X-ray crystal structure of C3d: a C3 fragment and ligand for complement receptor 2.

Activation and covalent attachment of complement component C3 to pathogens is the key step in complement-mediated host defense. Additionally, the antigen-bound C3d fragment interacts with complement receptor 2 (CR2; also known as CD21) on B cells and thereby contributes to the initiation of an acquired humoral response. The x-ray crystal structure of human C3d solved at 2.0 angstroms resolution reveals an alpha-alpha barrel with the residues responsible for thioester formation and covalent attachment at one end and an acidic pocket at the other. The structure supports a model whereby the transition of native C3 to its functionally active state involves the disruption of a complementary domain interface and provides insight into the basis for the interaction between C3d and CR2.

Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity.

It has long been known that mouse C4 has unusually low hemolytic activity relative to the C4 of other mammalian species (e.g. human and guinea pig), the measurements being done in most cases using a C4-deficient guinea pig serum reagent in a one-step assay with EA. This low activity for mouse C4 previously had been attributed to "technical" difficulties such as lability of the protein during blood collection and partial species incompatibilities with guinea pig components. Recently, we presented evidence for the involvement of human C4 beta-chain residues 455-469, a putatively exposed hydrophilic segment, in contributing to a C5 binding site in the C4b subunit of the classical pathway C5 convertase, C4b3b2a. Given that there were five sequence differences between the human and mouse protein within this segment, we hypothesized that these substitutions may have compromised the C5 convertase subunit activity of mouse C4, thereby resulting in its low hemolytic activity. Using a multi-step hemolytic assay which was totally dependent upon C5 cleavage by the classical pathway, we found that mouse C4 was completely devoid of classical pathway C5 convertase subunit activity. We have been able to rule out the most obvious potential species incompatibilities (e.g. between C4mo and C5gp) as being responsible for this lack of activity. Moreover, we found that the low level of hemolytic activity of mouse C4 measured in the one-step assay can be ascribed totally to C5 cleavage, and subsequent terminal component assembly, by the alternative pathway C5 convertase, (C3b)2Bb. However, the assembly of the latter enzyme complex is dependent upon the presence of C3b molecules deposited initially via the classical pathway C3 convertase in which mouse C4b is a subunit. Finally, whereas conversion of human residues 458RP to the mouse-like sequence PL was sufficient to abrogate classical pathway C5 convertase subunit activity in human C4, the five substitutions which "humanized" the 452-466 segment of mouse C4 (corresponding to human residues 455-469) were on their own insufficient to impart this activity to mouse C4. This implies that, in addition to the 455-469 beta-chain segment of human C4, there are other regions of the molecule contributing to C5 binding which are also non-conserved between human and mouse C4.

A reexamination of the role of magnesium in the human alternative pathway of complement.

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.

Biosynthesis of the third component of complement by the human monocyte-like cell line, U-937.

The human monocyte-like cell line, U-937, was shown to synthesize and secrete C3 by hemolytic assays, radioimmunoassays and metabolic labeling experiments. The daily synthesis of antigenic C3 by unstimulated U-937 cells was low (about 3 ng/10(6) cells/24 hr) over a 3 day period. Induction of the cells to differentiate into macrophage-like cells with phorbol myristate acetate (PMA), resulted in 5-fold augmentation of C3 synthesis and secretion into the culture medium. Using a plaque assay for enumerating C3 production by single cells, approx. 5% of unstimulated U-937 cells were found to secrete hemolytically active C3. The proportion of C3-plaque forming cells was increased about 6-fold in PMA stimulated cells. The synthesis of C3 by U-937 cells was reversibly inhibited by cycloheximide. Data from SDS-PAGE analyses showed that U-937 cells synthesized C3 as a precursor polypeptide chain and was capable of processing this pro-molecule into the secreted two chain form. C3 antigen immunoprecipitated from stimulated U-937 cell lysates showed an increased amount of low mol. wt material as compared to C3 antigen immunoprecipitated from the lysates of unstimulated cells. This may be attributable to increased intracellular proteolytic activity in the PMA stimulated cells. The studies show that the U-937 cell line provides a useful model for studies on the synthesis and processing of complement proteins and the physiological regulation of complement production.

Processing of human factor I in COS-1 cells co-transfected with factor I and paired basic amino acid cleaving enzyme (PACE) cDNA.

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed form of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.

Structural basis for the preferential association of autologous immunoglobulin subunits: role of the J region of the light chain.

We have used a series of sequenced V kappa 21 L-chains produced from murine myelomas to determine whether the V or J segment of the V region is responsible for dictating preferential recombination. In each competitive recombination, equimolar amounts of two different L-chains, selected on the basis of common V or J segments, were allowed to compete for a limiting amount of H-chain. It was found that the J segment of the L-chain was primarily responsible for dictating the ability of a chain to compete and that the nature of residue 96, the first residue of the J segment, was particularly important. Specifically, charged residues caused the L-chain to compete poorly against L-chains with hydrophobic side chains at this position. Furthermore, if Phe or, to a lesser extent, Tyr were present at position 96, the L-chain competed more successfully than chains with Trp-96 or Leu-96. This suggests that both the aromaticity and size of this residue were important factors in determining preferential recombination. It was also found that all other residues in VL were secondary to residue 96 in contributing to the ability of a chain to compete. Finally, unlike all previous studies, we observed a substantial number (64%) of preferred heterologous recombinations. These results are consistent with the hypothesis that the VL and VH gene rearrangements occur independently, thus resulting in random pairing of VH and VL domains.

The role of the thioester bond in C3 and C4 in the determination of the conformational and functional states of the molecule.

The numerous ligand binding properties expressed by the activated forms of C3 (C3b) and C4 (C4b) are best explained as arising from proteolytic-cleavage-induced conformational changes. The studies described above provide direct physical evidence for such conformational transitions in both complement proteins. Significantly, however, a virtually identical conformational end state was also produced by direct nucleophilic scission of the internal thioester bond in C3 and C4 in the absence of any proteolysis. Clearly, it is the integrity of this thiolactone structure that is the determining factor in maintaining the native conformation in C3 and C4. Recent studies suggest a similar conformational role for the thioester in alpha 2-macroglobulin. Proteolytic activation in all three thioester-containing molecules renders this structure more susceptible to nucleophilic or solvolytic attack. Whether this effect is mediated by a peptide-cleavage-induced increase in the electrophilicity of the reactive carbonyl, or simply by increasing the accessibility of the solvent to this structure, is as yet unknown. In the case of C3 and C4, removal of the activation peptide also has a profound effect on the kinetics of the conformational change initiated by the loss of the thioester. This kinetic constraint has made it possible to correlate the acquisition of ligand binding properties with the spectroscopically detectable conformational changes.

Philosophical assumptions in Freud, Jung and Bion: questions of causality.

The historical development of concepts of causality in philosophy is described. Since the Enlightenment and the growth of science, exponents of the two most important concepts, determinism and teleology, have been in conflict. At the inception of psychoanalysis at the end of the nineteenth century this conflict was particularly intense. It was the cause of the first major schism in psychoanalysis between Jung and Freud. Psychoanalytic theorists have continued to disagree over this issue. Post-modernist philosophy has abolished all metaphysics and therefore called into question concepts of psychic causality. Parallel to, but uninfluenced by this development, Bion has developed a psychoanalytic conceptualization which may be seen as transcending causality. The clinical and theoretical implications of these developments are described.