Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression

Abstract

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

Kinetic Analysis of the Interactions of Complement Receptor 2 (CR2, CD21) with Its Ligands C3d, iC3b, and the EBV Glycoprotein gp350/220

“…Similarly, differential expression of various epitopes on fluid-phase vs surface-bound C3 has already been proposed. Nilsson and coworkers have found that the reactivity of several mAbs recognizing the C3 ␣-chain differ when the C3 fragments are in fluid phase or surface-bound (60).…”

Control of C3b and C5b deposition by CD46 (membrane cofactor protein) after alternative but not classical complement activation

“…The following Ab were used: mouse anti-CD46 mAb MCI20.6 directed against CCP I [21,22] and GB24 directed against CCP III-IV [15], mAb anti-C3b, iC3b and C3c WM1 (European Collection of Animal Cell Cultures, no. 92021211) and 7D326 [23], mAb anti-iC3b and C3dg 7D26 [23], polyclonal rabbit anti-C3b, iC3b and C3c (L440 M. Fontaine, unpublished) and polyclonal goat anti-human C5 (Quidel, San Diego, CA). PE-labeled anti-mouse IgG(H+L) and antigoat Ig(H+L) were purchased from Immunotech (Marseille, France) and Quidel, respectively.…”

Evidence for iC3 generation during cardiopulmonary bypass as the result of blood-gas interaction

“…7D6I5.1 and 7D33I.1 (against neoepitopes expressed in C3d, in the 20-kD and 40-kD fragments of C3c. respectively) [24]. 7D323.1 (against a neocpitope in C3g) [25] and 7DI69.7 (against epitopes in the /^-chain of C3) [20].…”