Human Alternative Complement Pathway: Membrane-Associated Sialic Acid Regulates the Competition between B and β1H for Cell-Bound C3b

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Abstract
The relative affinity of B and β1H for particle-bound C3b is influenced by the relative abundance of cell surface sialic acid moieties and determines whether or not the particle has the capacity to activate the human alternative complement pathway. The effect of membrane sialic acid on the binding of B and β1H to particle bound C3b was determined with radiolabeled proteins and a technique for separating bound from free ligand by centrifugation of the particles through an oil/aqueous interphase. The affinity constants at equilibrium and the effective number of C3b binding sites for B and β1H on the particles were determined by Scatchard analysis of the binding data. The sheep erythrocyte (Es) served as a nonactivating particle, whereas the activating particles included Es from which 80% of the membrane sialic acid residues had been removed by sialidase or 80 to 85% had been converted to heptulosonic acid by NaIO4 and NaBH4 treatment and zymosan particles that lack sialic acid. The affinity constants at equilibrium for C3b on EsC3b were 1 × 107 M-1 for β1H and 2.1 × 106 M-1 for B, in the presence of 5 mM Mg++, indicating an approximately 5-fold greater affinity of β1H for the binding sites that were present in approximately equal numbers for the two ligands as determined by Scatchard analysis. The affinity of B at equilibrium for C3b bound to sialic acid-deficient sheep erythrocytes and to zymosan was the same as that for C3b on Es, and the calculated number of effective binding sites for B agreed with the number of sites estimated by the prior uptake of radiolabeled C3b. In contrast, the number of C3b binding sites with an affinity for β1H of approximately 107 M-1 on sialic acid-deficient EsC3b or ZyC3b was only ⅕ to ⅙ the number interacting with B. The presence of additional binding sites for β1H with an affinity so low that they were not revealed by the Scatchard analysis because of the dominant effect of the high affinity sites was determined in competitive binding studies between B and β1H for C3b on sialic acid-deficient surfaces. The affinity constant of β1H for these sialic acid independent C3b sites is approximately 7 × 105 M-1, a Kβ1H at least 10-fold less than that of the sialic acid-dependent high affinity C3b sites. The relative inefficiency of β1H in competing with 125I-B for uptake by C3b on the sialic acid-deficient erythrocytes correlated with the decreased capacity of β1H to decay-dissociate C3b,Bb from such particles. Thus a deficiency of sialic acid diminishes the number of high affinity C3b sites for β1H and creates an activating particle on which binding of B by C3b is favored relative to β1H and decay-dissociation of C3b,Bb by β1H is impaired.

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