Complement C3 Convertase: Cell Surface Restriction Offi1h Control and Generation of Restriction on Neuraminidase-Treated Cells
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Proc. Natl. Acad. Sci. USA Vol. 75, No. 5, pp. 2416-2420, May 1978 Immunology Complement C3 convertase: Cell surface restriction of fi1H control and generation of restriction on neuraminidase-treated cells (alternative pathway/properdin/cell membranes/protein-protein interactions/C3b inactivator) MICHAEL K. PANGBURN AND HANS J. MOLLER-EBERHARD Department of Molecular Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037 Contributed by Hans J. Muiller-Eberhard, February 27,1978 ABSTRACT The alternative or properdin pathway of The purpose of this communication is to show that restricted complement is primarily controlled by the endopeptidase C3b control of CS convertase is entirely due to reduced accessibility inactivator (C3bINA) and the nonproteolytic glycoprotein j1H. The molecular mechanisms of control were investigated by of ,B1H to surface-bound C3b, and to report an example of performing binding studies of radiolabeled complement pro- generation of restriction by enzymatic modification of the outer teins to C3b bearing sheep erythrocytes (EsC~b) C3b was found membrane of intact cells with neuramninidase (acylneuraminyl to have distinct binding sites for P1H, C3bINA, Factor B, and hydrolase; EC 3.2.1.18). The neuraminidase-treated cells also pro erdin. jB1H binding increased C3bINA binding 30-fold, acquired the ability to activate the alternative pathway. These while Factor B binding prevented C3bINA action on C3b and studies were presented in part at the Seventh International was competitive with P1H binding. Properdin binding, which facilitates Factor B interaction with C3b, had no effect on the Complement Workshop (1977), St. Petersburg Beach, FL (7). #1H and C3bINA sites. Activators such as rabbit erythrocytes (ER) have previously been shown to interfere with the effec- MATERIALS AND METHODS tiveness of the control by C3bINA and P1H, thereby allowing unrestricted formation of C3 convertase. Such restriction of Preparation of Alternative Pathway Components. Highly control does not occur on the surface of Es, a nonactivator of the purified human CS (8), Factor B (9), Factor D (10), activated alternative pathway. On the basis of comparative binding properdin (P) (10), C3bINA, and j1H (4) were isolated as de- studies, restriction of control is explained entirely by reduced binding of P1H to ERC3b relative to EsC3b. Access of properdin, scribed. (31H, C3bINA, Factor B, P, and anti-C3 Fab were ra- Factor B, C3bINA, and the Fab fragment of anti-C3 to the two diolabeled by using insolubilized lactoperoxidase at 00 (11) or cell types was unrestricted. Restriction of j1H control could be by the Bolton-Hunter method (New England Nuclear, Gar- generated on the surface of Es by removal of cell-surface sialic dena, CA) (12). These proteins were labeled to specific activities acid with neuraminidase (acylneuraminyl hydrolase; EC ranging between 0.4 and 1.5 Ci/g. The IgG fraction of a 3±2.1.18) This enzymatic treatment converted Es from a non- monospecific rabbit anti-CS was used to prepare anti-CS Fab activator to an activator of the alternative pathway. (13). The alternative pathway C3 convertase (C3bb) is formed by Buffers and Reagents. The following buffers were used (14): the proteolytic action of Factor D on the bimolecular complex VB, veronal-buffered saline, pH 7.2; VB2+, VB containing 0.15 consisting of C3b and Factor B (CMb,B). Upon cleavage of mM CaCl2 and 0.5 mM MgCl2; GVB2+, VB2+ containing 0.1% Factor B, the Ba fragment is dissociated, while the active-site- gelatin; Mg-GVB, VB containing 0.5 mM MgCl2 and 0.1% bearing Bb fragment is retained in the complex. CS convertase gelatin; GVBE, GVB2+ containing 10 mM EDTA; DGVB2+, into CMa and C&b. one-half diluted VB2+ containing 0.1% gelatin, 2.5% (wt/vol) cleaves its substrate, native CS, the fragments dextrose, and 0.01% sodium azide. C4-depleted normal human A C3b molecule thus produced can combine with a molecule serum was prepared by passing serum containing 20mM EDTA of Factor B and, with the aid of Factor D, form a second mol- over an immune adsorbent column prepared by coupling the ecule of CS convertase (1). As this process of positive feedback IgG fraction of a monospecific goat antiserum to human C4 to continues, the originally small signal provided by the first C3b CNBr-activated Sepharose 4B according to the manufacturers molecule is greatly amplified. directions (Pharmacia Fine Chemicals, Inc., Piscataway, NJ). Since C3b can anchor itself firmly on the surface of particles, was concentrated to its including cells, the entire process of amplification can take place The eluting protein pooled, original molecules volume, and dialyzed exhaustively against VB. This C4-de- on surfaces. As the multiplicity of surface-bound CMb pleted serum contained no detectable C4 and did not lyse he- increases, immune adherence reactivity and C5 convertase molysin-sensitized sheep erythrocytes (Es), indicating an in- activity are generated. Through the action of C5 convertase on operative classical pathway. It contained normal levels of CS, C5, complement-dependent cytolysis is initiated. Factor B, P, (31H, and C3bINA, as assessed by radial immu- Regulation of the amplification phase of the alternative nodiffusion. pathway is exerted by multiple mechanisms: (i) intrinsic decay Preparation of EC3b and Neuraminidase-Treated EC3b. of CS convertase, (Hi) stabilization of this enzyme by properdin, EC3b cells were prepared by incubating 1010 erythrocytes with (iii) disassembly of this enzyme by the serum glycoprotein ,B1H 5 mg of purified CS and 50 ,ug of trypsin in 1 ml of GVB2+ for (2, 3), (iv) inactivation of C3b by the CMb inactivator (CMbINA) cells were washed the ac- 3 min at 37°. The resulting C3b-bearing and ,B1H (4), and (v) protection of C3 convertase from thoroughly in cold Mg-GVB and stored at 00 in DGVB2+. EC3b tion of these control proteins afforded by the surface properties cells possessing varying amounts of C3b per cell were prepared of certain cells and other activators of the alternative pathway by incubating 1010 EC3b cells prepared with trypsin with 100 (5, 6). ,ug of Factor B, 2 Mg of Factor D, and excess partially purified The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Abbreviations: CMbINA, C3b inactivator; VB, veronal-buffered saline; "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate P. properdin; P. activated properdin; ER, rabbit erythrocytes; ES, sheep this fact. erythrocytes. 2416 Downloaded by guest on September 28, 2021 Immunology: Pangburn and Mfiller-Eberhard Proc. Natl. Acad. Sci. USA 75 (1978) 2417 60 120 50' 100- , 40 0 V .0 'D 0 I 30 .0 C0?. I 20- UP0 44 )00 50,000 75,000 100,000 Number of C3b per cell FIG. 1. Dependence of jT1H binding on the number of C3b mol- ecules per cell. Bolton-Hunter labeled 1256-#lH (0.4 Ci/g) was incu- 1i2 4 8 bated at 0.8 Mg/ml with 5 X 107 Es bearing various numbers of C3b 1gg P or B added on their surface in a final volume of 100 Al of DGVB2+. The cells plus FIG. 2. Competition between P, Factor B, and #1H for binding bound #1H were then centrifuged through 20% sucrose. The amount to cell-bound C3b. EsC3b cells (5 X 107) were added to a solution of C3b per cell was determined by the amount ofpurified, soluble C3b of Fab (DGVB2+) containing the indicated amounts ofP or Factor B and 0.08 required to inhibit by 50% the binding 125I-anti-C3 fragment ,ug of 125I-ft1H in a final volume of 100 Ml. The reaction mixtures were to the EC3b cells. This number was consistently higher than the es- allowed to reach equilibrium at room temperature and the cells were timate of C3b deposited calculated from 125I-C3 uptake. centrifuged through 20% sucrose. The percent bound was calculated by taking as 100% the proportion of #1H bound in the absence of P nephritic factor (15) in 1 ml of Mg-GVB for 10 min at 37'. The or Factor B (54% ofthe total 125I-01H added). A, EC3b + P; 0, EC3b washed cells possessing the nephritic-factor-stabilized C3 + Factor B; 0, EC3b P + Factor B. convertase on their surface were incubated with varying amounts of purified C3 in 1 ml of Mg-GVB for 2 hr at 37°. After conditions that cleave and inactivate C3b, indicating that #I1H overnight decay of the nephritic-factor-stabilized enzyme at cannot bind to inactivated C3b (CMbj). Prior treatment of EC~b 00 in GVBE, the cells were washed and stored in DGVB2+. In with #l1H alone had no effect on subsequent 125I-fl1H binding. some experiments a small amount of 125I-labeled C3 was added Similar results of ,B1H binding studies were obtained inde- to unlabeled C3 in order to compare the amount of C3b uptake pendently by Ruddy et al. (17). by sheep and rabbit erythrocytes. #1H accelerates the rate of dissociation of Bb from EC3b,Bb Neuraminidase (Behring Diagnostics, Somerville, NJ) as well as from the properdin-stabilized CS convertase treatment was accomplished by suspending cells in 50 mM EC3b,P,Bb (2, 3). Since Bb cannot rebind once dissociated, sodium acetate/0.9% NaCl/0. 1% CaC12, pH 5.5, containing 10 competition studies could not be performed with the active units of neuraminidase per ml. The reaction was complete after enzyme. Therefore, the nonactivated complex was used, which 60 min at 37°. The treated cells were washed and stored in is formed by the interaction of EC3b and native Factor B in the DGVB2+. Sialic acid determinations on supernatants and cells absence of Factor D (18).