar to human DDD, whereas factor I–deficient mice, as a-chain was covalently linked to other molecules (38). Another well as factors H and I double knockout animals, do not develop study analyzed glomerular dense deposits by mass spectroscopy kidney disease (37). The authors conclude that to develop DDD, isolated by laser capture microdissection from patients with DDD factor I is required to generate C3 fragments in the circulation of and showed that deposits contain various complement components factor H–deficient mice (37). This study also showed that plasma and other plasma proteins such as ApoE, fibronectin, and fibrinogen The Journal of Immunology 1229

(39). These in vivo studies are consistent with the results reported in Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int. 82: 454–464. this article and suggest that under normal physiological conditions, 14. Mold, C., and M. E. Medof. 1985. C3 nephritic factor protects bound C3bBb covalent attachment of C3b to plasma proteins in the fluid-phase from cleavage by factor I and human erythrocytes. Mol. Immunol. 22: 507– may be a passive mechanism to minimize host cell deposition at sites 512. 15. Martı´nez-Barricarte, R., M. Heurich, F. Valdes-Can˜edo, E. Vazquez-Martul, of complement activation. However, excessive generation and/or E. Torreira, T. Montes, A. Tortajada, S. Pinto, M. Lopez-Trascasa, B. P. Morgan, defective clearance of these circulating C3b:plasma protein com- et al. 2010. Human C3 mutation reveals a mechanism of dense deposit disease plexes may have pathological consequences in conditions such as pathogenesis and provides insights into complement activation and regulation. J. Clin. Invest. 120: 3702–3712. the C3Gs. 16. Kim, Y. U., M. C. Carroll, D. E. Isenman, M. Nonaka, P. Pramoonjago, Currently, disease-specific treatment options for the C3Gs are J. Takeda, K. Inoue, and T. Kinoshita. 1992. Covalent binding of C3b to C4b within the classical complement pathway C5 convertase. Determination of minimal (40). On the basis of our current understanding of path- amino acid residues involved in ester linkage formation. J. Biol. Chem. 267: ogenesis, therapies that warrant consideration include drugs that 4171–4176. can prevent ongoing dysregulation of the C3 convertase or drugs 17. Whiteman, L. Y., D. B. Purkall, and S. Ruddy. 1995. Covalent linkage of C3 to properdin during complement activation. Eur. J. Immunol. 25: 1481–1484. that can potentially “mop up” the C3 breakdown products. Of in- 18. van Dam, A. P., and C. E. Hack. 1987. Formation of C3-IgG complexes in serum terest, i.v. Ig (IVIG) therapy has been used to treat autoimmune and by aggregated IgG and by non-immunoglobulin activators of complement. Im- inflammatory disorders, and IVIG is known to scavenge activated munology 61: 105–110. 19. Trujillo, G., D. M. Habiel, L. Ge, M. Ramadass, N. E. Cooke, and R. R. Kew. complement fragments, including C3b (41–43). This feature raises 2013. Neutrophil recruitment to the lung in both C5a- and CXCL1-induced the intriguing prospect that IVIG could interfere with the formation alveolitis is impaired in vitamin D-binding protein-deficient mice. J. Immunol. 191: 848–856. of C3b:plasma protein complexes, but this possibility remains to be 20. Law, S. K., N. A. Lichtenberg, and R. P. Levine. 1980. Covalent binding and investigated. In addition, a major question for future studies will be hemolytic activity of complement proteins. Proc. Natl. Acad. Sci. USA 77: 7194– Downloaded from to determine whether different C3b:protein complexes have differ- 7198. 21. Kew, R. R., and R. O. Webster. 1988. Gc-globulin (vitamin D-binding protein) ent clearance rates and associations with specific diseases. Fur- enhances the neutrophil chemotactic activity of C5a and C5a des Arg. J. Clin. thermore, the fate and function of plasma proteins with an attached Invest. 82: 364–369. C3 peptide tag remain to be investigated. We believe that further 22. Pangburn, M. K., R. D. Schreiber, and H. J. Mu¨ller-Eberhard. 1977. Human complement C3b inactivator: isolation, characterization, and demonstration of an work on characterizing C3b:plasma protein complexes is warranted, absolute requirement for the serum protein beta1H for cleavage of C3b and C4b and these complexes possibly could be useful as a marker of dys- in solution. J. Exp. 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