Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies.

Abstract

The reaction mechanisms of C3 are central to both the classical and alternative pathways of complement activation and have in consequence been extensively studied (see 1 and 2 for reviews). The activation of C3 by cleavage to C3b is brought about by the C3-converting enzymes of the classical and alternative pathways, as well as by a number ofnoncomplement enzymes. C3b is subsequently inactivated and prevented from further participation in the C3b feedback cycle and in the hemolytic reaction by cleavage to C3bi by factor I, which splits C3b when the latter is combined with factor H. C3bi is the C3 conversion product that appears in serum in vitro after complement activation by either pathway, and it is fairly clear that the C3 product, which was known as "beta 1A" in much of the earlier complement literature, is C3bi and not C3c, the fragment with which beta 1A has usually been identified (3). C3bi does, however, undergo further breakdown both in serum and when cell bound. It was originally shown by Lachmann and Mi.iller-Eberhard (4) that the conglutinable form of C3 bound on cells (cell-bound C3bi) could be cleaved by 1/~g/ml of trypsin to lose its conglutinability. This was accompanied by the elution of C3c from the cell, C3d remaining bound. In serum, too, C3 gave rise, on aging, to two fragments, one of which was described as beta 1A and the other, a fast-migrating fragment, as alpha 2D (5). Alpha 2D was generally equated with C3d (3). As will be pointed out in this paper, this identification is not wholly correct.Antigenic analysis with polyclonal antisera to C3 had shown the existence of distinct antigenic specificities in C3a, C3c, and C3d, as well as antigenic determinants found only in native C3 (5, 6). Studies with monoclonal anti-C3 antibodies (7) showed one that reacted with C3c, one with C3d, and one that showed an unusual pattern of reactivity that has now been found to react with the fragment here called C3g. By the use of the monoclonal antibodies, the breakdown of C3bi both on cells and in plasma has been reinvestigated, and the results of this investigation are presented herein. Materials and MethodsMonoclonal Anti-C3 Antibodies. The three monoclonal antibodies are described as clone 3, clone 4, and clone 9 and have been fully described (7). Unless stated to the contrary, ascites from tumor-bearing rats was used as the source of monoclonal antibodies without further purification.

Solvent Accessibility of Native and Hydrolyzed Human Complement Protein 3 Analyzed by Hydrogen/Deuterium Exchange and Mass Spectrometry

“…Several studies have indicated that C3 undergoes conformational changes upon its conversion to C3(H 2 O), but the regions involved in these changes are largely unknown (1,3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Using HDX and mass spectrometry, we have localized changes in solvent accessibility between native C3 and C3(H 2 O).…”

Structural insights into the central complement component C3

“…Nevertheless the C345C domain is exposed in both C3 and C3c, which suggests that it is also exposed in C3b and thus accessible for factor B binding. The segment formed by residues 933-942 (O' Keefe et al, 1988) on CUB is exposed in C3 and proteolytically removed by factor I in C3c (Lachmann et al, 1982). In the structure of C3 it is located close to the C345C domain and possibly even after conformational changes factor B can bind simultaneously to both regions.…”

“…A second cleavage occurs between Arg-1298 and Ser-1299, in strand ␤8 of CUB f generating C3f (2 kDa) and iC3b 2 (Davis and Harrison, 1982;Harrison and Lachmann, 1980). Third, factor I cleaves between Arg-932 and Ser-933 in loop ␤3'-␤4 of the CUB g part, resulting in the formation of C3dg (40 kDa) and C3c (135 kDa) (Lachmann et al, 1982). This indicates that factor I might bind in various ways to the CUB domain assisted by one of the cofactors which might be involved in the unraveling of the CUB domain enabling factor I to cleave.…”

Characterization of the complement sensitivity of paroxysmal nocturnal hemoglobinuria erythrocytes.

“…It has been reported that inactivation of cell-bound C3b is impaired on PNH erythrocytes, due to deficient factor I cofactor activity on the abnormal cells (8). For human erythrocytes, CR1 serves as the cofactor for the enzymatic cleavage of cell-bound C3b to iC3b and C3dg by factor I (44)(45)(46)(47). The number of CR1 sites on PNH III cells was well within the normal range, whereas the PNH II and CCAD erythrocytes had extremely low numbers of CRI sites (Table I).…”