William P. Kolb

Ba and Bb fragments of factor B activation: fragment production, biological activities, neoepitope expression and quantitation in clinical samples.

Factor B is a centrally important component of the alternative complement pathway. Alternative pathway activation results in factor B cleavage and production of the amino-terminal Ba and the carboxyl-terminal Bb fragments which have molecular weights of approximately 30,000 and 63,000 daltons, respectively. Both Ba and Bb fragments have been reported to express a variety of biological activities in vitro. Thus, binding of Ba and Bb fragments to specific B lymphocyte surface receptors modulates proliferation of prestimulated B cells. In addition, the enzymatically active Bb fragment induces activation and spreading of human and murine macrophages and monocytes as well as regulates C5a des Arg chemotactic activity. The fractional catabolic rate and metabolism of factor B in vivo is similar to that of C3, C4 and C5 complement proteins, which are among the most metabolically active plasma proteins in the circulatory system. Factor B hyperconsumption and increased catabolism, concomitant with factor B fragment production, occurs in a wide variety of diseases, including gram-negative sepsis, autoimmune diseases and burns. Measurement of alternative pathway activation in vivo has been attempted utilized a number of different techniques to quantitate factor B fragments in biological fluids. However, the recent development of enzyme immunoassays (EIA) employing monoclonal antibodies (MoAbs) reactive with factor B fragment neoepitopes provides the best approach currently available for the quantitation of factor B activation fragments. Results obtained using these new MoAb-based EIAs have indicated that factor B fragment concentrations were elevated, as compared with normal donor levels, in EDTA plasma samples obtained from patients with rheumatoid arthritis and systemic lupus erythematosus (SLE). Plasma concentrations of factor B fragments, especially Ba fragment levels, in these patients showed a positive correlation with disease activity scores. One of the highest disease activity correlations was obtained with Ba fragment measurements in SLE plasma samples. In fact, the results strongly suggested that quantitation of Ba fragment levels in SLE plasma samples more accurately reflected disease activity and was a more sensitive predictor of impending flare in these patients than any other test(s) currently available.

Activation of the fifth component of human complement (C5) induced by monosodium urate crystals: C5 convertase assembly on the crystal surface

Abstract Incubation of monosodium urate crystals (MSUC) with normal human serum (NHS) resulted in the production of a factor chemotactic for neutrophils which was inactivated by monospecific antiserum to human C5, indicating it to be a product of C5 activation. When MSUC were incubated with NHS containing radioiodinated C5, a significant portion of the label was incorporated into a 1 × 10 6 MW, fluid phase complex identified as SC5b-9. The complex had a sedimentation velocity of 23.5 S and contained C5b as demonstrated by radioautography of reduced SDS-polyacrylamide electrophoretic slab gels. In addition, urate crystals preincubated with C5-deficient human serum, followed by extensive washing, were able to convert highly purified C5 to C5b. While purified C5 readily adsorbed to urate crystals, it was not activated to C5b in the absence of a fully functional complement cascade. These data provide direct evidence for the assembly of a complement C5 convertase enzyme on the surface of MSUC during incubation of crystals with human serum. The convertase then mediates C5 activation to C5a and C5b with resultant assembly of the fluid phase SC5b-9 complex. These observations further clarify the nature of MSUC-complement interactions and provide a mechanism for complement-mediated neutrophil chemotaxis in gout.

Physicochemical characterization of fluid phase (SC5b-9) and membrane derived (MC5b-9) attack complexes of human complement purified by immunoadsorbent affinity chromatography or selective detergent extraction

Abstract Immunoadsorbent affinity chromatography, employing a monospecific caprine anti-human C5 (IgG)-agarose bead matrix, was applied to the purification of complement attack complexes from insulin activated normal human serum (SC5b-9). Gel filtration chromatography (Biogel A-15M) of the SC5b-9 complexes eluted from the anti-C5 column by guanidine-HCl was utilized as a second step to obtain a highly purified SC5b-9 preparation. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoretic (SDS-PAGE) analysis of SC5b-9 isolated by immunoadsorbent chromatography revealed the appropriate subunit composition. Complement attack complexes extracted from complement lysed erythrocyte membranes (MC5b-9) upon solubilization with either Triton X-100 (TX-100) or deoxycholate (DOC) were also purified by anti-C5 immunoadsorbent and gel filtration chromatography, but with somewhat less than satisfactory results. Thus, an alternative purification procedure was developed based upon the ability of zwitterionic detergents to selectively extract MC5b-9 from erythrocyte membranes. Zwitterionic detergent solubilized MC5b-9 complexes were isolated in high yield in a one-step purification procedure by gel filtration column chromatography (Biogel A-15M). SDS-PAGE analysis of MC5b-9 isolated in this manner revealed a subunit composition which was similar to SC5b-9 except for the complete absence of S protein. Sucrose density gradient ultracentrifugal analysis of MC5b-9 complexes solubilized by TX-100, DOC, or zwitterionic detergents revealed similar sedimentation profiles with the major portion of MC5b-9 expressing a sedimentation coefficient of29S with minor peaks of 23S and 34S or greater. Thus MC5b-9 complexes solubilized from biological membranes by nonionic, anionic, or zwitterionic detergents form a heterogeneous population of high molecular weight, ordered, oligomeric structures with the 23S form representing MC5b-9 monomer, 29S the MC5b-9 dimer and 34S the MC5b-9 trimer or possibly tetrameric structure. These results clearly suggest a possible relationship between the observed MC5b-9 complex physical heterogeneity and the heterogeneity in the effective size of functional complement lesions.

Immunoadsorbent affinity purification of the fifth component (C5) of human complement and development of a highly sensitive hemolytic assay

The fifth component of complement (C5) has been isolated from human serum in fully hemolytically active form by immunoadsorbent and anion exchange column chromatography. The immunoadsorbent column was prepared by the covalent coupling of the purified IgG fraction obtained from monospecific goat anti-human C5 antiserum to CNBr activated Sepharose 4B. Establishment of appropriate conditions for the dissociation and elution of functionally active C5 from the immunoadsorbent column was of central importance in the development of this purification procedure. The C5 preparations exhibited final yields of 20--50% with 570--710-fold purification factors based on recovery of specific hemolytic activity. These preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide slab gel electrophoretic and immunochemical criteria. A C5-depleted reagent (C5D) was generated from the non-adsorbed protein containing fractions obtained subsequent to the passage of freshly drawn NHS plus 10 mM EDTA through the monospecific anti-C5 Sepharose 4B column. Upon reconstitution of C5D with Ca2+, Mg2+, and C1q, this reagent was utilized for the detection and quantitation of C5 hemolytic activity. The purified C5 preparations contained 1.5--2.5 x 10(12) effective molecules/mg protein and NHS expressed 0.5--2.0 x 10(11) effective molecules/ml.

Fluorometric detection of low temperature thermal transitions in the Clq component of human complement

Abstract Fluorescence studies with the human complement component Clq were performed as a function of temperature and demonstrated the existence of low temperature, thermally induced structural transitions in the Clq molecule. Both intrinsic protein fluorescence and the fluorescence of the apolar probe 2-p-toluidinylnaphthalene-6-sulfonate independently showed thermal transitions at 15°C, 35°C and 48°C. Clq activity measurements indicated no loss of hemolytic activity at temperatures below 46°C. It is proposed that these structural transitions are a consequence of the internal flexibility of the native Clq molecule.