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PubMed1991

Study of HLA class I, class II and complement genes (C2, C4A, C4B and BF) in Japanese psoriatics and analysis of a newly-found high-risk haplotype by pulsed field gel electrophoresis.

Genetic polymorphisms of HLA antigens and HLA-linked serum complement components (C2, C4A, C4B and BF) were investigated in 79 Japanese patients suffering from psoriasis. HLA typing revealed increased frequencies of HLA-A1, A2, B39, Bw46, Cw6, Cw7 and Cw11. Among complement components, positive associations were obtained with C4A4 and C4B2 and a negative association with BFF. The major histocompatibility complex haplotype (supratype), HLA-A2-Cw11-Bw46-C2C-BFS-C4A4-C4B2-DRw8 is purported to be a new high-risk haplotype in Japanese patients with psoriasis. Analysis of patients with this supratype via pulsed field gel electrophoresis showed the existence of specific, extensive DNA deletions near HLA-DR genes, but no disease-specific patterns could be observed by means of this technique. The newly-found high-risk haplotype indicates racial and ethnic differences among psoriatic patients.

Alleles↗
PubMed1991

Synthesis of complement components C2 and C4 by human monocyte-derived macrophages during in vitro differentiation in serum-free culture conditions.

In order to analyze the kinetics of complement component synthesis by human monocytes/macrophages, we have developed a system of defined culture conditions in the absence of serum. Moreover, the use of the polymerase chain reaction (PCR) provides a high sensitivity for the detection of mRNAs and the study of the regulation of complement component synthesis by these cells. Human blood monocytes were collected and purified by cytapheresis and elutriation, and then cultured in nonadherent cell culture bags for up to 3 weeks. Cells were grown in Iscove's modified Dulbecco's medium supplemented with alpha-phosphatidylcholine, transferrin, insulin, glutamine and antibiotics. The phenotypic and functional properties of macrophages differentiated under these serum-free culture conditions have been previously analyzed [1]. Secretion of complement component was measured by a hemolytic assay. mRNA was prepared using guanidine isothiocyanate extraction followed by cesium gradient ultra-centrifugation. cDNA was obtained by reverse transcription, then amplified by PCR. Fresh monocytes did not display any secretion of C2 as measured by hemolytic assay. However, C2 secretion was detected on and after the 3rd day of serum-free cultured unstimulated monocytes. The rate of C2 production increased along with macrophage differentiation up to the 3rd week of culture. We were not able to detect any functional C4 secretion. In preliminary studies, C2 and C4 mRNAs were detected in macrophages and in unstimulated fresh monocytes. These preliminary studies show that the serum-free culture conditions we have developed allow very satisfactory survival and differentiation of human monocytes, and provide optimal conditions for the study of their secretory activity.

Cell Differentiation↗
PubMed1983

HLA-linked complement polymorphisms (C2, BF) in psoriasis.

Starting from the known association between psoriasis and several HLA antigens and from the fact that the HLA chromosomal region contains the structural genes for at least three complement components, the authors have looked for an association between psoriasis and allotypes of C2 and BF. C2 and BF polymorphism were examined in 230 psoriatic patients. Two rare complement genes were found to be significantly increased when compared with controls: the frequency of the C2*2 gene was 0.061 among patients and 0.035 among controls (P less than 0.05); for BF*SO7, the frequencies were 0.0304 in patients and 0.0092 in controls (P less than 0.0005). The BF*F gene frequency, however, was significantly decreased among patients: 0.1196 vs. 0.1743 (P less than 0.01). The relative risks were 1.79 for the C2 2, 3.44 for the BF SO7, and 0.6 for the BF F gene product. From previous studies, it is known that these three complement alleles (C2*2, BF*SO7, BF*F) are in linkage disequilibrium with HLA alleles that have also been found increased or decreased, respectively, in psoriasis.

Alleles↗
PubMed1996

Down-regulation of secretion of human complement component C2 by the product of an alternatively spliced C2 messenger RNA.

We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.

Alternative Splicing↗
PubMed1989

cDNA cloning and expression of human complement component C2.

A full-length cDNA clone for C component C2 was isolated from a human liver cDNA library in lambda gt11 by initially screening with an affinity-purified rabbit anti-C2 antibody and then using the isolated partial C2 cDNA as a probe for re-screening the library. The cDNA insert of clone lambda C2HL5-3 was sequenced in its entirety. It consisted of 2961 nucleotides including a 5' untranslated region of 388 nucleotides, followed by a 60 nucleotide region coding for a putative signal peptide, a 2196 nucleotide region coding for the 732 amino acids of the mature C2 polypeptide, and a 317 nucleotide long 3' untranslated region. Comparison of the nucleotide sequence to the previously reported C2 cDNA sequence showed two major differences. First, the 5' untranslated region of C2HL5-3 was 352 nucleotides longer and included four ATG followed by in-frame termination codons. Second, nucleotide residue 1987 was a C instead of a G, resulting in a change of amino acid residue 513 from Leu to Phe and in the appearance of an EcoRI site. The full-length C2 cDNA was cloned into the expression vector p91023(B). Transfection of the recombinant plasmid in COS cells resulted in the secretion of a protein with antigenicity and hemolytic activity similar to those of native C2. Western blot analysis indicated that secreted rC2 had the same apparent m.w. as C2 in human serum. Northern blot analysis of total RNA isolated from transfected COS cells showed two bands of C2 mRNA, both of which were longer than human liver C2 mRNA and represent transcripts generated by the vector-C2 construct.

Amino Acid Sequence↗
PubMed1988

Angioedema induced by a peptide derived from complement component C2.

Synthetic peptides that correspond to the COOH-terminal portion of C2b enhance vascular permeability in human and guinea pig skin. In human studies, 1 nmol of the most active peptide of 25-amino acid residues produced substantial local edema. A pentapeptide and a heptapeptide corresponding to the COOH-terminal sequence of C2b each induced contraction of estrous rat uterus in the micromole range; a peptide of 25 amino acids from this region induced a like contraction of rat uterus at a concentration 20-fold lower than the smaller peptides. The vascular permeability of guinea pig skin was enhanced by doses of these synthetic peptides in a similar fashion as that observed for the concentration of rat uterus. The induction of localized edema by intradermal injection in both the guinea pig and the human proceeds in the presence of antihistaminic drugs, suggesting that there is a histamine-independent component to the observed increase in vascular permeability. Cleavage of C2 with the enzymic subcomponent of C1, C1s, yields only C2a and C2b, and no small peptides, whereas cleavage of C2 with C1s and plasmin yields a set of small peptides. These plasmin-cleaved peptides are derived from the COOH terminus of C2b, and they induce the contraction of estrous rat uterus.

Amino Acid Sequence↗
PubMed1981

Linkage of total deficiency of the second component (C2) of the complement system and of genetic C2-polymorphism to the major histocompatibility complex of the guinea pig.

In the guinea pig six common phenotypes for hemolytically active C2 were detected. They resulted from three allotypic variants: a basic C2B and two more acidic variants C2A and C2A1. (The gene frequency for C2B in the outbred population was 0.36, 0.33 for C2A, and 0.31 for C2A1.) These variants were inherited as autosomal codominant traits. A strong linkage-disequilibrium between C2 and C4 allotypes was observed consisting of the pairs C2A1-C4F. C2A-C4S, whereas C2B was associated with either C4S or C4S1. To investigate the linkage of C2 to the MHC a family of strain 2 X strain OM3 was studied: the C2B allotype segregated with the strain haplotype of OM3 whereas C2A1 does the same with the strain 2 haplotype. During the C2-typing, an animal totally deficient in hemolytic C2 activity was discovered. Its C1, C4, C3, C5, factors B, D, and H were normal. Breeding yielded finally homozygous and heterozygous-deficient individuals. The latter ones exhibited 50 to 60% of the hemolytic activity of normal guinea pigs. Combined typing data of C4, C2 and GPLA showed that the deficient gene behaved like a rare, silent allele of C2, which is GPLA-linked and inherited like the normal C2-variants. An antiserum against C2 was raised in C2-deficient animals, which reacted with normal guinea pig serum but not with serum from C2-deficient animals. No cross-reactivity with human or mouse serum could be detected.

Alleles↗
PubMed1990

A new, simplified method for oxidation of human complement component C2.

Oxy C2 is a valuable immunologic reagent, particularly for investigators whose research or clinical assays require a stable classical pathway C3 and/or C5 convertase or a highly active plasma complement source. To date only one method for the conversion of serum C2 or purified C2 to their respective oxy C2 forms has been published. However, this method has several disadvantages. For example, handling and dissolving the iodine crystals required in this process are difficult and time consuming. Also, the enhancement procedure results in a significant dilution of the original C2 sample. We have, therefore, developed a new, simplified method for oxidation of plasma, serum, or pure C2 which circumvents the difficulties associated with the earlier method. Moreover, this method offers additional flexibility with regard to oxidative conditions (i.e., buffer pH, temperature, and C2 concentrations) and reagent handling and final C2 product stability.

Complement C2↗
PubMed1982

Reversal of immune complex inhibition of antibody-dependent cell-mediated cytotoxicity by normal human serum.

The results of this study demonstrate that inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) of human peripheral blood mononuclear cells (PBMC) by ovalbumin (OA)-IgG anti-OA immune complexes (IC) can be reversed by normal human serum (NHS) or serum from a patient with congenital deficiency of the second component of complement (C2 def-HS) lacking activity of the classical complement (C) pathway. On the other hand, NHS that had been inactivated by heating at 56 degrees C for 30 min (HI-NHS) or NHS depleted of the alternative C pathway activity by absorption with zymosan (Zy-NHS) did not restore the ADCC of IC-blocked PBMC. These results suggest that the alternative pathway of C plays a very important role in the re-establishment of ADCC of PBMC blocked with IC. The recovered activity was susceptible to a new inhibition when re-exposed to IC, demonstrating that the NHS effect depends on the recovery of the functional activity of the receptor for the FC fragment of IgG on PBMC and not on the induction of non-specific cytotoxicity. The ability of NHS to restore the cytolytic potential of IC-inhibited PBMC was dependent on the time of exposure of PBMC to IC before the addition of the unblocking agent (NHS). After prolonged reaction of PBMC with IC, the blocked cells were unable to recover their ADCC activity by incubation with NHS. Unblocking of the Fc receptor of PBMC by C may be a physiological way to prevent permanent impairment of the immune mechanisms that depend on its function in the free state.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

Structural analysis of chicken factor B-like protease and comparison with mammalian complement proteins factor B and C2.

Chicken complement factor B-like protease is a glycoprotein of 95 kDa. Activation of chicken serum complement with inulin cleaved the B-like protease into an N-terminal Ba fragment of 37 kDa and a C-terminal Bb fragment of 60 kDa. The whole protein and the two fragments were purified by affinity chromatography using mAb to chicken Ba or Bb followed by ion exchange chromatography. Amino acid sequencing showed that chicken B-like protease was cleaved at a site homologous to that cleaved in mammalian complement components B and C2 on activation. Limited tryptic digestion of the B-like protease generated fragments similar to Ba and Bb. More than 200 residues of the Ba sequence and two N-linked glycosylation sites were established by amino acid sequencing of peptides derived by digestion with four proteases. Comparison of human and mouse C2 and B sequences indicated a slower evolutionary rate for B (85% sequence identity) than for C2 (74% sequence identity). Comparison of chicken Ba to human and mouse C2b and Ba showed 42 to 45% sequence identity with respect to C2b fragments, and 46 to 49% sequence identity with respect to Ba fragments. Taking the slower evolutionary rate of factor B into account, chicken factor B-like protease seems to be equally related to mammalian complement components B and C2, and the B-like protease most likely represents the present-day descendant of a common ancestral protein for mammalian B and C2. This conclusion is in agreement with the requirement for the B-like protease in both classical and alternative activation pathways for chicken complement, and with the apparent lack of a chicken serum protein with exclusive C2 activity.

Amino Acid Sequence↗
PubMed1983

[B, C2 and C4 complement factors, the HLA system and diseases. Genetic relation].

Close linkage of the complement factor loci C2, C4 and Bf to HLA loci has been firmly documented. A review of the literature was made about genetic defects of these components associated with a variety of disorders, especially immunologic diseases. Some allotypes of these factors are also closely linked to several diseases. Relationship between these diseases and the genes controlling the synthesis of C2, C4 and Bf and their allelic variants is not yet known but this association suggested several pathogenic hypotheses.

Complement C2↗
PubMed1989

Isolation and characterization of a novel plasma protein which binds to activated C4 of the classical complement pathway.

We report here the isolation and partial characterization of a previously unrecognized protease-sensitive plasma protein identified during the development of a novel protocol for the purification of the second component of human complement (C2). This new protein is physicochemically similar to C2. It coprecipitates with C2 on polyethylene glycol fractionation and specifically binds, like C2, to Sepharose-bound iC4/C4b. Binding occurs both in the presence and absence of C2. The purified protein has a chain structure similar to C2 as determined by sodium dodecyl sulfate-gel electrophoresis in the presence or absence of reducing agent and has a molecular mass of 120 kDa, only somewhat greater than C2 at 95 kDa. Both proteins radioiodinate under similar conditions to the same specific activities with each of two different methods that yield 10-fold disparate results. Quantitative Mancini analysis identifies 300 micrograms/ml of the 120-kDa protein in plasma and serum. The protein is present at normal concentrations in serum from individuals genetically deficient in C2, has no C2 functional activity, and is not cleaved as is C2 when serum complement is activated. Potent monospecific polyclonal anti-serum to each do not cross-immunoprecipitate using standard gel techniques. However, these anti-sera identify epitopes in common by Western blotting. The data presented indicate that the 120-kDa protein is a distinct plasma component and suggest that the protein is not an "immature" form of C2. Initial experiments to delineate a functional role for the 120-kDa protein have demonstrated a consistent inhibition of C1 site generation on EAC4b which is dose-dependent and reversible. Thus, this protein appears to be a new complement regulatory factor.

Blood Proteins↗
PubMed1977

Influence of a deficiency of the second component of complement on the bactericidal activity of neutrophils in vitro.

Serum from three patients with a complete, selective deficiency of the second component of complement (C2) did not promote optimal killing of Staphylococcus aureus, 502A by neutrophilic polymorphonuclear leukocytes (PMN) in vitro. The addition of C2 reagent or the presence of heat-stable opsonin in the C2-deficient serum corrected the defective killing of S. aureus that was observed with patient or control PMN. PMN from the patients or control subjects killed bacteria with equal efficiency under conditions of optimal opsonization (normal pooled serum). However, twice-washed control PMN were better than patient PMN in killing S. aureus under circumstances of suboptimal opsonization (C2-deficient serum, heated C2-deficient serum, heated normal pooled serum, or no replacement of serum). The latter finding was due to residual C2 on the surface of twice-washed control cells. As repeated washing of control PMN progressively removed cell-associated C2, the staphylocidal effectiveness of the control RMN decreased to the level of patient PMN. In contrast to the findings with S. aureus, triply-washed PMN from patients or controls killed normal numbers of Escherichia coli, ON2, in C2-deficient serum.

Animals↗
PubMed1984

HLA-B27 and allotypes of complement components in ankylosing spondylitis.

Thirteen families were studied for the frequency of HLA-B27 and allotypes of the components of complement, C2, C4 and Bf. The 13 probands all suffered from definite ankylosing spondylitis (AS) as defined by the New York criteria. The results showed that HLA-B27 positive haplotypes in AS patients possessed different complement allotypes. Thus, it is postulated that the gene conferring predisposition to the development of AS is either HLA-B27 itself or another gene in very close proximity to the HLA-B locus.

Alleles↗