RESUMEN
BACKGROUND: Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs. METHODS: The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients. RESULTS: ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects. CONCLUSIONS: Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection.
Asunto(s)
Anticuerpos , Receptor de Asialoglicoproteína/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Carcinoma Hepatocelular/patología , Queratinas/metabolismo , Neoplasias Hepáticas/patología , Células Neoplásicas Circulantes/patología , Animales , Receptor de Asialoglicoproteína/inmunología , Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Carcinoma Hepatocelular/sangre , Línea Celular Tumoral , Femenino , Humanos , Queratinas/inmunología , Neoplasias Hepáticas/sangre , Ratones Endogámicos BALB C , Células Neoplásicas Circulantes/inmunologíaRESUMEN
The wound repair process is a highly ordered sequence of events that encompasses haemostasis, inflammatory cell infiltration, tissue regrowth and remodelling. Wound healing follows tissue destruction so we hypothesized that antibodies might bind to wounded tissues, which would facilitate the engulfment of damaged tissues by macrophages. Here, we show that B cells, which produce antibodies to damaged tissues, are engaged in the process of wound healing. Splenectomy delayed wound healing, and transfer of spleen cells into splenectomized mice recovered the delay in wound healing. Furthermore, wound healing in splenectomized nude mice was also delayed. Transfer of enriched B220(+) cells by magnetic beads accelerated wound healing in splenectomized mice. We detected immunoglobulin G1 (IgG1) binding to wounded tissues by using fluorescein isothiocyanate-labelled anti-IgG1 6-24 hr after wounding. Splenectomy reduced the amount of IgG1 binding to wounded tissues. Immunoblotting studies revealed several bands, which were reduced by splenectomy. Using immunoprecipitation with anti-IgG bound to protein G we found that the intensity of several bands was lower in the serum from splenectomized mice than in that from sham-operated mice. These bands were matched to myosin IIA, carbamoyl-phosphate synthase, argininosuccinate synthase, actin and alpha-actinin-4 by liquid chromatography tandem mass spectrometry analysis.
Asunto(s)
Complejo Antígeno-Anticuerpo/metabolismo , Linfocitos B/metabolismo , Inmunoglobulina G/metabolismo , Piel/metabolismo , Cicatrización de Heridas/inmunología , Actinina/inmunología , Actinina/metabolismo , Actinas/inmunología , Actinas/metabolismo , Traslado Adoptivo , Animales , Complejo Antígeno-Anticuerpo/inmunología , Argininosuccinato Sintasa/inmunología , Argininosuccinato Sintasa/metabolismo , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos B/trasplante , Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Células Cultivadas , Femenino , Inmunoglobulina G/inmunología , Antígenos Comunes de Leucocito/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Miosina Tipo IIA no Muscular/metabolismo , Piel/inmunología , Piel/lesiones , Bazo/patología , Bazo/cirugía , EsplenectomíaRESUMEN
Hepatocyte paraffin 1 (Hep Par 1), a murine monoclonal antibody, is widely used in surgical pathology practice to determine the hepatocellular origin of neoplasms. However, identity of the antigen for Hep Par 1 is unknown. The aim of this study was to characterize the Hep Par 1 antigen. To identify the antigen, immunoprecipitation was used to isolate the protein from human liver tissue, and a distinct protein band was detected at approximately 165 kDa. The protein band was also present in small intestinal tissue, but was not present in several other non-liver tissues nor in three human hepatocellular carcinoma cell lines, Huh-7, HepG2, and LH86. The protein was purified and analyzed by mass spectrometry. It was identified as carbamoyl phosphate synthetase 1 (CPS1). CPS1 is a rate-limiting enzyme in urea cycle and is located in mitochondria. We demonstrated that hepatoid tumors (gastric and yolk sac) were immunoreactive with both Hep Par 1 antibody and anti-CPS1 antibody, further confirming the results of mass spectrometric analysis. We found that the three human hepatocellular carcinoma cell lines do not express either CPS1 RNA or protein. We confirmed that the gene was present in these cell lines, suggesting that suppression of CPS1 expression occurs at the transcriptional level. This finding may have relevance to liver carcinogenesis, since poorly differentiated hepatocellular carcinomas exhibit poor to absent immunoreactivity to Hep Par 1. In conclusion, we have identified the antigen for Hep Par 1 antibody as a urea cycle enzyme CPS1. Our results should encourage further investigation of potential role that CPS1 expression plays in liver pathobiology and carcinogenesis.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Secuencia de Bases , Western Blotting , Cromatografía Liquida , Cartilla de ADN , Femenino , Humanos , Inmunoprecipitación , Neoplasias Hepáticas/enzimología , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.
Asunto(s)
Anticuerpos Monoclonales , Hibridomas/inmunología , Mitocondrias Hepáticas/inmunología , Proteínas Mitocondriales/metabolismo , Proteómica , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo , Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Electroforesis en Gel Bidimensional , Biblioteca de Genes , Humanos , Inmunoprecipitación , Proteínas Mitocondriales/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones SubcelularesRESUMEN
Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.
Asunto(s)
Envejecimiento/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/análisis , Mitocondrias Hepáticas/enzimología , Factores de Edad , Animales , Anticuerpos Monoclonales , Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Inmunoensayo , Immunoblotting , Inmunohistoquímica , Masculino , Mitocondrias Hepáticas/ultraestructura , Ratas , Ratas Endogámicas , Conservación de TejidoRESUMEN
A partial carbamylphosphate synthetase (CPS: EC 6.3.4.16) deficiency (McKusick 23730) was found in a male child who presented with generalized convulsions, rickets and apnoeic attacks at six months of age. By his second year he showed serious developmental delay and a gut biopsy revealed an absence of CPS activity with an elevated ornithine transcarbamylase activity. Analysis of the gut biopsy sample on SDS-polyacrylamide gels, followed by electrophoretic transfer to a nitrocellulose filter probed with monospecific antibodies to CPS showed that the child had normal levels of immunoreactive enzyme, but instead of one band corresponding to normal CPS with a subunit size of 165,000 u, the patient had three immunoreactive bands, one larger and two smaller than that found in normal controls. The genetic defect in this child therefore results in an unusual form of CPS being made which has markedly reduced enzyme activity.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/deficiencia , Ligasas/deficiencia , Errores Innatos del Metabolismo/enzimología , Amoníaco/sangre , Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Carbamoil-Fosfato Sintasa (Amoniaco)/aislamiento & purificación , Humanos , Inmunoquímica , Lactante , Intestino Delgado/enzimología , Masculino , Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/genética , Peso Molecular , Conformación ProteicaRESUMEN
Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Ligasas/inmunología , Mitocondrias/enzimología , Neurospora crassa/enzimología , Neurospora/enzimología , Animales , Especificidad de Anticuerpos , Evolución Biológica , Carbamoil-Fosfato Sintasa (Amoniaco)/análisis , Carbamoil-Fosfato Sintasa (Amoniaco)/aislamiento & purificación , Núcleo Celular/enzimología , Precursores Enzimáticos/análisis , Escherichia coli/enzimología , Sueros Inmunes/inmunología , Técnicas de Inmunoadsorción , Riñón/enzimología , Hígado/enzimología , Peso Molecular , Mutación , Ratas , Saccharomyces cerevisiae/enzimologíaRESUMEN
Four types of carbamyl phosphate synthetase have been previously distinguished on the basis of catalytic properties and metabolic role. Immunoblot assay has now demonstrated cross-reactivity between rat liver carbamyl phosphate synthetase I and the following other three types of synthetases: carbamyl phosphate synthetase II from SV40-transformed baby hamster kidney cells, carbamyl phosphate synthetase III from spiny dogfish liver and from largemouth bass liver, and Escherichia coli carbamyl phosphate synthetase. The strongest cross-reactivity was observed between carbamyl phosphate synthetases I and III. These findings indicate at least partial structural homology among the various synthetases and constitute the first demonstration of such a relationship among the enzymes.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/inmunología , Ligasas/inmunología , Animales , Bovinos , Cricetinae , Reacciones Cruzadas , Escherichia coli/enzimología , Peces , Humanos , Técnicas de Inmunoadsorción , Riñón/enzimología , Hígado/enzimología , Ratas , Especificidad de la EspecieRESUMEN
A method has been developed to establish the degree of cross-reactivity of an antiserum raised against purified carbamoyl-phosphate synthase (ammonia) from adult rat liver, toward a homologous enzyme from another species without purification of the latter enzyme. For that purpose the ratio between enzyme activity and enzyme protein, i.e., the molecular specific activity in crude liver extracts, was determined by two independent methods. When the molecular specific activity was determined by means of radioimmunoassay using a specific antiserum raised against rat liver carbamoyl-phosphate synthase this ratio was a factor four higher for the axolotl than for the rat. Both axolotl and rat liver carbamoyl-phosphate synthase appear as a very prominent band with an apparent molecular weight of 165,000 after sodium dodecyl sulphate-polyacrylamide gelelectrophoresis. Therefore, the amount of enzyme protein could be determined by means of densitometry of this band using purified rat liver carbamoyl-phosphate synthase as a standard. The ratio between enzyme activity and enzyme protein calculated from this method appeared to be the same for axolotl and rat. From these results it can be deduced that the degree of cross-reactivity between rat and axolotl liver carbamoyl-phosphate synthase is approximately 25% when using the antiserum raised against the rat liver antigen.
Asunto(s)
Carbamoil-Fosfato Sintasa (Amoniaco)/inmunología , Ligasas/inmunología , Hígado/enzimología , Ambystoma mexicanum , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/análisis , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Técnicas para Inmunoenzimas , Peso Molecular , Ratas , Especificidad de la EspecieRESUMEN
Carbamoylphosphate synthetase (ammonia) isolated from human liver has been characterized. It is composed of monomers (constituted by a single polypeptide of 160,000 +/- 5,000 Mr) that can associate into dimers, although in the native state the monomer predominates. Physical and kinetic properties and amino acid composition are very similar to those found for the rat liver enzyme. There is extensive immunological cross-reactivity between the enzymes from both species. These results are discussed in the light of a possible regulatory role of carbamoylphosphate synthetase. The implications of these findings on a number of pathological states are also discussed.