Interaction of S-sulfonated human IgG with human complement and its components.

S-sulfonated human IgG (S-sIgG) was prepared by treating IgG with sodium sulfite and sodium tetrathionate. The treatment resulted in the selective cleavage of interchain disulfide bonds of the IgG to give S-sulfonate groups. Complement fixing activities of aggregated S-sIgG and the immune complex formed with the S-sIgG antibody were very weak. S-sIgG at a high dose reduced the activity of the first complement component (C1) in normal human serum without any reduction of other complement components activites, but S-alkylated IgG at the same dose did not. Loss of C1 activity was not caused by either S-sulfonated myeloma proteins (IgA and IgE) or urea-treated S-sIgG, in which both inter- and intra-chain disulfide bonds were cleaved. These results suggest that the selective reduction of C1 by S-sIgG is due to a conformational change of the immunoglobulin.     

Calcium-linked self-association of human complement C1s.

The weight-average molecular weight of C1s, an activated serine protease subcomponent of human complement C1, has been measured by means of sedimentation equilibrium over a wide range of both protein and calcium ion concentrations. The combined data may be accounted for quantitatively by a simple model for Ca(2+)-dependent self-association of C1s to a dimer. According to this model, the monomer contains a single Ca2+ binding site with K approximately equal to 3 x 10(5) M-1, and the dimer contains three independent Ca binding sites, two having a Ca2+ affinity lower than that of the monomer (K approximately equal to 3 x 10(4) M-1). The third binding site in the dimer, which presumably lies at the interface between the two amino-terminal alpha domains, has a higher Ca2+ affinity (K approximately equal to 1 x 10(8) M-1) and provides the driving force for C1s dimerization in the presence of calcium.     

C'3 polymorphism of human complement in North-East England.

The C'3 polymorphism of human complement was investigated in a sample of 268 unrelated individuals in North-East England. The two common genes were comparable in frequency with other European populations investigated so far. Two individuals were found to have rare variant C'3 types.     

Identification of a human non-interferon lymphokine activating monocyte complement biosynthesis.

A monocyte-stimulating activity produced by mitogen-induced mononuclear cells has been defined by its ability to enhance the synthesis in vitro of complement C1 subcomponents, C2 and C3. A lymphokine responsible for this activity was purified from culture supernatants of peripheral blood mononuclear cells activated by staphylococcal enterotoxin A. From 0.5 litre of supernatant the purification procedure [(NH4)2SO4 precipitation, phenyl-Sepharose chromatography and preparative electrofocusing] yielded about 100 pmol of purified lymphokine. Its pI is 7.9 and its Mr, estimated by SDS/polyacrylamide-gel electrophoresis, is 14,600, 27,000 and 56,000, the high-Mr species representing oligomeric forms of the Mr-14,600 molecule. Its amino acid analysis reveals a high percentage of hydrophobic amino acids (34%); the absence of histidine residues suggests that it is a novel monocyte-activating lymphokine. It enhances C1r and C1s biosynthesis at a pretranslational level. From its structure and activity this lymphokine appears different from gamma-interferon.     

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.     

The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.     

The sequence and topology of human complement component C9.

A partial nucleotide sequence of human complement component C9 cDNA representing 94% of the coding region of the mature protein is presented. The amino acid sequence predicted from the open reading frame of this cDNA concurs with the amino acid sequence at the amino-terminal end of three proteolytic fragments of purified C9 protein. No long stretches of hydrophobic residues are present, even in the carboxy-terminal half of the molecule which reacts with lipid-soluble photoaffinity probes. Monoclonal antibody epitopes have been mapped by comparing overlapping fragments of C9 molecule to which the antibodies bind on Western blots. Several of these epitopes map to small regions containing other surface features (e.g., proteolytic cleavage sites and N-linked oligosaccharide). The amino-terminal half of C9 is rich in cysteine residues and contains a region with a high level of homology to the LDL receptor cysteine-rich domains. A model for C9 topology based on these findings is proposed.     

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.     

Receptors for IgG and complement in human spleen lymphoid cells. Preferential binding of particulate immune complexes through complement receptors.

Human lymphoid spleen cells attached to Petri dishes by poly-L-lysine bind 51Cr-labeled erythrocytes coated with IgG antibodies or complement but not uncoated erythrocytes or those coated with IgM antibodies. The number of erythrocytes bound through complement receptors is several times larger than that bound through IgG receptors. Increasing up to five times the number of IgG molecules on the red blood cells only leads to a slight increase of binding. However, the addition of complement to the IgG-coated erthrocytes increases 10 times the binding to spleen cells, even in the presence of an excess of normal IgG. These results can be explained by postulating that there is a larger number (or greater affinity) of spleen cell receptors for complement than that of spleen cell receptors for IgG.     

Purification and structural analysis of the fourth component of human complement.

The fourth component of human complement (C4) has been purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which had been depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on diethylaminoethylcellulose, QAE-Sephadex, and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieving separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37 degrees C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A three-chain structure for C4 was confirmed, and molecular weight estimates of 93 000 +/- 9300, 75 000 +/- 7500, and 30 000 +/- 3000 determined for the alpha, beta, and gamma chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by C1s and trypsin were accompanied by the fragmentation of the alpha chain. Inactivation of C4 by hydrazine, on the other hand, produced no detectable change in chain size. Separation of the chains was accomplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains have been performed, and N-terminal sequences of the latter established by automated Edman degradation.     

The purification and properties of the second component of human complement.

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.     

Components of human complement system. Testing and isolation of C4

A modified reagent for testing the hemolytic activity of human complement component C4 has been obtained. Reagent R4 was obtained by treatment of human blood serum pools with 0.075 M solution of hydrazine hydrate. This reagent was found to be rich in the serum fraction obtained by chromatography on DEAE-cellulose DE-52 and containing an active complement C3 component. To test the sensitivity and specificity of the reagent, component C4 was subjected to purification. This procedure resulted in a hemolytically active, electrophoretically and immunoelectrophoretically homogeneous component C4. DEAE-cellulose DE-52, DEAE-Sephacel, Ultragel AcA-34 and, again, DEAE-Sephacel were used consecutively as purification agents. The activity yield of component C4 with regard to the initial serum level was 20%.     

Interactions of C-reactive protein with the first component of human complement.

The monocyte-macrophage cell line is an important member of the host defense system. This report describes a series of assays that can be applied routinely in the evaluation of human monocyte function and gives information as to the activity of normal monocytes in these systems. Tests were chosen to assess various aspects of monocyte function that give some insight into the host defense status and the degree of "activation" of the monocyte. The assays outlined in this report are relatively simple to perform and include measurements of human monocyte chemotaxis, phagocytosis, fungal and bacterial killing, adhesion, and spreading.