RESUMEN
Activated complement component C4 (C4b) is the nonenzymatic component of the classical pathway (CP) convertases of the complement system. Preparation of C4 and C4b samples suitable for structural biology studies is challenging due to low yields and complexity of recombinant C4 production protocols reported so far and heterogeneity of C4 in native sources. Here we present a purification protocol for human C4 and describe sample preparation methods for structural investigation of C4 and its complexes by crystallography, small angle X-ray scattering, and electron microscopy.
Asunto(s)
Complemento C4/química , Complemento C4/aislamiento & purificación , Proteómica/métodos , Biología/métodos , Cromatografía en Gel/métodos , Cristalografía por Rayos X/métodos , Humanos , Intercambio Iónico , Microscopía Electrónica/métodos , Coloración Negativa/métodos , Conformación Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodosRESUMEN
During classical complement pathway activation, the internal thio-ester of both C3 and C4 becomes exposed which enables C3 and C4 to bind covalently to nearby molecules. Recently, we described that C3 and C4 bind to C1q, the recognition molecule of the classical pathway, upon activation of this pathway. Covalently linked complexes between C1q and activated C4 (C1q-C4 complexes) are specific markers for classical complement pathway activation. In the present study we further investigated the molecular characteristics of complexes between C1q and activated C3 or C4 that occur in vivo. In human serum only complexes of C1q with C3d or C4d fragments were detected but not those with the larger C3b/bi or C4b/bi fragments. We identified that C1q-C4 complexes circulate as part of the intact C1 complex instead of as free C1q. Finally, we investigated whether deposited C3d or C4d affect C1 haemolytic activity. We observed that both C1q-C3 and C1q-C4 complexes are significantly (P<0.05) less active in a C1q-haemolytic assay than non-complexed C1q. Thus, the dominant types of C1q complexes that circulate in vivo are C1q-C3d and C1q-C4d complexes. These complexes are still able to interact with C1r and C1s to form a C1 complex, but seem to have a reduced activity as compared to C1q not carrying C3- or C4-fragments.
Asunto(s)
Complemento C1q/inmunología , Complemento C3/inmunología , Complemento C4/inmunología , Hemólisis , Animales , Cromatografía de Afinidad , Activación de Complemento/efectos de los fármacos , Complemento C1q/aislamiento & purificación , Complemento C3/aislamiento & purificación , Complemento C3d/inmunología , Complemento C4/aislamiento & purificación , Humanos , Polietilenglicoles/farmacología , OvinosRESUMEN
Duplication and diversification of several complement components is a striking feature of bony fish complement systems. It gives an interesting insight into an evolutionary strategy for the possible enhancement of the repertoire of innate immunity. The present study is aimed at examining diversity in bony fish C4, a member of the thioester-containing complement components. Two diverged cDNA sequences sharing only approximately 32% identity at the amino acid level were isolated from the common carp and designated C4-1 and C4-2. C4-1 and C4-2 share a number of C4-like structural signatures, such as the thioester site and a disulfide-linked three-chain structure. Interestingly, they differ at the residue corresponding to the thioester-catalytic histidine, as seen in the human C4A and C4B isotypes, suggesting their distinct substrate specificities in the binding reaction of the thioester. Phylogenetic analysis indicates that the divergence of C4-1 and C4-2 predated the separation of the cartilaginous and bony fish lineages. Genomic Southern hybridization suggests the presence of single copy genes each encoding C4-1 and C4-2 in the carp genome. An activation fragment, C4a, was shown to be released from each isotype in carp serum activated via the classical and/or lectin pathways. Synthetic peptides representing a putative C2 binding site on C4-1 and C4-2 inhibited the classical pathway-mediated hemolytic activity of carp serum in a dose-dependent manner. The results suggest that C4-1 and C4-2 represent two major lineages of C4 that are present in carp serum, have distinct binding specificities, and are functional in the classical/lectin pathways of complement activation.
Asunto(s)
Carpas/genética , Carpas/inmunología , Complemento C4/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Complemento C4/genética , Complemento C4/fisiología , Dosificación de Gen , Duplicación de Gen , Hemólisis/genética , Hemólisis/inmunología , Humanos , Datos de Secuencia Molecular , Péptidos/genética , Isoformas de Proteínas/sangre , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 A resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process.
Asunto(s)
Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/química , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Sitios de Unión , Rastreo Diferencial de Calorimetría , Complemento C4/química , Complemento C4/aislamiento & purificación , Complemento C4/metabolismo , Cristalografía por Rayos X , Activación Enzimática , Estabilidad de Enzimas , Semivida , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/aislamiento & purificación , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Termolisina/farmacologíaRESUMEN
We have investigated the ability of HIV-1 protease to cleave human complement proteins of the classical complement pathway: C1q, C2 and C4 as well as the regulatory protein, C1-inhibitor. Purified complement proteins were incubated with recombinant HIV-1 protease in vitro and analyzed by SDS-PAGE and immunoblotting assay. The only cleavage site was found in N-terminal region of C1-inhibitor, and it was located between residues Leu-32 and Phe-33 as determined by amino acid sequence analysis of the 85 kDa proteolytic fragment after 12 Edman degradation cycles. The HIV-1 protease cleavage sites were not found in C1q, C2 and C4 protein. HIV-1 protease-susceptible site in N-terminal region of C1-inhibitor is very close to the cleavage sites of some other proteases that are able to induce N-terminal proteolysis of the protein.
Asunto(s)
Proteasa del VIH/metabolismo , Serpinas/metabolismo , Secuencia de Aminoácidos , Proteínas Inactivadoras del Complemento 1 , Proteína Inhibidora del Complemento C1 , Complemento C1q/aislamiento & purificación , Complemento C1q/metabolismo , Complemento C2/aislamiento & purificación , Complemento C2/metabolismo , Complemento C4/aislamiento & purificación , Complemento C4/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Serpinas/química , Serpinas/aislamiento & purificaciónRESUMEN
To clarify the evolutionary origin of the linkage of the MHC class III complement genes with the MHC class I and II genes, we isolated C4 cDNA from the banded hound shark (Triakis scyllium). Upon phylogenetic tree analysis, shark C4 formed a well-supported cluster with C4 of higher vertebrates, indicating that the C3/C4 gene duplication predated the divergence of cartilaginous fish from the main line of vertebrate evolution. The deduced amino acid sequence predicted the typical C4 three-subunits chain structure, but without the histidine residue catalytic for the thioester bond, suggesting the human C4A-like specificity. The linkage analysis of the complement genes, one C4 and two factor B (Bf) genes, to the shark MHC was performed using 56 siblings from two typing panels of T. scyllium and Ginglymostoma cirratum. The C4 and one of two Bf genes showed a perfect cosegregation with the class I and II genes, whereas two recombinants were identified for the other Bf gene. These results indicate that the linkage between the complement C4 and Bf genes, as well as the linkage between these complement genes and the MHC class I and II genes were established before the emergence of cartilaginous fish >460 million years ago.
Asunto(s)
Clonación Molecular , Activación de Complemento/genética , Complemento C4/química , Complemento C4/genética , Complejo Mayor de Histocompatibilidad/genética , Tiburones/genética , Tiburones/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular/métodos , Complemento C4/aislamiento & purificación , Factor B del Complemento/genética , Evolución Molecular , Genes MHC Clase I , Genes MHC Clase II , Ligamiento Genético/inmunología , Datos de Secuencia Molecular , Filogenia , Subunidades de Proteína/química , Subunidades de Proteína/clasificación , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificaciónRESUMEN
Contrary to infective anticardiolipin (aCL) antibodies, autoimmune aCL antibodies react with phospholipids (PL) mainly via binding to the plasma glycoprotein cofactor beta2-Glycoprotein I (beta2GPI). While there is a well-documented link between the risk of thrombosis and the presence of beta2GPI-dependent anticardiolipin antibodies, the pathological impact of other antiphospholipid antibodies is less clear. By means of cardiolipin affinity-chromatography, we isolated and identified 3 CL-binding proteins, complement component C4, complement factor H and a kallikrein-sensitive glycoprotein, and tested for the presence of autoantibodies against these proteins in patients with antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and other autoimmune diseases. High titers of autoantibodies to C4 as compared to age- and sex-matched healthy controls were present in 3 of 26 patients with APS, and weak titers were found in 2 of 26 patients with SLE and in none of 26 patients with other autoimmune diseases. Autoantibodies to complement factor H were found in 4 APS, 3 SLE and none of the other autoimmune patients. Autoantibodies to kallikrein-sensitive glycoprotein were detected in 6 APS patients, 1 SLE patient, and 1 patient with another autoimmune disease. A close relationship between these antibodies was found, suggesting their origin from a common macromolecular complex. However, no relationship with anti-beta2GPI antibodies was found, with the three patients with higher levels of autoantibodies having a low titer of anti-beta2GPI antibodies. In conclusion, some patients with APS harbor circulating antibodies to other CL-binding proteins which might be useful to further characterize these patients.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Autoanticuerpos/sangre , Cardiolipinas/metabolismo , Proteínas Portadoras/inmunología , Adolescente , Adulto , Factores de Edad , Secuencia de Aminoácidos , Anticoagulantes/inmunología , Síndrome Antifosfolípido/etiología , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/inmunología , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Complemento C4/inmunología , Complemento C4/aislamiento & purificación , Complemento C4/metabolismo , Factor H de Complemento/inmunología , Factor H de Complemento/aislamiento & purificación , Factor H de Complemento/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicoproteínas/inmunología , Humanos , Masculino , Análisis por Apareamiento , Persona de Mediana Edad , Datos de Secuencia Molecular , Factores Sexuales , beta 2 Glicoproteína IRESUMEN
Human intestinal epithelial cells have been established as local sites for complement biosynthesis. In this study, we investigated the effects of IFN-gamma and sodium butyrate on biosynthesis of MHC class III gene products (complement C4 and factor B) in the human fetal intestinal epithelial cell line INT-407. IFN-gamma induced a dose- and time-dependent increase in C4 and factor B secretion. However, sodium butyrate dose-dependently inhibited IFN-gamma-induced C4 and factor B secretion. These effects were also observed at the mRNA level. Immunoblotting indicated that IFN-gamma induced a rapid activation of Stat1alpha, and fluorescence immunohistochemistry detected a translocation of Stat1alpha into the nucleus within 1 h. However, the translocation of Stat1alpha was not affected by the addition of sodium butyrate. Nuclear run-on assay indicated that IFN-gamma induced a weak increase in the transcription rate of factor B gene, and sodium butyrate did not affect this response. IFN-gamma and sodium butyrate induced a counter-regulatory effect on C4 and factor B secretion: IFN-gamma acted as a potent inducer, but sodium butyrate potently abrogated these responses. These are mainly regulated through the post-transcriptional mechanism.
Asunto(s)
Butiratos/farmacología , Complemento C4/biosíntesis , Factor B del Complemento/biosíntesis , Interferón gamma/farmacología , Mucosa Intestinal/efectos de los fármacos , Northern Blotting , Línea Celular , Complemento C4/química , Complemento C4/genética , Complemento C4/aislamiento & purificación , Factor B del Complemento/química , Factor B del Complemento/genética , Factor B del Complemento/aislamiento & purificación , Medios de Cultivo Condicionados/química , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Mucosa Intestinal/metabolismo , Fosforilación , ARN Mensajero/biosíntesis , Factor de Transcripción STAT1 , Factores de Tiempo , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Previous studies based on the use of human serum as a source of C have provided evidence for the C-dependent enhancement of cell infection by HIV-1. The present study was undertaken to distinguish C from other serum factors and to identify the proteins and the mechanisms involved in C-dependent cell infection by HIV-1. The classical C activation pathway was reconstituted from the proteins C1q, C1r, C1s, C4, C2, C3, factor H, and factor I; each were purified to homogeneity. A mixture of these proteins at physiological concentrations was shown to reproduce the ability of normal human serum to enhance the infection of MT2 cells by HIV-1 at low doses of virus. This enhancing effect was abolished when heat-inactivated serum and C2- or C3-depleted serum were used, and was restored upon addition of the corresponding purified proteins. A mixture of two synthetic peptides corresponding to positions 10-15 and 90-97 of human C receptor type 2 (CD21) as well as soluble CD4 both inhibited the C-dependent infection process. These data provide unambiguous evidence that HIV-1 triggers a direct activation of the classical C pathway in vitro and thereby facilitates the infection of MT2 cells at low doses of virus. These findings are consistent with a mechanism involving increased interaction between the virus opsonized by C3b-derived fragment(s) and the CD21 cell receptors and subsequent virus entry through CD4 receptors.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , VIH-1/inmunología , Modelos Inmunológicos , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Línea Celular , Complemento C2/deficiencia , Complemento C2/fisiología , Complemento C3/deficiencia , Complemento C3/fisiología , Complemento C4/aislamiento & purificación , Complemento C4/fisiología , Factor H de Complemento/aislamiento & purificación , Factor H de Complemento/fisiología , Factor I de Complemento/aislamiento & purificación , Factor I de Complemento/fisiología , Relación Dosis-Respuesta Inmunológica , Sinergismo Farmacológico , Humanos , Receptores de Complemento 3d/química , Receptores de Complemento 3d/metabolismoRESUMEN
Complement components C3 and C4 have been isolated from the serum of the nurse shark (Ginglymostoma cirratum) and of the channel catfish (Ictalurus punctatus). As in the higher vertebrates, the fish C4 proteins have three-chain structures while the C3 proteins have two-chain structures. All four proteins have intra-chain thioesters located within their highest molecular mass polypeptides. N-terminal sequence analysis of the polypeptides has confirmed the identity of the proteins. In all cases except the catfish C3 alpha-chain, which appears to have a blocked N-terminus, sequence similarities are apparent in comparisons with the chains of C3 and C4 from higher vertebrates. We have confirmed that the activity/protein previously designated C2n is the nurse shark analogue of mammalian C4. This is the first report of structural evidence for C4 in both the bony and cartilaginous fish.
Asunto(s)
Complemento C3/química , Complemento C4/química , Ictaluridae/inmunología , Tiburones/inmunología , Secuencia de Aminoácidos , Animales , Complemento C3/aislamiento & purificación , Complemento C4/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A method is described for the affinity chromatographic purification of thiol ester proteins. These comprise the complement proteins C3 and C4 and the protease inhibitor alpha 2-macroglobulin (alpha 2M) and are known to contain an internal beta-cysteinyl-gamma-glutamyl thiol ester. The method employs aminoalkyl ligands coupled to a divinylsulfonyl-derivatized agarose matrix, and the length of the aminoalkyl spacer arm was found to be important for the effectiveness of the matrix. Optimal results were obtained with diaminododecyldivinylsulfonyl-agarose. Employing this matrix the thiol ester proteins C3, C4 and alpha 2M were isolated from human pregnancy serum. Application of the method to chicken and rainbow trout serum gave rise to isolation of several proteins including chicken and rainbow trout alpha 2M.
Asunto(s)
Cromatografía de Afinidad/métodos , Complemento C3/aislamiento & purificación , Complemento C4/aislamiento & purificación , alfa-Macroglobulinas/aislamiento & purificación , Animales , Western Blotting , Complemento C3/química , Complemento C4/química , Electroforesis en Gel de Poliacrilamida , Ésteres/química , Femenino , Hemólisis , Humanos , Embarazo , Ovinos , Compuestos de Sulfhidrilo/química , alfa-Macroglobulinas/químicaRESUMEN
Intoducción. Objetivo: evaluar las modificaciones en los niveles de IgA y la fracción 3 y 4 del complemento (C3 y C4) y las algutininas en la leche humana al ser sometida a calentamiento rápido empleando el horno de microondas. Material y métodos. Mediante un ensayo clínico (antes, después) en muestras de leche humana elegidas al azar, se estudiaron 46 muestas de leche, recolectadas en los primeros 5 días del puerperio, proveniente de madres con embarazo y parto sin complicaciones. Se fraccionaron las muestras para formar 2 grupos, la muestra de leche del primer grupo se mantuvo en temperatura ambiente (grupo 1) y la segunda se incluyó para ser calentada durante 30 segundos a temperatura máxima en un horno de microondas de marca comercial (grupo 2). En ambas muestras se determinaron las concentraciones de IgA total, y la aglutinación contra Candida albicans. Resultados. La temperatura a la que fueron sometidas las muestras del grupo 2 fue de 55.5 ºC (límite de 32 a 72 ºC). Al comparar los resultados entre ambos grupos se observaron que los valores de IgA fueron de 0.95 y 0.63 g/L (P< 0.001), C3 de 2.22 y 0.49 g/L (P< 0.01), C4 de 3.7 y 1.21 g/L (P< 0.0003) y las aglutininas con dilución de 1467 y 1156 (P< 0.04), para los grupos 1 y 2 respectivamente. La temperaturar crítica fue diferente para cada variable. Para la IgA y C3 fue de 50 ºC; para C4 y las aglutininas fue de 56 ºC. Conclusiones. Se documentó el efecto negativo en la concentración de IgA, C3 C4 y aglutininas por efecto del calentamiento en el horno de microondas. La temperatura generada por este medio es alta y difícilmente controlada. Por lo que no se recomienda el clentamiento de la leche humana en el horno de microondas
Asunto(s)
Aglutininas/análisis , Aglutininas/aislamiento & purificación , Atención , Complemento C3/aislamiento & purificación , Complemento C4/aislamiento & purificación , Inmunoglobulina A/análisis , Microondas/efectos adversos , Leche Humana , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Among the goals of the primary care workup are detection of serious disease and determination of whether referral--such as to a urologist or nephrologist--is indicated. Diagnosis is facilitated when hematuria is accompanied by other findings; isolated hematuria presents a more complex challenge. Measurement of serum complement levels may be helpful in narrowing the differential.
Asunto(s)
Glomerulonefritis Membranoproliferativa/complicaciones , Hematuria/etiología , Adulto , Factor Nefrítico del Complemento 3/aislamiento & purificación , Complemento C4/aislamiento & purificación , Diagnóstico Diferencial , Glomerulonefritis Membranoproliferativa/diagnóstico , Glomerulonefritis Membranoproliferativa/patología , Hematuria/diagnóstico , Humanos , Hipertensión Renal/etiología , Fallo Renal Crónico/etiología , Masculino , PronósticoRESUMEN
The fourth component of the complement system, C4, is encoded by two highly homologous MHC-linked genes expressing the two isotypes C4A and C4B. A gene size polymorphism (either 22.5 or 16 kb) has been described which depends on the presence or absence of a 6.5-kb insertion in intron 9 of the C4 gene. By sequencing a C4A-specific lambda clone from a human genomic library containing the long intron 9 as well as PCR-amplified DNA containing the short intron, the DNA sequences of both introns were determined. The long and short introns have lengths of 6,787 bp and 415 bp, respectively. The sequence of the short intron is almost identical (96%) to the corresponding parts of the long intron. At position 282 of the short intron, a 6,372-bp insertion is present in the long intron which has all characteristics of a full-length endogenous retrovirus. The proviral DNA is flanked by two 6-bp target site repeats. The orientation of the proviral sequence is opposite to that of the C4 coding strand. Long terminal repeats (LTRs) of 548 bp were found at both ends of the provirus. A TATA box and an SV40 enhancer core as well as a polyadenylation signal are present in the LTR. A 5' primer binding site for lysine tRNA was identified. The strongest sequence homologies were found in comparison to human endogenous retrovirus (HERV-K): between 65-88% for gag, pol and env genes. However, a search for open reading frames in these regions indicated the presence of multiple stop codons in all three reading frames.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Complemento C4/genética , Polimorfismo Genético , Retroviridae/genética , Integración Viral/genética , Composición de Base , Secuencia de Bases , Clonación Molecular , Complemento C4/aislamiento & purificación , Humanos , Intrones/inmunología , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos/genética , Retroviridae/química , TATA Box/genéticaRESUMEN
We have recently demonstrated that sex-limited protein (Slp) plays a key role in an EDTA-resistant mouse complement activation pathway. A rapid procedure, utilizing classical chromatography methods on an FPLC system, was developed for the isolation of functionally active Slp. The method is based on the fractionated precipitation of serum by polyethylene glycol 6000, followed by heparin Sepharose Cl-6B affinity chromatography, Mono Q anion exchange and Superose 12 gel filtration. The isolation of Slp was monitored by a hemolytic assay. The procedure resulted in the purification of Slp, which by SDS-PAGE gave a single band of M(r) 2000,000 under non-reducing conditions, and under reducing conditions three bands corresponding to M(rs) of 105,000, 76,000 and 37,000.
Asunto(s)
Proteínas Sanguíneas/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Antígenos H-2/aislamiento & purificación , Animales , Precipitación Química , Cromatografía de Afinidad , Complemento C4/aislamiento & purificación , Ensayo de Actividad Hemolítica de Complemento , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Peso Molecular , ConejosRESUMEN
Thiol compounds have been investigated as inhibitors of the covalent binding reaction of human complement protein C4 using Sepharose-C1s as a combined activating and binding surface. o- and p-substituted aminothiophenols are equally effective inhibitors, whereas the m-substituted compound is a less potent inhibitor. The anti-hypertensive drug captopril is also shown to inhibit the covalent binding reaction. A comparison of the effects of these compounds on the covalent binding reaction of isolated C4A and C4B has been made. Results suggest that a Pro-to-Leu substitution in C4B is likely to account for the differences in inhibitory potency of C4B compared with C4A observed with the aromatic inhibitors.
Asunto(s)
Aminofenoles/metabolismo , Complemento C4/metabolismo , Vía Clásica del Complemento/fisiología , Penicilamina/análogos & derivados , Compuestos de Sulfhidrilo/metabolismo , Aminofenoles/farmacología , Unión Competitiva , Captopril/metabolismo , Captopril/farmacología , Complemento C1s/metabolismo , Complemento C4/química , Complemento C4/efectos de los fármacos , Complemento C4/aislamiento & purificación , Complemento C4a/efectos de los fármacos , Complemento C4a/metabolismo , Complemento C4b/efectos de los fármacos , Complemento C4b/metabolismo , Humanos , Isomerismo , Penicilamina/metabolismo , Penicilamina/farmacología , Sefarosa/metabolismo , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/farmacologíaAsunto(s)
Complemento C3/aislamiento & purificación , Complemento C4/aislamiento & purificación , Animales , Anticuerpos Monoclonales , Cromatografía de Afinidad/métodos , Cromatografía por Intercambio Iónico/métodos , Complemento C3/análisis , Complemento C4/análisis , Electroforesis en Gel de Poliacrilamida/métodos , Cobayas , Hemólisis , Humanos , Indicadores y Reactivos , Sustancias Macromoleculares , Peso Molecular , OvinosRESUMEN
Disruption of the thiolester in native C4 yields a 'C4b-like C4' molecule (iC4) that functionally resembles C4b and is therefore probably accompanied by conformational changes in the C4 molecule. In most purified C4 preparations, iC4 and C4b are present to a variable extent. In this study we evaluated the use of fast protein liquid chromatography (FPLC) to resolve and isolate these various forms of C4. C4 was purified from fresh human plasma in a 4-step procedure that included barium citrate adsorption, polyethylene glycol 6000 (PEG) precipitation, Q-Sepharose Fast Flow and mono Q ion exchange chromatography. The final preparation appeared to be homogeneous on SDS-PAGE and under reducing conditions consisted of three bands that corresponded to the intact alpha, beta and gamma chains of C4. In some preparations the alpha' chain of C4b was also observed. On a Mono Q column the purified C4 preparations could be separated into three peaks that by hemolytic assay and SDS-PAGE were characterized as representing native C4, and monomeric and dimeric iC4 (or monomeric and dimeric C4b). Finally, the apparent KA of the various forms of C4 for C4b-binding protein (C4BP) was investigated. The monomeric iC4 and C4b species demonstrated similar C4BP binding affinity with an apparent KA of 5.6-6.4 x 10(8) M-1, whereas their dimeric forms demonstrated a higher affinity for C4BP with an apparent KA: 0.9-2.3 x 10(9) M-1. Binding of native C4 to C4BP was undetectable.
Asunto(s)
Complemento C4/aislamiento & purificación , Animales , Proteínas Portadoras/metabolismo , Cromatografía Liquida , Complemento C3/metabolismo , Complemento C4/análisis , Complemento C4/metabolismo , Complemento C4b/análisis , Complemento C4b/aislamiento & purificación , Humanos , Integrina alfaXbeta2 , ConejosRESUMEN
The fourth component of human complement (C4) is encoded at two separate but closely linked loci within the MHC on the short arm of chromosome 6. Thus, there are two types of C4 protein in most individual and pooled normal human sera (NHS): C4A and C4B. Incubation of individual sera, pooled NHS, or purified heterogeneous C4 (C4A/C4B) with bacterial sialidase at 37 degrees C increased C-mediated hemolysis of antibody-sensitized sheep erythrocytes 1.54- to 1.93-fold. Comparative studies of Tmax of human C2, using asialo-C4 or buffer-treated C4 on EAC1gp and extrapolation to time 0 indicated a z value 4-fold higher with asialo-C4. This indicated that more hemolytically active C42 complexes are available with sialidase-treated C4 compared to untreated C4. There was no appreciable difference in the % 125I-C4 bound to EAC1gp (sialidase- or buffer-treated). Sera from two different blood donors with C4A3 phenotype (C4BQ0), two different donors with C4B1 phenotype (C4AQ0), and serum from an individual heterozygous deficient at both C4A3 and C4B1 regions (A3, AQ0; B1, BQ0) were investigated. The C4 allotypes, purified from these sera, were treated with sialidase; the C4A3 was enhanced in hemolytic assays by sialidase-treatment (1.52- to 2.3-fold), whereas the C4B1 allotype was not enhanced. Fluorometric determinations revealed that approximately the same percentage of sialic acid was released from sialidase-treated C4A3 and C4B1. Therefore, the increase in hemolytic titer observed after treatment of NHS or purified heterogeneous C4 with sialidase is a property of C4A3 but not a property of C4B1.
Asunto(s)
Asialoglicoproteínas/fisiología , Complemento C4/fisiología , Asialoglicoproteínas/aislamiento & purificación , Tampones (Química) , Complemento C2/efectos de los fármacos , Complemento C2/fisiología , Complemento C4/efectos de los fármacos , Complemento C4/aislamiento & purificación , Complemento C4a/fisiología , Complemento C4b/fisiología , Relación Dosis-Respuesta Inmunológica , Humanos , Neuraminidasa/farmacocinética , Neuraminidasa/farmacologíaRESUMEN
Adsorbents from four commercially available devices, Protein A-Sepharose (Immunosorba Protein A-62,5; Excorim KB, Lund Sweden), Tryptophan-PVA (Immusorba TR-350; Asahi Medical Co., Tokyo, Japan), Phenylalanine-PVA (Immusorba PH-350; Asahi Medical Co., Tokyo, Japan), and Dextran sulfate (Liposorber LA-15; Kanegafuchi Chemical Co. Ltd, Osaka, Japan) were tested under optimal in vitro conditions to determine their adsorption capability for several plasma constituents which are usually the target of plasma therapy. The parameters of interest were: double stranded DNA-antibodies (anti-dsDNA), antiglomerular basement membrane antibodies (anti-GBM), anti-acetylcholin receptor antibodies (AChRAb), circulating immune complexes (CIC), rheumatoid factor (RF), IgA, IgG, IgM, IgE, C3c, C4, LDL-cholesterol, total cholesterol, erythropoietin (EPO) and beta 2-microglobulin (beta 2M). The IgG auto antibodies, CIC and RF can be removed by Protein A-Sepharose, Try-PVA and Phe-PVA. IgG is best adsorbed by Protein A-Sepharose, while IgE can be removed efficiently by Try-PVA. Dextran sulfate is without doubt the best adsorbent for LDL-cholesterol. All four adsorbents bind also complement components C3c and C4. No significant adsorption was found for EPO and beta 2M. The four devices exhibit a quite different adsorption profile which can be used as a guide for the optimal selection of an adsorption column in clinical apheresis.