RESUMEN
The regulators of complement activation (RCA) gene cluster in 1q31-1q32 includes most of the genes encoding complement regulatory proteins. Genetic variability in the RCA gene cluster frequently involve copy number variations (CNVs), a type of chromosome structural variation causing alterations in the number of copies of specific regions of DNA. CNVs in the RCA gene cluster often relate with gene rearrangements that result in the generation of novel genes, carrying internal duplications or deletions, and hybrid genes, resulting from the fusion or exchange of genetic material between two different genes. These gene rearrangements are strongly associated with a number of rare and common diseases characterized by complement dysregulation. Identification of CNVs in the RCA gene cluster is critical in the molecular diagnostic of these diseases. It can be done by bioinformatics analysis of DNA sequence data generated by massive parallel sequencing techniques (NGS, next generation sequencing) but often requires special techniques like multiplex ligation-dependent probe amplification (MLPA). This is because the currently used massive parallel DNA sequencing approaches do not easily identify all the structural variations in the RCA gene cluster. We will describe here how to use the MLPA assays and two computational tools to analyze NGS data, NextGENe and ONCOCNV, to detect CNVs and gene rearrangements in the RCA gene cluster.
Asunto(s)
Activación de Complemento/genética , Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Aberraciones Cromosómicas , Enzimas Activadoras de Complemento/genética , Vía Alternativa del Complemento/genética , Vía Clásica del Complemento/genética , Proteínas del Sistema Complemento/genética , Variaciones en el Número de Copia de ADN , Reordenamiento Génico , Pruebas Genéticas/métodos , Humanos , Familia de Multigenes/genética , Análisis de Secuencia de ADNRESUMEN
Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a ß-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.
Asunto(s)
Enzimas Activadoras de Complemento/química , Activación de Complemento , Proteínas Inactivadoras de Complemento/química , Diseño de Fármacos , Modelos Moleculares , Ingeniería de Proteínas , Animales , Sitios de Unión , Enzimas Activadoras de Complemento/genética , Enzimas Activadoras de Complemento/metabolismo , Factor H de Complemento/química , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Inactivadores del Complemento/química , Inactivadores del Complemento/metabolismo , Inactivadores del Complemento/farmacología , Proteínas Inactivadoras de Complemento/genética , Proteínas Inactivadoras de Complemento/metabolismo , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
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.
Asunto(s)
Activación de Complemento/genética , Proteínas del Sistema Complemento/genética , Genes Letales , Mutación , Sustitución de Aminoácidos , Animales , Colectinas/genética , Enzimas Activadoras de Complemento/química , Enzimas Activadoras de Complemento/genética , Humanos , Ratones , Polimorfismo Genético , Conformación ProteicaRESUMEN
Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host's immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity approximately 200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.
Asunto(s)
Enzimas Activadoras de Complemento/antagonistas & inhibidores , Enzimas Activadoras de Complemento/metabolismo , Virus de la Viruela/inmunología , Proteínas de la Matriz Viral/antagonistas & inhibidores , Proteínas de la Matriz Viral/fisiología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión/genética , Sitios de Unión/inmunología , Sitios de Unión de Anticuerpos , Células CHO , Enzimas Activadoras de Complemento/genética , Complemento C3b/metabolismo , Cricetinae , Cricetulus , Glicosaminoglicanos/antagonistas & inhibidores , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Humanos , Hibridomas , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Virus de la Viruela/genética , Virus de la Viruela/patogenicidad , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
A fully active complement system deriving from the maternal circulation as well as from local production by various cell source is present in the placenta. The role of this system at the placental level, as in any other tissue in the body, is to protect both the fetus and the mother against infectious and other toxic agents. As fetal tissues are semi-allogeneic and alloantibodies commonly develop in the mother, the placenta is potentially subject to complement-mediated immune attack at the feto-maternal interface with the potential risk of fetal loss. Uncontrolled complement activation is prevented in successful pregnancy by the three regulatory proteins DAF, MCP and CD59 positioned on the surface of trophoblasts. The critical role played by these complement regulators is supported by the embryonic lethality observed in mice deficient in the complement regulator Crry. Excessive complement activation in the placenta places the fetus at risk for growth restriction or death. The role played by the complement system in the fetal damage induced by anti-phospholipid antibodies in a mouse model will be examined.
Asunto(s)
Activación de Complemento/inmunología , Proteínas Inactivadoras de Complemento/inmunología , Transfusión Fetomaterna/inmunología , Isoanticuerpos/inmunología , Intercambio Materno-Fetal/inmunología , Trofoblastos/inmunología , Animales , Anticuerpos Antifosfolípidos/inmunología , Enzimas Activadoras de Complemento/genética , Enzimas Activadoras de Complemento/inmunología , Activación de Complemento/genética , Proteínas Inactivadoras de Complemento/genética , Femenino , Muerte Fetal/genética , Muerte Fetal/inmunología , Muerte Fetal/patología , Transfusión Fetomaterna/genética , Transfusión Fetomaterna/patología , Humanos , Intercambio Materno-Fetal/genética , Ratones , Ratones Noqueados , Embarazo , Receptores de Complemento/deficiencia , Receptores de Complemento/inmunología , Receptores de Complemento 3b , Trofoblastos/patologíaRESUMEN
Analysis of the human MASP-1/3 gene, which encodes two proteases of the lectin-triggered complement cascade, has revealed alternatively used serine-protease-encoding regions for the gene's two protein products. Phylogenetic studies indicate that one arose by retrotransposition early in vertebrate evolution, supporting the idea that the lectin branch of the complement cascade arose earlier than the 'classical' pathway.
Asunto(s)
Enzimas Activadoras de Complemento/genética , Evolución Molecular , Serina Endopeptidasas/genética , Empalme Alternativo , Animales , Activación de Complemento/genética , Genes/genética , Humanos , Isoenzimas/genética , Serina Proteasas Asociadas a la Proteína de Unión a la ManosaRESUMEN
Although subcomponents C1r and C1s of the first complement component, C1, have been established to be in the same linkage group as the proline-rich protein gene cluster on chromosome 12p13.2, no direct analysis of linkage between the C1R and C1S structural gene loci has been available. We have detected through a population screening study 5 families which are heterozygous at the structural loci for both C1R and C1S. Three of the 5 families, 21 individuals, were informative for linkage. A maximum lod score of 1.505 at theta = 0.00 was found in a two-point analysis between C1R and C1S. Ten populations were screened for structural variation at the C1S locus. Only US Whites and a Kodiak Island Eskimo group expressed heterogeneity. The frequencies of the designated alleles, C1S*1 and C1S*2, were 0.992 and 0.007, respectively, in the US White population and 0.998 and 0.002, respectively, in the Kodiak Island Eskimo population. In addition, the product of a putative new allele, designated C1S*4, was observed in a single US White individual but segregation of this variant was not observed in the limited family data available.
Asunto(s)
Proteínas Sanguíneas/genética , Enzimas Activadoras de Complemento/genética , Complemento C1s/genética , Ligamiento Genético , Genética de Población , Complemento C1/genética , Complemento C1r , Femenino , Frecuencia de los Genes , Humanos , Masculino , Linaje , Fenotipo , Grupos Raciales , Estados UnidosRESUMEN
Isoelectric focusing and immunoblotting reveals considerable biochemical and genetic variation in the C1R subcomponent of the first complement component. The nature of the intraindividual biochemical variation can be explained by differences in sialic acid content because after digestion with neuraminidase the terminal sialic acids are removed to yield a single major band corresponding to the C1R polypeptide. Plasma samples from a large number of different ethnic groups, consisting of U.S. whites, U.S. blacks, Nigerian blacks, and Inuit, Aleut, and Amerindian populations from the Western Hemisphere have revealed genetically determined charge variation with heterozygous phenotypes consisting of two major asialo bands, indicating that the underlying variation is not due to variation in sialic acid content. Two previously reported common alleles, C1R*1 and CIR*2, have been observed in all studied populations, the notable exception being the Dogrib Indian population, which is devoid of the C1R*2 allele. Several new alleles--designated C1R*3, C1R*4, C1R*5, C1R*6, and C1R*7-have been observed, with variable frequencies ranging from the occurrence in a single individual and related family members to the polymorphic occurrence of certain alleles in several populations. Of these new alleles, the C1R*5 is of considerable interest in population and anthropological genetics studies. The C1R*5 allele is widely distributed, at a frequency of .03 to .17, in all of the North American aboriginal populations screened. This allele is not present in U.S. whites but is present at a polymorphic frequency in U.S. and Nigerian blacks.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Enzimas Activadoras de Complemento/genética , Complemento C1/genética , Polimorfismo Genético , Alelos , Complemento C1r , Humanos , Focalización Isoeléctrica , FenotipoRESUMEN
Complementary DNA clones for human C1s were isolated from cDNA libraries that were prepared with poly(A)+ RNAs of human liver and HepG2 cells. A clone with the largest cDNA insert of 2664 base pairs (bp) was analyzed for its complete nucleotide sequence. It contained 202 bp of a 5' untranslated region, 45 bp of coding for a signal peptide (15 amino acid residues), 2019 bp for complement component C1s zymogen (673 amino acid residues), 378 bp for a 3' untranslated region, a stop codon, and 17 bp of a poly(A) tail. The amino acid sequence of C1s was 40.5% identical to that of C1r, with excellent matches of tentative disulfide bond locations conserving the overall domain structure of C1r. DNA blotting and sequencing analyses of genomic DNA and of an isolated genomic DNA clone clearly showed that the human genes for C1r and C1s are closely located in a "tail-to-tail" arrangement at a distance of about 9.5 kilobases. Furthermore, RNA blot analyses showed that both C1r and C1s genes are primarily expressed in liver, whereas most other tissues expressed both C1r and C1s genes at much lower levels (less than 10% of that in liver). Multiple molecular sizes of specific mRNAs were observed in the RNA blot analyses for both C1r and C1s, indicating that alternative RNA processing(s), likely an alternative polyadenylylation, might take place for both genes.
Asunto(s)
Enzimas Activadoras de Complemento/genética , Complemento C1/genética , Complemento C1s/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , Complemento C1r , ADN/análisis , Humanos , Hígado/análisis , Datos de Secuencia Molecular , Poli A/análisis , ARN/análisis , ARN MensajeroRESUMEN
Genetic polymorphism of the C1R subcomponent of human complement component C1 has been investigated in neuraminidase treated EDTA plasma samples of 440 healthy Japanese individuals living in Tokyo by means of thin-layer polyacrylamide gel isoelectric focusing (PAGIEF) at pH 3.5-9.5 in the presence of 8.0 M urea followed by an electroblotting with enzyme immunoassay. Three common and three rare alleles were detected in the Japanese population. Of these, two common alleles were identical to C1R*1 and C1R*2 and other new alleles were tentatively designated C1R*3, C1R*4, C1R*5 and C1R*6, respectively. The results of the family studies suggested that the genetic model for C1R polymorphism assumed autosomal codominant Mendelian inheritance. The allele frequencies were estimated as C1R*1 = 0.4216, C1R*2 = 0.3602, C1R*3 = 0.2068, C1R*4 = 0.0091 and C1R*R(C1R*5 and C1R*6) = 0.0023, respectively. The distribution of allotypes fitted the Hardy-Weinberg equilibrium. The C1R system provides a useful genetic marker for human genetics, anthropologic studies and forensic science.
Asunto(s)
Enzimas Activadoras de Complemento/genética , Complemento C1/genética , Genética de Población , Polimorfismo Genético , Alelos , Mapeo Cromosómico , Complemento C1r , Frecuencia de los Genes , Humanos , JapónRESUMEN
Our study was undertaken to determine if native DNA (dsDNA), which is known to bind to collagen, also binds to the collagen-like segment of Clq (CLS). Three methods were employed to demonstrate the binding of dsDNA to CLS: (1) Six human sera and a mouse monoclonal antibody to dsDNA were employed in an enzyme linked immunosorbent assay to detect CLS bound dsDNA. When applied to CLS bound dsDNA, sera with antibodies to dsDNA and monoclonal antibody to DNA yielded mean OD values of greater than or equal to 0.6, significantly higher than those values obtained from control experiments (mean OD less than or equal to 0.2, p less than or equal to 0.003). (2) In a solid phase immunoassay radiolabelled DNA (3H DNA) was exposed to increasing amounts of solid phase adherent CLS. The binding of 3H DNA to the solid phase was substantially greater (more than a 30-fold increase) when the solid phase had been precoated with CLS. (3) dsDNA binding to CLS was demonstrated further by incubating electrophoretically resolved CLS with dsDNA and localizing the bound dsDNA by ethidium bromide staining. Our results indicate the dsDNA binds to CLS. Since CLS is the binding site for Clr and then Clr2Cls2, molecules which bind to CLS, such as dsDNA, could be important factors affecting Cl activation.
Asunto(s)
Colágeno/genética , Enzimas Activadoras de Complemento/metabolismo , Complemento C1/metabolismo , ADN/metabolismo , Enzimas Activadoras de Complemento/análisis , Enzimas Activadoras de Complemento/genética , Activación de Complemento , Complemento C1/análisis , Complemento C1/genética , Complemento C1q , Difusión , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Colagenasa Microbiana/farmacología , Peso MolecularRESUMEN
C1r and C1s are distinct, but structurally and functionally similar, serine protease zymogens responsible for the enzymatic activity of the first component of complement (C1). Recent comparisons indicate a significant degree of sequence similarity between C1r and C1s and support the hypothesis that they are related by gene duplication. Complementary DNA probes for human C1r and C1s do not cross-hybridize even at mild stringency conditions and are therefore gene-specific. Using a panel of 25 human-rodent cell hybrids, we have independently assigned the C1r and the C1s genes to chromosome 12. In situ hybridization analyses were consistent with these assignments, showing in addition that both C1r and C1s are located on the short arm of the chromosome in the region p13. These data suggest that the homologous C1r and C1s genes have remained closely linked after duplication of a common ancestor. The C1r and C1s loci also provide useful polymorphic DNA markers for the short arm of chromosome 12.
Asunto(s)
Cromosomas Humanos Par 12 , Enzimas Activadoras de Complemento/genética , Complemento C1/genética , Complemento C1s/genética , Animales , Mapeo Cromosómico , Complemento C1r , Cricetinae , Cricetulus , ADN/genética , Marcadores Genéticos , Humanos , Células Híbridas/análisis , Ratones , Hibridación de Ácido NucleicoRESUMEN
In humoral defence, pathogens are cleared by antibodies acting as adaptor molecules: they bind to antigen and trigger clearance mechanisms such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and complement lysis. The first step in the complement cascade is the binding of C1q to the antibody. There are six heads on C1q, connected by collagen-like stems to a central stalk, and the isolated heads bind to the Fc portion of antibody rather weakly, with an affinity of 100 microM (ref. 3). Binding of antibody to multiple epitopes on an antigenic surface, aggregates the antibody and this facilitates the binding of several C1q heads, leading to an enhanced affinity of about 10 nM (ref. 1). Within the Fc portion of the antibody, C1q binds to the CH2 domain. The interaction is sensitive to ionic strength, and appears to be highly conserved throughout evolution as C1q reacts with IgG from different species (for example see ref. 8). By systematically altering surface residues in the mouse IgG2b isotype, we have localized the binding site for C1q to three side chains, Glu 318, Lys 320 and Lys 322. These residues are relatively conserved in other antibody isotypes, and a peptide mimic of this sequence is able to inhibit complement lysis. We propose that this sequence motif forms a common core in the interactions of IgG and C1q.
Asunto(s)
Enzimas Activadoras de Complemento/inmunología , Complemento C1/inmunología , Inmunoglobulina G/inmunología , Animales , Sitios de Unión , Enzimas Activadoras de Complemento/genética , Complemento C1/genética , Complemento C1q , Hemólisis , Humanos , Fragmentos Fc de Inmunoglobulinas , Ratones , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
We have isolated cDNA clones covering the complete B chain of the complement subunit C1q from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide, all of the 228 amino acids of the mature protein and 140 nucleotides of the 3' untranslated region, including a poly A addition signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of C1q. By comparing the nucleotide sequence of the mouse B chain of C1q with the B chain from human (Reid, K. B. M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.
Asunto(s)
Enzimas Activadoras de Complemento/genética , Complemento C1/genética , ADN/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Complemento C1q , Humanos , Ratones , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/genética , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Southern blot analysis of the B-chain genes in one of eight C1q-deficient individuals revealed an abnormal banding pattern. The defect, which was homozygous, could be localized by restriction mapping to a single Taq I site within residue 150 in the coding region of the B-chain gene. DNA sequencing across the site revealed a stop codon that would cause premature termination of the protein product. No material corresponding to the A or C chains, or a truncated B chain, could be identified by antigenic analysis of the patient's serum, indicating that a complete B chain is required for secretion of a C1q molecule.
Asunto(s)
Enzimas Activadoras de Complemento/genética , Complemento C1/genética , Síndromes de Inmunodeficiencia/genética , Codón , Enzimas Activadoras de Complemento/deficiencia , Complemento C1/deficiencia , Complemento C1q , Femenino , Humanos , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , Regiones Terminadoras GenéticasAsunto(s)
Antígenos CD59 , Proteínas Portadoras , Proteínas del Sistema Complemento/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/fisiología , Enzimas Activadoras de Complemento/genética , Enzimas Activadoras de Complemento/fisiología , Proteínas Inactivadoras del Complemento 1/genética , Proteínas Inactivadoras del Complemento 1/fisiología , Proteínas del Sistema Complemento/fisiología , Humanos , Biología Molecular , Relación Estructura-ActividadRESUMEN
We have isolated cDNA clones covering the complete B chain of the complement subunit Clq from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide; all of the 228 amino acids of the mature protein; and 140 nucleotides of the 3' untranslated region, including a poly A additional signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of Clq. By comparing the nucleotide sequence of the mouse B chain of Clq with the human B chain (Reid, K.B.M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.
Asunto(s)
Enzimas Activadoras de Complemento/genética , Complemento C1/genética , ADN/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enzimas Activadoras de Complemento/análisis , Complemento C1/análisis , Complemento C1q , Ratones , Datos de Secuencia MolecularRESUMEN
The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s.
Asunto(s)
Clonación Molecular , Enzimas Activadoras de Complemento/genética , Complemento C1s/genética , ADN/análisis , Secuencia de Aminoácidos , Secuencia de Bases , Complemento C1s/análisis , Humanos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Overlapping molecular clones encoding the complement subcomponent Cls were isolated from a human liver cDNA library. The nucleotide sequence reconstructed from these clones spans about 85% of the length of the liver Cls messenger RNAs, which occur in three distinct size classes around 3 kilobases in length. Comparisons with the sequence of Clr, the other enzymatic subcomponent of Cl, reveal 40% amino acid identity and conservation of all the cysteine residues. Beside the serine protease domain, the following sequence motifs, previously described in Clr, were also found in Cls: (a) two repeats of the type found in the Ba fragment of complement factor B and in several other complement but also noncomplement proteins, (b) a cysteine-rich segment homologous to the repeats of epidermal growth factor precursor, and (c) a duplicated segment found only in Clr and Cls. Differences in each of these structural motifs provide significant clues for the interpretation of the functional divergence of these interacting serine protease zymogens. Hybridizations of Clr and Cls probes to restriction endonuclease fragments of genomic DNA demonstrate close physical linkage of the corresponding genes. The implications of this finding are discussed with respect to the evolution of Clr and Cls after their origin by tandem gene duplication and to the previously observed combined hereditary deficiencies of Clr and Cls.
Asunto(s)
Enzimas Activadoras de Complemento/genética , Complemento C1/genética , Complemento C1s/genética , ADN/genética , Genes , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Complemento C1r , Ligamiento Genético , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Plásmidos , Homología de Secuencia de Ácido NucleicoRESUMEN
Charge-based structural variation has been observed in the C1s subcomponent of the first complement component C1 after isoelectric focusing and immunoblotting. One common and two uncommon autosomal co-dominantly expressed alleles, designated C1S*1, C1S*2 and C1S*3, have been recognized at the C1S structural locus. The frequency of these alleles was 0.979, 0.016 and 0.005, respectively, in a U.S. white population. No variation at the C1S locus was observed in a U.S. black sample (n = 95).