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PubMed1991

Human complement factor H. Tissue specificity in the expression of three different mRNA species.

Using cDNA clones H-19 and H-46, we have shown previously that three different mRNA species (4.3 kb, 1.8 kb and 1.4 kb) for complement factor H are expressed constitutively in human liver. Here we report data suggesting that the expression of these different factor-H mRNA species is regulated by tissue-specific control mechanisms. Total RNA and poly(A)-enriched RNA from various human tissues (heart, lung, temporal cortex, kidney, spleen, bone marrow and muscle) various cell lines (HepG2, HepG3, HepG4, Hep3B, H-4, Jurkat, Molt4, H-9, KHos24Os, A-431, U937, Mono Mac 6 and Raji) and from primary cultures of peripheral blood monocytes, fibroblasts and human umbilical vein endothelial cells (HUVEC) were investigated for the expression of factor-H mRNA. In RNA preparations from extrahepatic tissue, factor-H mRNA was only detected in biopsies from the lung. Using 20 micrograms total RNA isolated from all 13 cell lines it was not possible to detect any factor-H mRNA, while mRNA for factor H was expressed in monocytes, HUVEC and fibroblasts. When expressed in extrahepatic tissues, only the 4.3-kb and the 1.8-kb mRNA species were detected, while the 1.4-kb mRNA is expressed abundantly in liver. Interferon-gamma did not induce the expression of factor-H mRNA in any of the cell lines tested. On the other hand, tumour necrosis factor-alpha induced the expression of the 4.3-kb mRNA species in U937 cells. In HUVEC and fibroblasts the relative quantities of the 4.3-kb and the 1.8-kb mRNA species and the regulatory effects of interferon-gamma, interleukin-1, dexamethasone and retinoic acid on their expression showed significant tissue specificity.

Blotting, Northern↗
PubMed1988

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 Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma.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.

Amino Acid Sequence↗
PubMed1992

A new biological activity of the complement factor H: identification of the precursor of the major macrophage-chemotactic factor in delayed hypersensitivity reaction sites of guinea pigs.

The guinea pig complement factor H(FH) and the plasma precursor(PMCFS-1) of the major monocyte-chemotactic factor(MCFS-1) found in the skin site of delayed hypersensitivity reaction(DHR) induced in the guinea pigs were compared in the antigenicity and the function. Both anti-FH-IgG and anti-MCFS-1-IgG formed a single precipitation line against FH, PMCFS-1, MCFS-1 and guinea pig plasma, and these lines fused one another without any spur formation. The inhibition activity of FH for C3bBb was absorbed by anti-MCFS-1-F(ab')2 in a dose-dependent manner. PMCFS-1 inhibited C3bBb activity dose-dependently as FH. These results show that FH is identical to PMCFS-1 and imply that FH, converted to MCFS-1 plays as a monocyte-chemotactic factor in the site of DHR.

Animals↗
PubMed1991

Localization of the heparin-binding site on complement factor H.

Factor H is a regulator of complement activation and, in this capacity, it prevents activation of the alternative pathway on host cells and tissues when it recognizes markers on these surfaces. This report describes the binding characteristics and location of the site on factor H that is responsible for host recognition. Factor H was found to bind a variety of polyanions, including heparin, heparan sulfate, dextran sulfate, and clusters of sialic acid. In heparin-agarose binding assays it exhibited an affinity for heparin only 2-fold weaker than that of antithrombin III. Factor H exhibited little or no affinity for polyaspartic acid or bacterial colominic acid (polysialic acid). Factor H (Mr 150,000 with approximate dimensions of 30 x 600 A) is composed of 20 highly homologous domains (SCRs) that are arranged as beads on a string. Polyanions were found to block a tryptic cleavage site in domain 15, and a photoaffinity-tagged heparin probe labeled the region between domains 12 and 15. Affinity chromatography of tryptic fragments on heparin-Sepharose confirmed that this region contained the heparin-binding site. CNBr cleavage at Met787 located between SCRs 13 and 14 split the photoaffinity-tagged region. Sequence analysis strongly suggests that domain 13 contains the primary site of polyanion binding. Factor H expresses its complement regulatory function through a site located in domains 4-6 where C3b binds. Thus, the polyanion-binding site that regulates the affinity of factor H for C3b appears to reside more than 200 A away from the C3b-binding site.

Amino Acid Sequence↗
PubMed1989

Human complement factor H: an additional gene product of 43 kDa isolated from human plasma shows cofactor activity for the cleavage of the third component of complement.

In addition to the 150-kDa factor H protein, we have previously described a 43-kDa factor H molecule in human plasma, which probably represents a translational product of the additional 1.8-kb mRNA for factor H. This factor H was isolated from human plasma by means of immunoaffinity chromatography and high-performance liquid chromatography. When tested for its functional activity, this purified 43-kDa H protein was shown to act as cofactor for factor I- mediated cleavage of fluid-phase C3b to iC3b.

Antibodies, Monoclonal↗
PubMed1992

Location of the mouse complement factor H gene (cfh) by FISH analysis and replication R-banding.

A technique for replication R- and G-banding of mouse lymphocyte chromosomes was developed, and the replication R-banding pattern was analyzed. Optimal banding patterns were obtained with thymidine- and BrdU-treatment of lymphocytes in the same cell cycle. This produced replication R-band patterns that were the complete reverse of the G-band patterns on all chromosomes. Replication R-banding methods can be used in conjunction with nonisotopic, fluorescence in situ hybridization (FISH) to localize cloned probes to specific chromosomal bands on mouse chromosomes. with these methods the mouse complement factor H gene (cfh) was localized to the terminal portion of the F region of Chromosome 1. Q-banding patterns were also obtained by the replication R-banding method and may be useful for rapid identification of each chromosome.

Animals↗
PubMed1985

Expression of complement factor H on the cell surface of the human monocytic cell line U937.

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.

Antibodies, Monoclonal↗
PubMedDate not supplied

Differential modulation of complement factor H and C3 expression by TNF-alpha in the rat. In vitro and in vivo studies.

The biosynthesis of alternative regulatory complement protein factor H was investigated using both an in vivo rat model and an in vitro rat hepatocyte culture system, and compared to that of C3 component. Subcutaneous injection of a single dose of 20 micrograms of recombinant murine tumor necrosis factor-alpha (rmTNF-alpha) had no effect on factor H liver mRNA levels, while it increased C3 mRNA levels. In correlation with this, serum factor H levels remained unchanged after rmTNF-alpha injection, whereas C3 levels were increased. In contrast in vitro studies showed that rmTNF-alpha had no effect on factor H and C3 expression by rat hepatocytes. Recombinant human interleukin-1 alpha (rhIL-1 alpha) did not alter the expression of factor H, whereas it increased C3 expression, and recombinant human interleukin-6 (rhIL-6) stimulated expression of both proteins. This study shows that TNF-alpha is not directly responsible for the increased levels of factor H observed in vivo during induced inflammation in the rat. Its in vivo effect on C3 secretion might be secondary to the TNF-alpha-induced release of IL-1 and/or IL-6.

Animals↗
PubMed1991

Effect of complement-protein-C3b density on the binding of complement factor H to surface-bound C3b.

Various amounts of the activation fragment C3b of the complement (C) protein C3 were coupled to Sepharose 4B by catalysis with the C3 convertase of the alternative pathway of C. The binding of radioactively labelled C proteins B and H (= factor H) to the C3b-carrying particles was assayed. It was found that the relative binding of H, but not of B, fell rapidly with decreasing densities of solid-phase C3b, suggesting a sigmoidal relationship between C3b density and binding of H. To study the phenomenon in more detail, preformed C3b was coupled to activated thiopropyl-Sepharose 6B at various densities. By using this model system, it was shown that the binding of H/unit amount of C3b was positively correlated to C3b density up to a C3b concentration of about 0.5 mg/ml of gel, whereas binding of B was independent of C3b density. The results show that accumulation of high densities of C3b on a surface creates high-affinity binding sites for H. Because H has recently been shown to form dimers in solution, the interaction of dimeric H with neighbouring C3b molecules is a likely explanation for the phenomenon. The C3b density effect may be a regulatory mechanism keeping the activation of the alternative pathway of C on activating surface within reasonable limits.

Complement C3-C5 Convertases↗
PubMed1991

Oligomeric domain structure of human complement factor H by X-ray and neutron solution scattering.

Factor H is a regulatory component of the complement system. It has a monomer Mr of 150,000. Primary structure analysis shows that the polypeptide is divided into 20 homologous regions, each 60 amino acid residues long. These are independently folding domains and are termed "short consensus repeats" (SCRs) or "complement control protein" (CCP) repeats. High-flux synchrotron X-ray and neutron scattering studies were performed in order to define its solution structure in conditions close to physiological. The Mr of factor H was determined as 250,000-320,000 to show that factor H is dimeric. This structure is maintained at concentrations between 1 and 11 mg/mL in the pH range 5-9. Zn2+ ions are an inhibitor of C3b cleavage by factor I, a reaction in which factor H acts as a cofactor. Additions of Zn2+ to factor H caused it to form oligomers containing 4-10 monomers. The radius of gyration RG of native factor H by X-rays or by neutrons in 0% or 100% 2H2O buffers is not measurable but is greater than 12.5 nm. Two cross-sectional radii of gyration RXS-1 and RXS-2 were determined as 3.0-3.1 and 1.8 nm, respectively. Analyses of the cross-sectional intensities show that factor H is composed of two distinct subunits. The RXS-1 corresponds to the cross-sectional properties of both subunits and exhibits an unusual radiation dependence on the X-ray flux. Since RXS-2 is close to the corresponding RXS of C4b binding protein (91% of which is formed from SCR/CCP domains), it is inferred that the SCR/CCP domains of factor H and C4b binding protein have similar solution structures. The use of hydrodynamic spheres to reproduce literature sedimentation coefficients of 5.5-5.6 S showed that these were compatible with a V-shaped arrangement of two rods (36 spheres each, length 87 +/- 5 nm) joined at an angle of 5 degrees. The use of a similar arrangement of 244 spheres arranged in two rods (length 77 nm) to fit the experimental X-ray and neutron scattering curves showed that the two rods are joined at an angle of 5 degrees. This model corresponds to an actual RG of 21-23 nm. The separation between each SCR/CCP in factor H is close to 4 nm. In the solution structure of factor H, the SCR/CCP domains are in a highly extended conformation.

Complement C3b↗
PubMed1989

Analysis of complement factor H mRNA expression: dexamethasone and IFN-gamma increase the level of H in L cells.

Murine complement protein H is encoded by a 100-kb gene on chromosome 1. A 3.2-kb fragment of the 5' flanking region of the H gene was sequenced, and two transcription start sites for this gene were identified by RNase protection and S1 nuclease analyses, each of which had upstream TATA and CAAT boxes. This region shares sequence homology with known regulatory elements, including the SV40 enhancer consensus, the Sp1 binding site, and two glucocorticoid-responsive core elements (GRE). Tissue and cell-line specificity has been examined by Northern analysis, and the 4.4-kb full-length H messenger RNA was identified in liver, kidney, spleen, thymus, liver cell line 1469, and L cells. IFN-gamma did not induce H mRNA expression in the macrophage cell line P388D.1 but had a positive effect on both the mRNA and protein levels of H in L cells. PMA, LPS, and vitamin D did not increase H mRNA levels in L cells. Pursuant to the discovery of two GRE in the 5' regulatory region of the H gene, we examined the effects of glucocorticoids on H mRNA expression. Dexamethasone (10(-7) M) was found to increase markedly the levels of H mRNA and protein after 24 h of incubation, and the effect on the mRNA was detectable by 30 min. The fact that H is a down-regulator of complement activation is consistent with the known immunosuppressive role of glucocorticoids. To our knowledge, this is the first time that dexamethasone has been shown to increase the levels of a complement protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

Structure of the murine complement factor H gene.

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.

Animals↗
PubMed1986

Human complement factor H: isolation of cDNA clones and partial cDNA sequence of the 38-kDa tryptic fragment containing the binding site for C3b.

We isolated cDNA clones coding for the functionally important tryptic N-terminal 38-kDa fragment of human complement control protein factor H using polyclonal and monoclonal antibodies to screen a human liver cDNA library cloned in a bacterial expression vector, PEX-1. By testing the reactivity of antibodies specific for the recombinant proteins produced by individual clones with proteolytic fragments of serum H the exact position of these cDNA clones within H was mapped. One clone, H-19, coding for the 38-kDa fragment of H was sequenced and found to code for 289 amino acids derived from the 38-kDa N-terminal fragment as well as for the first 108 amino acids belonging to the complementary 142-kDa tryptic fragment. The derived protein sequence could be arranged in 6 highly homologous repeats of about 60 amino acids each, the homology between the repeats being determined by the characteristic position of cysteine, proline, glycine, tyrosine and tryptophane residues. The region coding for the epitope recognized by one of our monoclonal antibodies was localized by subcloning restriction fragments of H-19 into the expression plasmid and testing for the expression of this epitope.

Amino Acid Sequence↗
PubMed1989

Membrane-associated complement factor H on lymphoblastoid cell lines Raji expresses a co-factor activity for the factor I-mediated cleavage of C3b.

Evidence of factor H was found on the surface of Raji cells derived from two different lines; a 150,000 MW species identical to 150,000 MW plasma factor H was detected. Rosette experiments performed at physiological ionic strength using EAC3b or EC3b were negative. In contrast, Raji cells expressed a co-factor activity in the factor I-mediated cleavage of fluid-phase C3b. This activity was cell number dependent and was inhibited by anti-factor H but not by anti-CR1. Raji cells did not secrete factor H in the extracellular medium.

B-Lymphocytes↗
PubMed1990

Genetic polymorphism of complement factor H in cattle.

Bovine factor H was found to be polymorphic by the combined techniques of SDS-polyacrylamide electrophoresis of bovine plasma and immunoblotting. Three phenotypes (S, SF, F) were identified in a sample population of 149 cattle. Variant S and F differed by an apparent molecular weight of 5000 daltons. Family studies demonstrated Mendelian segregation of variants S and F. The data indicate that these genetic variants of bovine factor H are encoded by two codominant alleles at a single autosomal locus.

Alleles↗
PubMed1991

Cloning of the 1.4-kb mRNA species of human complement factor H reveals a novel member of the short consensus repeat family related to the carboxy terminal of the classical 150-kDa molecule.

Three factor H mRNA species of 4.3 kb, 1.8 kb, and 1.4 kb are constitutively expressed in human liver. Having previously characterized full-length cDNA clones derived from the 4.3-kb and 1.8-kb factor mRNA, we report here the isolation and eucaryotic expression of full-length cDNA clones coding for the 1.4-kb mRNA species. The 1266-bp cDNA codes for a polypeptide of 330 amino acids and contains two polyadenylation signals and a short poly(A)+tail. The protein is composed of a leader peptide followed by five short consensus repeat domains. It shows a hybrid structure with the last three domains being almost identical to the carboxy-terminal of the classical 150-kDa factor H molecule and the two first domains representing unique short consensus repeat structures. Eucaryotic expression in COS7 cells revealed two polypeptides derived from one cDNA clone that are also found in human serum. Differences between the cDNA clones within the last three domains indicate two distinct, possibly allelic sequences that, in addition, differ from the authentic 150-kDa factor H sequence. Southern blot results support the notion that the 4.3-kb factor H and the 1.4-kb factor H-related mRNA are transcribed from two separate but highly homologous genes.

Amino Acid Sequence↗