Molecular cloning of a human serum protein structurally related to complement factor H.

Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H.     

Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, P _meta = 6.6×10⁻⁸, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, P _meta = 2.9×10⁻⁷, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ∼146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (P _meta = 3.2×10⁻⁷, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (P _meta = 3.5×10⁻⁴, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.     

Two major serum components antigenically related to complement factor H are different glycosylation forms of a single protein with no factor H-like complement regulatory functions.

Factor H is a 150-kDa serum glycoprotein with key regulatory functions in the alternative pathway of complement activation. Two glycoproteins with a molecular mass of approximately 42 and 37 kDa that react with an antiserum against factor H were purified from human plasma. The two glycoproteins have identical N-terminal amino acid sequences but differ in glycosylation. Sequence comparisons indicated that they both correspond to a 1.4-kb mRNA recently cloned from human liver cDNA. The serum concentration of the two glycoproteins together was estimated to be approximately 40 mg/liter. They were found not to exert factor H-like regulatory functions in the alternative pathway. Thus, the 42-kDa glycoprotein described here appears to be distinct from the previously characterized factor H-related protein of similar size, suggesting that human serum contains two factor-H related molecules which both have a molecular mass of 41 to 43 kDa but which differ largely in structure.     

Characterization of proteins structurally related to human N-acetyl-beta-D-glucosaminidase.

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000--200000) were present both in the unadsorbed fraction and in the 0.05--0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000--70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.     

The Plasmodium falciparum blood stages acquire factor H family proteins to evade destruction by human complement

The acquisition of regulatory proteins is a means of blood‐borne pathogens to avoid destruction by the human complement. We recently showed that the gametes of the human malaria parasite Plasmodium falciparum bind factor H (FH) from the blood meal of the mosquito vector to assure successful sexual reproduction, which takes places in the mosquito midgut. While these findings provided a first glimpse of a complex mechanism used by Plasmodium to control the host immune attack, it is hitherto not known, how the pathogenic blood stages of the malaria parasite evade destruction by the human complement. We now show that the human complement system represents a severe threat for the replicating blood stages, particularly for the reinvading merozoites, with complement factor C3b accumulating on the surfaces of the intraerythrocytic schizonts as well as of free merozoites. C3b accumulation initiates terminal complement complex formation, in consequence resulting in blood stage lysis. To inactivate C3b, the parasites bind FH as well as related proteins FHL‐1 and CFHR‐1 to their surface, and FH binding is trypsin‐resistant. Schizonts acquire FH via two contact sites, which involve CCP modules 5 and 20. Blockage of FH‐mediated protection via anti‐FH antibodies results in significantly impaired blood stage replication, pointing to the plasmodial complement evasion machinery as a promising malaria vaccine target.     

Polymorphism and deficiency of human factor H-related proteins p39 and p37

We described previously cDNA clones representing a novel factor H-related 1.4 kilobase mRNA. This mRNA species codes for a doublet of serum proteins of M _r 39 000 and 37 000 (p39/p37). The respective recombinant proteins of the three clones H-69, pFH1.4a, and pFH1.4b differ in the expression of the epitope recognized by the monclonal antibody (mAb) 3D11. This probably reflects the difference of three amino acid residues of the deduced protein sequence. Here we report evidence for corresponding alterations in the native proteins p39/p37 in human sera. Employing mAb 3D11 and a polyclonal factor H-specific antiserum we detected three different patterns in western blot analyses of human sera which we provisionally termed FH1.4p+m+, FH1.4p+m–, and FH1.4p–m–. In the first pattern, p39/p37 were recognized by both antibodies, while in the second pattern the two proteins reacted only with the polyclonal antiserum. Both antibodies failed to detect p39/p37 in the third pattern. These phenotypes are found in the healthy population with frequencies of 0.556, 0.40, and 0.044, respectively. The frequencies of the alleles FH1.4*p+m+, FH1.4*p+m–, and FH1.4*p–m–were estimated to be 0.33, 0.46, and 0.21, respectively, assuming the gene distribution to be in Hardy-Weinberg equilibrium. Studies of 98 members from 27 families revealed an autosomal Mendelian inheritance. Southern blot data support our assumption of a polymorphism of the factor H-related proteins p39 and p37.This work is part of E. Feifel's and M. Mölgg's Ph.D. thesis.     

A new subfamily of structurally related human F-box proteins

F-box proteins, a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box), recruit substrates for ubiquitination and consequent degradation through their specific protein-protein interaction domains. Here, we report the identification of full-length cDNAs encoding three novel human F-box proteins named FBG3, FBG4 and FBG5 which display similarity with previously identified NFB42 (FBX2) and FBG2 (FBX6) proteins. All five proteins are characterized by an approximately 180-amino-acid (aa) conserved C-terminal domain and thus constitute a third subfamily of mammalian F-box proteins. Analysis of genomic organization of the five FBG genes revealed that all of them consist of six exons and five introns. FBG1, FBG2 and FBG3 genes are located in tandem on chromosome 1p36, and FBG4 and FBG5 are mapped to chromosome 19q13. FBG genes are expressed in a limited number of human tissues including kidney, liver, brain and muscle tissues. Expression of rat FBG2 gene was found related to differentiation/proliferation status of hepatocytes. Specifically, FBG2 mRNA was expressed in foetal liver, decreased after birth and re-accumulated in adult liver. Expression of FBG2 was strongly inhibited in hepatoma cells by okadaic acid.     

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.     

Zebrafish complement factor H and its related genes: identification, evolution, and expression

The complement system consists of a number of serum and membrane-bound proteins that play a crucial role in protecting the host organism against microbial infection. Complement factor H (CFH) regulates the alternative pathway of complement in plasma and mediates discrimination of cellular surfaces to alternative pathway activators and non-activators. Although complement system of zebrafish has been extensively studied, the information regarding CFH and its related genes in this important model species remains lacking. In this study, we report the molecular cloning and identification of CFH and complement factor H-like 1–4 (CFHL1–4) in Danio rerio. Sequence comparison and phylogenetic, syntenic, as well as genomic structure analyses demonstrated that the scaffold encompassing CFH and CFHLs region was conserved during evolution from bony fish to humans, and CFH and CFHL1–4 originated by intra-chromosome duplication on chromosome 22. Besides, quantitative real-time polymerase chain reaction revealed that both zebrafish CFH and CFHLs were predominantly expressed in the liver in a tissue-specific manner, and their expression was inducible by lipopolysaccharide, an inducer of immune responses, suggesting that they are possibly involved in acute phase responses. These are the first such data in bony fish, laying a foundation for further study of their physiological functions.     

Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H-related 1 (CFHR1) and complement factor H-related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of approximately 84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.     

Two populations of complement factor H differ in their ability to bind to cell surfaces.

Using hydrophobic affinity chromatography on phenyl-Sepharose, human complement factor H can be separated into two subpopulations, phi 1 and phi 2. Although phi 1 and phi 2 are known to differ in their aggregation properties under non-physiological low ionic strength conditions, no difference in aggregation state was detected under the conditions used for cell-binding experiments. We have investigated these two subpopulations further to determine whether functional differences exist between them. The subpopulation phi 2 was found to bind specifically and saturably to the surface of Raji cells. The binding of the other subpopulation, phi 1, was low, and essentially non-specific. A monoclonal anti-factor H antibody, BGH-1, was raised which recognizes preferentially the phi 2 subpopulation and inhibits the binding of factor H to cell surfaces.     

The group B streptococcal beta and pneumococcal Hic proteins are structurally related immune evasion molecules that bind the complement inhibitor factor H in an analogous fashion

Complement evasion by different mechanisms is important for microbial virulence and survival in the host. One strategy used by pathogenic bacteria is to bind the soluble complement inhibitor factor H (fH) to their surfaces. In group B streptococci and pneumococci, fH binding has been shown to be mediated by the surface proteins beta and Hic, respectively. We showed previously that Hic binds to the middle region of fH and protects the pneumococcus from opsonophagocytosis. As the beta protein and Hic are structurally closely related, we wanted to compare the fH binding characteristics of these two proteins. By using direct binding assays with radiolabeled proteins and surface plasmon resonance analysis we show that both beta and Hic bind to the short consensus repeats 8-11 and 12-14 in the middle region of fH. Peptide mapping analysis suggested that the fH-binding sites on beta and Hic were composed of discontinuous and partially homologous sequences. Thus, the bacterial virulence proteins use multiple binding sites on fH to secure high avidity. Also, the functionally active sites on fH are thereby left free to inhibit C3b deposition and opsonophagocytosis. These results reveal the evolutionary conservation of an analogous immune evasion strategy in different types of pathogenic streptococci. Importantly, the respective virulence factors could be exploited in the development of protein-based vaccines against these pathogens.     

Site-specific N-glycan characterization of human complement factor H

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.     

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.     

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins,a family of putative kinase regulators

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.