Relationships between complement activation, complement binding, and EBV absorption by human hematopoietic cell lines.

Complement consumption (C.C.) and C3 deposition on the cell membrane, visualized by membrane fluorescence (CMF), were compared in a collection of established human lymphoid lines. C.C. was independent of the presence of C3 receptors. A positive CMF reaction was seen only in lines that expressed C3 receptors, however. Trypsin treatment abolished CMF and EAC rosetting but had virtually no influence on C.C. EBV absorptive capacity correlated with both C3-receptor expression, as measured by EAC rosetting, and CMF but not with C.C. This is in line with our previous finding on the association of EBV and C3 receptors.     

Volcanic ash and human complement

Volcanic ash shows slight conversion of C3 with little effect on hemolytic complement $\text{in vitro}$. No conversion of the alternative pathway component Factor B was noted. Complement activation should play only a minor role in any acute respiratory response to inhaled ash.     

Structure of human complement C8, a precursor to membrane attack

Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector. C8 initiates membrane penetration and coordinates MAC pore formation. High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly. We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density. Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible.     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.     

Diamine-induced dissociation of the first component of human complement, C1

Lysine has been shown to inhibit spontaneous and antibody-dependent C1 activation. This paper demonstrates that lysine does not prevent autoactivation of purified C1r. 20 mM lysine, 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane or 1,5-diaminopentane are able to dissociate C1 into its two entities, C1q and the calcium-dependent C1r2-C1s2 complex. Ig-ovalbumin insoluble complexes bearing C1 are also dissociated by lysine and the above-mentioned diamines used at the same concentration: C1q remains bound to the complexes whereas the C1r2-C1s2 complex is partially solubilized. The effect of lysine or diamines is not due to a competition with calcium for calcium-binding sites, as increasing concentrations of calcium even slightly increase the dissociation due to the amines. The dissociative effect is dependent on the carbon chain length of the diamines, with an optimum for 1,3-diaminopropane. It is also dependent on the relative 'cis-position' of the amino groups in the diamines. Polyamines such as spermine and spermidine are also able to dissociate C1 with even a higher efficiency than lysine and putrescine. Thus, a diamine-induced 'structural inhibition' of C1 is demonstrated, of potential interest for a pharmacological control of complement activation.     

Polymorphism of human complement component C4

An assessment has been made of the polymorphism of human complement component C4 by comparing derived amino acid sequences of cDNA and genomic DNA with limited amino acid sequences. In all, one complete and six partial sequences have been obtained from material from three individuals and include two C4A and two C4B alleles. Differences were found between the 4 alleles from 2 loci in only 15 of the 1722 amino acid residues, and 12 lie within one section of 230 residues, which in 1 allele also contains a 3-residue deletion. In three variable positions, an allelic difference in one C4 type was common to the other types. Three nucleotide differences were found in four introns. In spite of marked differences in their chemical reactivity, the many allelic forms appear to differ in less than 1% of their amino acid residue positions. This unusual pattern of polymorphism may be due to recent duplication of the C4 gene, or may have arisen by selection as a result of the biological role of C4, which interacts in the complement sequence with nine other proteins necessitating conservation of much of the surface structure.     

Phenotyping of human complement component C4, a class-III HLA antigen.

The plasma complement protein C4 is encoded at two highly polymorphic loci, A and B, within the class-III region of the major histocompatibility complex. At least 34 different polymorphic variants of human C4 have been identified, including non-expressed or 'null' alleles. The main method of identification of C4 polymorphic allotypes is separation on the basis of charge by agarose-gel electrophoresis of plasma. On staining by immunofixation with anti-C4 antibodies, each C4 type gives three major bands, but, since individuals can have up to five allotypes, the overlapping banding pattern is difficult to interpret. We show that digestion of plasma samples with carboxypeptidase B, which removes C-terminal basic amino acids, before electrophoresis, produces a single, sharp, distinct band for each allotype and allows identification of the biochemical basis of the multiple banding pattern previously observed in C4 phenotype determination.     

Omegacoeur, a Mediterranean nutritional complement, stimulates Na,K-ATPase activity in human endothelial cells.

Fatty acids are known as modulators of the vasoactive properties of the vessel wall and can influence the physical and functional properties of cell membrane. The membrane-bound enzyme Na,K-ATPase plays a central role in endothelial function such as vasoconstriction. In a previous study, we have shown that omega3 fatty acids inhibited Na,K-ATPase activity in human endothelial cells. As Mediterranean diet is known to protect from cardiovascular diseases, we have investigated the effects of Omegacoeur, a Mediterranean nutritional complement consisting of omega3, omega6, omega9 fatty acids, garlic and basil, on Na,K-ATPase activity in human endothelial cells (HUVECs). Cells were incubated for 18 hr with pure lecithin liposomes or Omegacoeur-enriched emulsions (4 mg lecithin/ml). Na,K-ATPase and 5'-nucleotidase activities were determined using coupled assay methods on microsomal fractions obtained from HUVECs. Cell fatty acid composition was evaluated by gas chromatography after extraction of lipids and fatty acids methylation. The results showed that Omegacoeur (0.1 mM) increased Na,K-ATPase activity by 40% without changes in 5'-nucleotidase activity. Cells incubated with Omegacoeur preferentially incorporated linoleic acid. Therefore, linoleic acid or others constituents of Omegacoeur could be responsible of the stimulation of the Na,K-ATPase activity that might be related to changes in endothelial membrane fluidity.     

Genetic variability of complement receptor on human erythrocytes

Erythrocytes from healthy men were examined for the presence of complement (C3b) receptor using haemagglutination assay with human aggregated IgG (aggIgG) and guinea pig complement. The results were expressed as the intensity of haemagglutination that corresponded to the C3b receptor sites density as evidenced by radioimmunobinding results. Among normal men three phenotypes of complement receptor (CR) were distinguished: high (CR^h/CR^h) phenotype corresponding to strong agglutination, an intermediate (CR^h/CR^I) producing weak agglutination and low phenotype (CR^I/CR^I) that gave no agglutination. In a population of 517 normal men these three phenotypes occurred in 63.8, 30.6 and 5.6%, respectively. Frequencies of the genes responsible for high (CR^h) and low (CR^I) expression of erythrocyte C3b receptor were 0.791 and 0.209, respectively.     

Membranolysis by the ninth component of human complement

The lytic property of isolated C9 was shown by using heat (48°C) to induce polymerization of C9, which was then exposed to egg lecithin vesicles containing carboxyfluorescein. In this environment, C9 penetrated the vesicles and released the marker. C5b-9 and C5b-8 also released carboxyfluorescein at 48°C, as did C5b-7 to a lesser extent. However, neither isolated C5b-6, C7, C8 nor albumin caused such release. At 30°C, the C9 remained in the monomeric form and could not lyse the vesicles.C9 polymers formed at 48°C were ring-shaped with an internal diameter of approximately 100 Å and an outer diameter of approximately 180 Å. These rings of C9 polymers strikingly resembled the ultrastructures formed by the membrane attack complex of complement, viewed from the top.Our results indicate that polymeric C9 causes the membranolysis of phospholipid vesicles and, hence, that C9 alone is a cytotoxic molecule.     

Polymorphism of the third component of human complement

Genetic polymorphism of the third component of human complement (C3) has been considered as a powerful marker for population genetics. Some studies on the distribution of gene frequencies have been performed among numerous populations all over the world. This review takes stock of population genetic studies reported up to now and points out some remarks on the distribution of the observed allelic frequencies.     

The human histone H3 complement anno 2011

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.