Genomic view of the evolution of the complement system

The recent accumulation of genomic information of many representative animals has made it possible to trace the evolution of the complement system based on the presence or absence of each complement gene in the analyzed genomes. Genome information from a few mammals, chicken, clawed frog, a few bony fish, sea squirt, fruit fly, nematoda and sea anemone indicate that bony fish and higher vertebrates share practically the same set of complement genes. This suggests that most of the gene duplications that played an essential role in establishing the mammalian complement system had occurred by the time of the teleost/mammalian divergence around 500 million years ago (MYA). Members of most complement gene families are also present in ascidians, although they do not show a one-to-one correspondence to their counterparts in higher vertebrates, indicating that the gene duplications of each gene family occurred independently in vertebrates and ascidians. The C3 and factor B genes, but probably not the other complement genes, are present in the genome of the cnidaria and some protostomes, indicating that the origin of the central part of the complement system was established more than 1,000 MYA.     

Synthesis and regulation of the two human complement C4 genes in stable transfected mouse fibroblasts

The major histocompatibility complex-linked human complement C4 genes are highly homologous in primary structure but give rise to products which differ in complement-activating function. In order to examine the synthesis, function, and regulation of these two genes independently, cloned C4A and C4B genes were transfected into mouse fibroblast L-cells. In the stable transfected cell lines, C4A and C4B are synthesized, undergo a complex series of post-translational modifications, and each functions appropriately in activation of the classical complement pathway. A marked difference in the kinetics of complement component C1-mediated cleavage of the C4A- and C4B-alpha chains was demonstrated in the transfectants and may contribute to the differences in the intrinsic functional activity of the two C4 isotypes. In contrast to the expression of other complement genes which are affected during the hepatic "acute phase response" (factor B, C3), the expression of C4 was not regulated by interleukin-1 or tumor necrosis factor. Interferon-gamma, however, mediated a dose- and time-dependent increase in the expression of the C4 genes. Moreover, interferon had a significantly greater and longer-lasting effect on the synthesis of C4A than that of C4B. Differences in the expression and regulation of these two genes provide insight into the control of complement activation during inflammation.     

A complement receptor locus: genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to 1q32

The alternative or classical pathways for complement system component C3 may be triggered by microorganisms and antigen-antibody complexes. In particular, an activated fragment of C3, C3b, covalently attaches to microorganisms or antigen-antibody complexes, which in turn bind to the C3b receptor, also known as complement receptor 1. The genes encoding the proteins that constitute the C3-activating enzymes have been cloned and mapped to a "complement activation" locus in the major histocompatibility complex, and we demonstrate in this study such a locus on the long arm of chromosome 1 at band 1q32.     

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: Implications for population susceptibility to diseases

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.     

Complement system of bony and cartilaginous fish

Accumulating evidence indicates that the complement system experienced a discontinuous development at an early stage of vertebrate evolution. Invertebrates such as echinoderms and ascidians, and the most primitive extant vertebrates, the cyclostomes, seem to have a primitive complement system equipped only with the alternative and lectin pathways. In contrast, cartilaginous fish and higher vertebrates seem to have a modern complement system which has two additional pathways, namely the classical and lytic pathways. Recent molecular analyses of the complement system of bony and cartilaginous fish have not only confirmed the above conclusion, but also revealed a unique characteristic of the complement system of fish, where certain key component genes are duplicated. The complement system seems to play a more pivotal role in body defence in fish, whose adaptive immunity is considered to be at a relatively undeveloped state.     

The possible role of immunogenetic factors in the pathogenesis of HIV infection

In this review the data on the presence of associations between immunogenetic markers and the development of HIV infection are presented. Special attention is given to spatial relationships of genetic determinants, responsible for the synthesis of the components of the complement system on one hand and the genes controlling resistance to the causative agents of opportunistic infections and malignant growth on the other hand. Suggestion is made on close relationship between the specific features of the course of HIV infection and the genetic control, responsible for the synthesis of complement components, in particular the genes making up the HLA system.     

Tissue-specific regulation of inflammation.

Proteins of the complement system are important effectors and modulators of inflammation. The complement cascade is triggered by microbes, tissue debris, and specific antibodies. Serum complement proteins are derived primarily from liver, but extrahepatic complement synthesis is important in homeostasis and in local host defenses. Tissue-specific regulation of expression of complement genes is governed by mechanisms similar to those that regulate other "acute phase reactants." That is, tissue injury or infection elicit changes in expression of these acute phase proteins, which, although variable in kinetics, magnitude, and direction, are a consequence of an elaborate system of cell-to-cell communication. This communication is mediated via a complex network of cytokines, including the interferons, interleukins, several growth factors, and sex hormones. The cell biological and molecular biological details of these mechanisms are now under active investigation. An understanding in molecular terms of the balance between proinflammatory and counterregulatory forces on complement gene expression should provide new insight into the functions of complement and the design of novel therapies for disorders of inflammation.     

Regulation of Alternative Pathway Activation and C3a Production by Adipose Cells

Adipose tissue is a major source of adpisin/factor D of the alternative pathway of complement. Adipose tissue also expresses the two other complement components which are involved in the first activation step of the alternative pathway, factor B and C3, and this step is activated in adipose tissue, producing C3a/Acylation Stimulating Protein (C3a/ASP), a stimulator of triglyceride synthesis. Complement activation is a highly regulated process, however, nothing is known about regulation of complement activation in adipose tissue. To gain insight into the nature of adipose complement activation and its regulation, we have now examined the expression of several complement activation regulatory genes, and analyzed the production of C3a/ASP in lean vs. obese, adipsin-deficient mice. We found that undifferentiated preadipocytes expressed the mRNAs encoding the negative regulatory proteins Crry and factor H, but expression of both genes was decreased upon differentiation. The positive regulator properdin, as well as Crry and factor H, were found in adipose tissue. None of these genes was regulated in murine genetic obesity. To investigate the relative levels of complement activation in lean vs. adipsin-deficient obese mice, we developed a radioimmunoassay for measurement of murine C3a/ASP in plasma. We report that there was no significant difference in the level of C3a in lean vs. obese plasma; however, we found a positive correlation between C3a and plasma triglyceride levels in normal lean mice.     

Efficiency and specificity of gene isolation by exon amplification

Exon amplification is an increasingly popular approach to the identification of transcribed sequences and will complement other strategies to isolate genes. We have used this system to amplify candidate exons from 32 cosmids, including 8 cosmids which span a well characterized 185-kb region of the human major histocompatibility class II region on Chromosome (Chr) 6. We have examined the efficiency, specificity, and reproducibility of the system in isolating exons from genes known to be present on particular cosmids and have determined the nature and frequency of artefact amplifications in routine cosmid screening. We were able to clone at least one exon from 88% (7/8) of all known genes tested (including exons which are differentially spliced) and obtained artefacts from 19% (6/32) of the cosmids tested. Such artefacts generally arise from the amplification of noncoding sequences flanked by regions with high homology to acceptor and donor splice junctions. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids which have different numbers of genes and gene structures and describe several approaches to the characterization of novel exons cloned in this study.     

Protein engineering to target complement evasion in cancer

The complement system is composed of soluble factors in plasma that enhance or “complement” immune-mediated killing through innate and adaptive mechanisms. Activation of complement causes recruitment of immune cells; opsonization of coated cells; and direct killing of affected cells through a membrane attack complex (MAC). Tumor cells up-regulate complement inhibitory factors – one of several strategies to evade the immune system. In many cases as the tumor progresses, dramatic increases in complement inhibitory factors are found on these cells. This review focuses on the classic complement pathway and the role of major complement inhibitory factors in cancer immune evasion as well as on how current protein engineering efforts are being employed to increase complement fixing or to reverse complement resistance leading to better therapeutic outcomes in oncology. Strategies discussed include engineering of antibodies to enhance complement fixation, antibodies that neutralize complement inhibitory proteins as well as engineered constructs that specifically target inhibition of the complement system.     

Characterization of responding cells in oxidative mitogen stimulation. III. Presence of I-A- and I-J, E, C-subregion gene products on the surface of required cells

Ia antigens coded by genes of the murine major histocompatibility complex are expressed on the surface of a population of cells critical to the proliferative response of murine spleen cells to the oxidative mitogen neuraminidase/galactose oxidase. By selective depletion with antiserum and complement, Ia antigens coded (or determined) by the I-A and I-J, E, C subregions of the Ir region can be detected on the surface of cells required for the response. In addition, I-A-subregion products have a functional significance in cellular activation which can be demonstrated by blocking experiments with anti-Ia serum in the absence of complement.     

Alternative complement pathway of channel catfish (Ictalurus punctatus): molecular characterization, mapping and expression analysis of factors Bf/C2 and Df

The complement system is important in both innate and adaptive host defense against microbial infection in vertebrates. It contains three pathways: the classical, alternative, and lectin pathways. Complement component factors B and D are two crucial proteases in the alternative pathway. In this study, the genes of complement factors Bf/C2 and Df from channel catfish, Ictalurus punctatus were identified and characterized. Two complement factor B-related genes, Bf/C2A and Bf/C2B, and factor D gene Df were identified. Phylogenetic analysis suggested that Bf/C2A and Bf/C2B is likely orthologous to factor B and factor C2, respectively. Southern blot results suggested that these three genes are all single-copy genes in the catfish genome. The catfish Bf/C2A, Bf/C2B and Df genes were genetically mapped on linkage group 3, 20 and 29, respectively. Bf/C2A and Bf/C2B are highly expressed in liver and kidney, while Df is highly expressed in gill and spleen. After infection with Edwardsiella ictaluri, the expression of Bf/C2A, Bf/C2B and Df genes were found to be remarkably induced in the gill, liver, spleen and kidney at some sampling times, indicating that these three complement factors play a pivotal role in immune responses after the bacterial infection in catfish.     

Lack of linkage between gene(s) controlling the synthesis of the seventh component of complement and the HLA region on chromosome No. 6 in man

Summary The family of an individual was studied who lacks the seventh component of complement in his serum (C7 homozygous deficiency). Both parents are C7 heterozygousdeficient. In this investigation, the following parameters were determined: complement components in functional and immunochemical tests; HLA-A,B antigens, HLA-D (MLC) determinants; the Bf system; glyoxalase I and B cell antigens. No evidence for linkage between the immunogenetic linkage group on chromosome 6 and gene(s) controlling the synthesis of the seventh component of complement was obtained. This is in accordance with the assumption that only genes controlling components of the initiating rather than the membrane attack unit of complement are linked to the HLA region.     

Disease-causing mutations in genes of the complement system

Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of “conventional” complement deficiencies with these newly described developmental roles.     

Beyond C4: Analysis of the complement gene pathway shows enrichment for IQ in patients with psychotic disorders and healthy controls

Variation in cognitive performance, which strongly predicts functional outcome in schizophrenia (SZ), has been associated with multiple immune‐relevant genetic loci. These loci include complement component 4 (C4A), structural variation at which was recently associated with SZ risk and synaptic pruning during neurodevelopment and cognitive function. Here, we test whether this genetic association with cognition and SZ risk is specific to C4A, or extends more broadly to genes related to the complement system. Using a gene‐set with an identified role in “complement” function (excluding C4A), we used MAGMA to test if this gene‐set was enriched for genes associated with human intelligence and SZ risk, using genome‐wide association summary statistics (IQ; N = 269 867, SZ; N = 105 318). We followed up this gene‐set analysis with a complement gene‐set polygenic score (PGS) regression analysis in an independent data set of patients with psychotic disorders and healthy participants with cognitive and genomic data (N = 1000). Enrichment analysis suggested that genes within the complement pathway were significantly enriched for genes associated with IQ, but not SZ. In a gene‐based analysis of 90 genes, SERPING1 was the most enriched gene for the phenotype of IQ. In a PGS regression analysis, we found that a complement pathway PGS associated with IQ genome‐wide association studies statistics also predicted variation in IQ in our independent sample. This association (observed across both patients and controls) remained significant after controlling for the relationship between C4A and cognition. These results suggest a robust association between the complement system and cognitive function, extending beyond structural variation at C4A.     

The sea urchin genome as a window on function

The emphasis on the sequencing of genomes seems to make this task an end in itself. However, genome sequences and the genes that are predicted from them are really an opportunity to examine the biological function of the organism constructed by that genome. This point is illustrated here by examples in which the newly annotated gene complement reveals surprises about the way Strongylocentrotus purpuratus, the purple sea urchin, goes about its business. The three topics considered here are the nature of the innate immune system; the unexpected complexity of sensory function implied by genes encoding sensory proteins; and the remarkable intricacy of the regulatory gene complement in embryogenesis.