RESUMO
Complement factor I is a serine proteinase that regulates the classical and alternative pathways of complement by cleaving C3b and C4b and preventing the assembly of C3 and C5 convertase enzymes. In order to understand the regulation of factor I gene expression in liver cells, 4kb of the 5' flanking region of the gene was cloned, and the 1474-bp 3'-end was sequenced and shown to contain a number of transcription factor consensus sequences. A major and two minor transcription start sites were identified, respectively, at 152, 178, and 198bp upstream of the translation start site by primer extension analysis. The transcriptional activity of the 1474-bp fragment was analyzed by fusion of 5' deletion constructs to a cat-encoding gene expression vector and transient transfections into Hep G2 cells. A 273-bp fragment located at -112 to +161 relative to the major transcription start site was sufficient for promoter activity. The 3' fragment spanning +3 to +161 and containing a TATA-like element did not demonstrate promoter activity, suggesting that the core promoter resides in a 115-bp sequence located between -112 and +3. This region contains an Inr-like element overlapping the major cap site and a CTF-NF1 element, two potential CCAAT boxes and an AP-2 element partially overlapping an Sp-1 site. Thus, factor I promoter may belong to the TATA-less Inr-driven class II promoters whose transcription is regulated by Sp-1. The transcriptional activity of the 1474-bp 5' flanking fragment was upregulated by PMA, IL-6 and TNF-alpha, suggesting that factor I may be an acute phase reactant.
Assuntos
Clonagem Molecular , Fator I do Complemento/genética , Regiões Promotoras Genéticas , Sequência de Bases , Humanos , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Sequências Repetitivas de Ácido Nucleico , TATA Box , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Regulação para CimaRESUMO
Utilizing a recently developed method (Boradeption) for transferring water-insoluble hydroxyorganoborane compounds into the cells, we observed inhibition of protein synthesis by three of these compounds and inhibition of secretion of plasma proteins by four of them in human hepatoma HepG2 cells. These effects were specific in that the cell viability was not affected and an increase in protein catabolism was not observed. Three compounds caused a compound-specific alterations in the electrophoretic mobility of secreted glycoproteins due to underlying changes in the N-linked carbohydrate moieties. Results presented suggest a potential new source of cellular probes.
Assuntos
Proteínas Sanguíneas/metabolismo , Boranos/farmacologia , Glicoproteínas/sangue , Proteínas Sanguíneas/biossíntese , Proteínas Sanguíneas/genética , Carcinoma Hepatocelular , Linhagem Celular , Humanos , Neoplasias Hepáticas , Peso Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Relação Estrutura-Atividade , alfa 1-Antitripsina/biossínteseRESUMO
Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.
Assuntos
Cromossomos Humanos Par 4 , DNA/análise , Endopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Fator I do Complemento , Humanos , Mosaicismo , Hibridização de Ácido NucleicoRESUMO
Interleukin-1 (IL-1) is a product of mononuclear phagocytes that mediates changes characteristic of the response to inflammation or tissue injury (the acute-phase response). One of two structurally and functionally homologous major histocompatibility complex (MHC) class III genes encodes a positive acute-phase protein, complement factor B. The closely linked complement C2 gene is not affected during the acute-phase response. Purified human IL-1, pH 7.0, and recombinant-generated murine IL-1, pH 5.0, increased the expression of factor B and other positive acute-phase proteins in human hepatoma cells but decreased the expression of albumin, a negative acute-phase reactant. Furthermore, in a murine fibroblast L-cell line transfected with cosmid DNA bearing the human C2 and factor B genes, IL-1 mediated a reversible dose- and time-dependent increase in factor B expression in the transfected cells. Expression of the C2 gene was not affected by IL-1. The effect of IL-1 on factor B expression involves a mechanism acting at a pre-translational level as demonstrated by an increase in specific messenger RNA content and a corresponding increase in biosynthesis and secretion of factor B. The structural basis and mechanism for selective and independent regulation of these genes provides insight into the molecular control of the inflammatory response.
Assuntos
Interleucina-1/farmacologia , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/metabolismo , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , CamundongosRESUMO
The net production of the complement protein C2, C4, and factor B differ among mononuclear phagocytes from peripheral blood and different tissues in experimental animals and in humans. To examine the mechanisms that regulate these differences in humans, the proportions of C2-producing cells, the average single-cell production rate of C2, the posttranslational glycosylation and kinetics of secretion of C2 and factor B, and the amounts of C2 and factor B mRNA were examined in freshly isolated peripheral blood monocytes, monocytes maintained up to 2 wk in culture, and freshly isolated tissue macrophages from breast milk and bronchoalveolar lavage. In addition, the biosynthesis of two other proteins synthesized and secreted by mononuclear phagocytes, C3 and lysozyme, were examined. We report that despite comparable rates of C3 and lysozyme synthesis and similar processing and kinetics of secretion of C2 and factor B, the freshly isolated tissue macrophage differs from the monocyte-derived macrophage in the proportion of C2-producing cells, in the average single-cell production rate of complement, and in the amounts of specific C2 and factor B mRNA. These differences are tissue specific, because C2-specific mRNA content in bronchoalveolar macrophages is considerably greater than in breast milk macrophages, although the amounts of factor B mRNA are comparable. These data suggest that tissue-specific regulation of complement production in human mononuclear phagocytes occurs at a pretranslational level. These studies now provide a basis for investigation of the molecular effects of agents that modulate the biologic functions of monocytes and macrophages.
Assuntos
Proteínas do Sistema Complemento/biossíntese , Macrófagos/metabolismo , Monócitos/metabolismo , RNA Mensageiro/biossíntese , Diferenciação Celular , Complemento C2/biossíntese , Complemento C2/metabolismo , Complemento C3/biossíntese , Complemento C3/metabolismo , Complemento C4/biossíntese , Complemento C4/metabolismo , Fator B do Complemento/biossíntese , Fator B do Complemento/metabolismo , Humanos , Macrófagos/citologia , Leite Humano/citologia , Monócitos/citologia , Oligossacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Alvéolos Pulmonares/citologia , RNA Mensageiro/fisiologiaRESUMO
The second component of complement (C2), is a class III major histocompatibility complex gene product and a glycoprotein in the classical complement activating system. Synthesis in the human hepatoma-derived cell line HepG2 results in three intracellular forms: an 84-kDa form secreted in 1-2 h; 79-kDa and 70-kDa forms that remain cell-associated for intervals up to 12 h. All three forms are C2 polypeptides as demonstrated by inhibition of immunoprecipitation with unlabeled C2 and the presence of common major peptide fragments following chymotryptic digestion. The cell-associated forms of C2 are not products of proteolysis as demonstrated by experiments with multiple proteinase inhibitors and by observations of the kinetics of synthesis. Inhibition of core glycosylation by tunicamycin and deglycosylation by acid hydrolysis indicate that the three intracellular C2 polypeptides are glycosylated to a similar extent. Although the 84-kDa form of C2 is susceptible to C1s cleavage, the two cell-associated forms are not. Cell-free biosynthesis by mRNA from HepG2 or human liver results in three primary translation products corresponding to the three unglycosylated forms of C2. These results indicate that HepG2 cells synthesize C2 protein in both secreted and cell-associated forms and that each form is derived from a separate primary translation product.
Assuntos
Complemento C2/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Carcinoma Hepatocelular , Linhagem Celular , Complemento C2/biossíntese , Complemento C2/metabolismo , Humanos , Cinética , Fígado/metabolismo , Neoplasias Hepáticas , Complexo Principal de Histocompatibilidade , Fragmentos de Peptídeos/análise , Tunicamicina/farmacologiaRESUMO
Human factor I is a two-chain plasma glycoprotein composed of disulfide-linked 50,000- and 38,000-dalton subunits. Analysis of its biosynthesis and postsynthetic processing demonstrated that factor I is synthesized as a single chain precursor (pro-I) that undergoes glycosylation and limited proteolysis to generate the native protein. One of three human hepatoma cell lines, HepG2 , secreted factor I predominantly (70-90%) in a single chain pro-I form. The other cell lines secrete factor I predominantly in its two chain native form. The defect in conversion of pro-I to I in HepG2 was protein specific since other multichain proteins, derived from single chain precursors, the third, fourth, and fifth components of complement were processed normally. Further analysis of the inefficient pro-I to I conversion by HepG2 revealed that Xenopus oocytes injected with HepG2 mRNA secreted factor I in a predominantly two-chain form. In addition, the apparent sizes of native factor I, transferrin, and alpha-1-antitrypsin secreted by the three hepatoma lines differed due to differences in postsynthetic processing.
Assuntos
Proteínas Sanguíneas/biossíntese , Carcinoma Hepatocelular/metabolismo , Proteínas Inativadoras do Complemento C3b/biossíntese , Complemento C4b , Neoplasias Hepáticas/metabolismo , Animais , Proteínas Sanguíneas/genética , Linhagem Celular , Proteínas Inativadoras do Complemento C3b/genética , Feminino , Fibrinogênio/isolamento & purificação , Humanos , Cinética , Substâncias Macromoleculares , Peso Molecular , Oócitos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , XenopusRESUMO
Northern blotting analysis has shown apo-E mRNA synthesis by human liver, HepG2 cells, and primary cultures of human monocyte macrophages but not by the macrophage-like cell line U937 and normal or transformed human fibroblasts. Cell-free translation has shown that the primary translation product of apo-E consists of one major and one minor isoprotein of apparent Mr = 28,500 and isoelectric points 6.20 and 6.02, respectively. These isoproteins differ by +1 and 0 charges from apo-E3 and have been designated preapo-E. Co-translational treatment of mRNA with dog pancreatic membranes converts both preapo-E isoproteins to a form which is undistinguishable by two-dimensional gel electrophoresis from plasma apo-E3. The isolation and nucleotide sequence analysis of a full length apo-E cDNA clone has shown that preapo-E contains an 18-amino acid NH2-terminal signal peptide compared to plasma apo-E. The signal peptide sequence is: MetLysValLeuTrpAlaAlaLeuLeuValThrPheLeuAlaGlyCysGlnAla. Comparison of co-translationally modified apo-E with intracellular, secreted, and plasma forms indicates that after the intracellular cleavage of the signal peptide, the protein is glycosylated with carbohydrate chains containing sialic acid, secreted as sialoapo-E (apo-Es), and subsequently desialated in plasma. These findings demonstrate that apo-E is synthesized as preprotein and undergoes intracellular proteolysis and glycosylation and extracellular desialation to attain the major asialoapo-E isoprotein form observed in plasma.
Assuntos
Apolipoproteínas/genética , Lipoproteínas HDL/genética , Fígado/metabolismo , Macrófagos/metabolismo , Peptídeos/metabolismo , Biossíntese de Proteínas , Transcrição Gênica , Sequência de Aminoácidos , Apolipoproteínas E , Carcinoma de Células Escamosas , Linhagem Celular , Células Cultivadas , Enzimas de Restrição do DNA , Fibroblastos/metabolismo , Humanos , Hibridização de Ácido Nucleico , Sinais Direcionadores de Proteínas , RNA Mensageiro/genéticaRESUMO
In order to characterize a genetic deficiency of C2 in guinea pigs, production of C2 by peritoneal macrophage cultures derived from four normal, four heterozygous deficient, and four homozygous deficient animals was measured functionally and immunochemically after metabolic labeling with 35S-methionine. Macrophage monolayers from homozygous deficient animals failed to secrete hemolytically detectable C2 up to 74 hr in culture. A single cell hemolytic plaque assay also failed to demonstrate any functional C2 production by cells from homozygous deficient animals. No C2 protein was detected in media from three of the four homozygous deficient animals, but in one, apparent C2 fragments were present. In contrast, intracellular C2 protein was identified in all four homozygous deficient cell cultures. Its mobility on SDS-PAGE was slightly faster than normal. Much less abnormal intracellular C2 protein was recovered from homozygous deficient macrophage monolayers than intracellular C2 protein from normal macrophage monolayers. Monolayers from heterozygous animals produced functional and immunochemical C2 at approximately 30% of the normal rate. Normal rates of biosynthesis and secretion of two other MHC-linked class III antigens, C4 and factor B, were detected in macrophage cultures from homozygous and heterozygous deficient animals. These data suggest that a specific defect, i.e. a structural abnormality in C2 protein, underlies C2 deficiency in guinea pigs.
Assuntos
Complemento C2/biossíntese , Macrófagos/metabolismo , Animais , Antígenos/análise , Complemento C2/deficiência , Complemento C2/imunologia , Complemento C4/análise , Complemento C4/biossíntese , Fator B do Complemento/biossíntese , Feminino , Cobaias , Hemólise , Macrófagos/enzimologia , Macrófagos/imunologia , MasculinoRESUMO
Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.
Assuntos
Complemento C2/metabolismo , Complemento C4/metabolismo , Fator B do Complemento/metabolismo , Precursores Enzimáticos/metabolismo , Animais , Líquido Ascítico/citologia , Metabolismo dos Carboidratos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Cobaias , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Precursores de Proteínas/metabolismo , Tunicamicina/farmacologiaRESUMO
A 180,000-dalton single-chain molecule (human pro-C3) is the precursor of the third component of human complement (C3), a disulfide-linked two-chain protein. The pro-C3 is converted by limited proteolysis to C3. The relationship between pro-C3 and C3 was established with the use of Hep G2, a cell line derived from a human hepatocellular carcinoma, which synthesizes at least 17 plasma proteins.
Assuntos
Complemento C3/biossíntese , Neoplasias Hepáticas Experimentais/metabolismo , Animais , Linhagem Celular , Humanos , Substâncias Macromoleculares , Peso Molecular , Precursores de Proteínas/metabolismoRESUMO
The NH2-terminal structures of the intracellular precursor of the third component of guinea pig complement (pro-C3), synthesized in macrophage cultures, and beta chain of C3 from guinea pig plasma were determined. Six of the first 16 residues of pro-C3 were identified by a microradiosequencing method. All six are nonpolar and are identical with the residues in beta chain of native plasma C3. This establishes beta chain as the NH2-terminal subunit in pro-C3. A comparison of primary structures of guinea pig and human C3 beta chains revealed identity of at least 8 residues within the NH2-terminal decapeptide. Incubation of plasmin with radiolabeled pro-C3 in macrophage lysate resulted in loss of pro-C3 and the generation of a two-chain molecule electrophoretically indistinguishable from native C3. These data indicate similarities between pro-C3 and the precursor of the fourth complement component, pro-C4, with respect to subunit organization, presence of phylogenetically conserved NH2-terminal regions, and the probable mechanism for generation of their respective native proteins.