Properdin : Initiation of alternative complement pathway ( properdin pathway / C 3 convertases ) DOUGLAS T . FEARON

Activation of the classical complement (C) system involves conversion of C1 to its active state with subsequent cleavae of C4 and C2 so as to form the classical C3 convertase, C42 (a bar indicates the activated form of a protein), which sequentially cleaves C3 and CS to initiate the cytolytic event associated with the complete reaction. An alternative, or properdin-dependent, pathway to complement activation generates a C3 convertase, CB, that is formed by cleavage of B with D in the presence of C3b, the major cleavage fragment of C3. C3b is capable of binding activated properidin (P) with resultant stabilization of CM, which otherwise rapidly decays by loss of B activity. Initial cleavage of C3, a prerequisite for formation of C3B, is demonstrated to occur through the interaction of native C3 and B in the presence of either Dor P alone, or together. The effect of P on the interaction of D, B, and C3 is attributed to stabilization of C3B as has been shown for C3B. Larger amounts of P and B with C3 in the absence of D form a C3 convertase that is designated (P)C3B to indicate that demonstrable cleavage of B does not occur although the active site is available. The generation of this initial convertase, as assessed by C3 inactivation, is dose-related to P and B inputs. The presence of both P and D greatly augments initial cleavage of C3 with D fully uncovering the active site of B and FP stabilizing that site. Activation of the classical complement system by an antigen-antibody complex containing immunoglobulin of the appropriate class involves conversion of C1 from its precursor form to its active state, C1 (8), with subsequent cleavage of C4 (9) and C2 so as to form the classical C3 convertase, C42 (10). The latter sequentially cleaves CS and C5, thereby initiating formation of the terminal C56789 complex (11) Abbreviations: ACD plasma, acid-citrate-dextrose plasma; VBS, isotonic Veronal-buffered saline; GVB++, VBS containing 5 X 10-4 M magnesium, 1.5 X 10-4 M calcium, and 0.1% gelatin; DGVB++, half-isotonic GVB++ with 2.5% dextrose; EDTA, ethylenediaminetetraacetate; GVB-EDTA, GVB++ from which cations were omitted and containing 0.04 M EDTA. Nomenclature: The nomenclature for the classical complement (C) components conforms to that agreed upon under the auspices of the World Health Organization (1968) Bull. W.H.O. 39, 395. The nomenclature for alternative pathway factors is that provisionally adopted at the Second International Congress of Immunology, 1974, Brighton, United Kingdom. B (1) is the symbol for a protein that has been termed C3 proactivator (2) and glycine-rich beta glycoprotein (GBG) (3); D (4, 5) is the symbol for the protein that has been termed precursor CS proactivator convertase (6); and P represents properdin (7). A bar over a letter indicates the activated form of a protein. The symbol over a letter is introduced to indicate activation of a protein without demonstrable cleavage. With respect to fragmentation, the larger fragment is designated by the suffix b and the lesser fragment by a. hu indicates human origin; SP, guinea pig; OXY indicates the oxidized form of C2. EA is a sheep erythrocyte sensitized with rabbit antibody. which mediates the cytolytic effect of the complement reaction. An alternative pathway to complement activation was described some 20 years ago as consisting of a group of serum factors which interacted with complex microbial polysaccharides to form a C3-cleaving enzyme distinct from CG2 (7). An alternative C3 convertase, C3B, was subsequently characterized as being formed by the interaction of C3b, the major cleavage fragment of C3, with B and D, two plasma proteins of the alternative pathway (6). D, a 25,000 molecular weight protein (4) whose active site is of the serine esterase class (5), cleaves B in the presence of C3b to form the labile bimolecular complex, C 9, which is similar to C42 in its capacity to activate C3-C9 (12). CSB decays by loss of B activity, which limits its potential for cytolytic activity, but can be regenerated by the action of D on additional B (12). Activated properdin, P, a gamma globulin of 223,000 daltons (13) with an isoelectric point of greater than 9.5, binds to C3b and retards decay of convertase function, thereby profoundly augmenting activation of C3-C9 by CSB (14). Formation of C3B requires the presence of C3b, indicating that an additional C3 convertase must be formed during initial activation of the alternative pathway. The finding that the introduction of P to a mixture of C3, B and D results in C3 cleavage (15) does not distinguish between a contribution to generation of the initial C3 convertase or a stabilizing effect on the C3B subsequently formed. Although interaction of C3, B, and D is sufficient to result in initial C3 cleavage, B or D can be limited to the extent that C3 cleavage is not sustained unless P is present. The further finding that the interaction of C3 with larger amounts of P and B in the absence of D leads to C3 cleavage without B cleavage establishes a role for P in the formation of an initial convertase of the alternative pathway distinct from C3B. MATERIALS AND METHODS Preparation of Alternative Pathway Factors and Classical Complement Components from Human Plasma. B (4) and D (5, 14) were purified to homogeneity so that each preparation presented a single band on alkaline disc gel electrophoresis corresponding to the position in a replicate, unstained gel from which activity was eluted. B was measured by radial immunodiffusion (4) and D by Folin analysis (16) with human serum albumin as the reference standard. P. purified from the pH 6.0 euglobulin of fresh acid-citratedextrose (ACD) plasma by sequential chromatography on quaternary aminoethyl Sephadex (Pharmacia Fine Chemicals, Piscataway, N.J.), sulfopropyl Sephadex, and Sephadex G-200, yielded a single band on acid disc gel electrophoresis (17). On Ouchterlony analysis, P presented no precipitin