Heterogeneity, Polypeptide Chain Composition and Antigenic Reactivity of C3 Nephritic Factor

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Abstract
C3 nephritic factor (C3NeF), recognized by its capacity to stabilize the cell-bound amplification C3 convertase, C3b,Bb, was purified from sera of three patients with hypocomplementemic glomerulonephritis and of two patients with partial lipodystrophy by QAE-A50 Sephadex and SP C-25 Sephadex chromatography, affinity for the fluid phase amplification C3 convertase, and QAE-A50 Sephadex chromatography. Each C3NeF preparation exhibited heterogeneity during cation exchange chromatography and the isoelectric points of the eluted fractions ranged from pI 8.3 to 8.9. The chromatographic fractions were interacted with purified B, D̄ and C3 to form fluid phase C3b,Bb(C3NeF) which sedimented as a 10S complex on sucrose density gradient ultracentrifugation; the isolated convertase was decayed with release of C3NeF, which was separated from C3b and Bi by anion exchange chromatography. Purified preparations of C3NeF radiolabeled with 125I were bound from 92 to 98% by 109 erythrocytes bearing C3b,Bb whereas erythrocytes carrying C3b bound from 0.6 to 18% and EA engaged in no specific uptake. Analysis of all 125I-C3NeF preparations by SDS-PAGE demonstrated an apparent m.w. of 150,000. After reduction in the presence of 8 M urea each 125I-C3NeF preparation revealed polypeptide chains of 54,000 and 23,500 m.w. which corresponded with the positions of the heavy and light chains of reduced IgG. The reaction of 125I-C3NeF from four patients was positive with Sepharose-bound antisera to IgG, γ1, γ2, κ and λ and negative with antisera to µ, α, δ, γ3 and γ4, while C3NeF from the fifth patient differed in not reacting with antiserum to κ. These studies indicate that C3NeF is an autoantibody directed against the antigens expressed by the amplification C3 convertase, C3b,Bb.