RESUMO
The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos/imunologia , Basófilos/imunologia , Basófilos/metabolismo , Imunoglobulina E/metabolismo , Antagonistas de Leucotrienos , Fragmentos de Peptídeos/imunologia , Receptores de IgE/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Basófilos/enzimologia , Sítios de Ligação de Anticorpos/genética , Ligação Competitiva/genética , Células CHO , Cálcio/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Cricetinae , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Antagonistas dos Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/farmacologia , Liberação de Histamina/imunologia , Humanos , Imunoglobulina E/genética , Imunoglobulina E/fisiologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Líquido Intracelular/metabolismo , Cinética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucotrienos/metabolismo , Camundongos , Dados de Sequência Molecular , Nitrofenóis/imunologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Fenilacetatos , Ratos , Receptores de IgE/metabolismo , Receptores de IgE/fisiologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais Cultivadas , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
BACKGROUND: Tuberculous peritonitis is a rare event which can mimic advanced stage ovarian cancer. A pelvic mass and an elevated CA-125 is suggestive of an ovarian malignancy; however, benign conditions may be discovered, especially in the premenopausal patient. CASE: A patient with a pelvic mass, ascites, and an elevated CA-125 underwent an exploratory laparotomy for presumed ovarian cancer. Final pathology revealed pelvic tuberculosis without any pulmonary involvement. Acid-fast bacilli were confirmed with polymerase chain reaction in the surgical specimen. DISCUSSION: Pelvic tuberculosis is an uncommon gynecologic condition that presents with ascites, a pelvic mass, and fever. An elevated CA-125 is not specific for ovarian malignancy.
Assuntos
Antígeno Ca-125/sangue , Febre/etiologia , Neoplasias Ovarianas/diagnóstico , Peritonite Tuberculosa/diagnóstico , Adulto , Ascite/etiologia , Diagnóstico Diferencial , Feminino , Humanos , Peritonite Tuberculosa/patologiaRESUMO
BACKGROUND: Because of inaccuracies in clinical staging, endometrial adenocarcinoma is now a surgically staged disease. This study was done to determine the safety and efficacy of a laparoscopically assisted approach in the treatment and staging of this disease. METHODS: Using a retrospective chart review, we identified demographic characteristics, mean blood loss, operative findings, and complications of patients who had laparoscopically assisted staging and treatment for endometrial carcinoma from 1992 to 1997. RESULTS: Of 34 patients, 28 had laparoscopic surgical staging that included pelvic and para-aortic lymph node assessment, peritoneal washings, bilateral salpingo-oophorectomy, and total vaginal hysterectomy; 23 patients (82%) had stage I disease, 2 (7%) had stage II disease, and 3(11%) had stage III disease. Complications included herniation through a 5 mm port site, necessitating small bowel resection, and a fatal myocardial infarction 10 days postoperatively. CONCLUSION: Laparoscopic staging and treatment of endometrial carcinoma is appropriate in a select group of patients.
Assuntos
Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/cirurgia , Laparoscopia , Perda Sanguínea Cirúrgica , Transfusão de Sangue , Feminino , Humanos , Laparoscopia/efeitos adversos , Tempo de Internação , Metástase Linfática , Prontuários Médicos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos RetrospectivosRESUMO
Molecular chemotherapy strategies have been developed for a number of epithelial malignancies based on selective delivery and expression of a toxin-encoding gene into the cancer cells. To date, these strategies have not been explored in the context of carcinoma of the cervix, despite the fact that a variety of factors suggest this as an appropriate disease for this gene therapy approach. One limitation in this respect is that appropriate tissue-specific promoters for selective toxin gene expression have not been defined for cervical carcinoma. In this regard, the secretory leukoprotease inhibitor (SLPI) gene has been shown to be constitutively expressed in many epithelial carcinoma cells including the uterine cervix. Thus, we investigated the utility of the SLPI gene as a tissue-specific promoter for regulatory control of the herpes simplex virus thymidine kinase gene for in vitro treatment of cervical carcinoma cells. For this analysis, a gene construct was derived with the herpes simplex virus thymidine kinase gene under regulatory control of the 5' upstream regions of the SLPI gene. Transient transduction of three human cervical carcinoma cell lines with the SLPI-thymidine kinase (TK) construct was followed by treatment with the prodrug ganciclovir. Crystal violet staining was subsequently used to assess cell viability. In this analysis, it was shown that the SLPI-TK construct directed TK-mediated killing in two of three tested cervical cell lines, with the two cell lines being positive for SLPI. In addition, mixing experiments established that cervical carcinoma cells could exhibit a bystander effect which potentially augments the efficacy of molecular chemotherapy approaches. These findings may allow for the development of efficacious, target-specific, toxin gene therapy strategies for cervical carcinoma in human patients.
Assuntos
Carcinoma/genética , Proteínas/genética , Simplexvirus/enzimologia , Timidina Quinase/genética , Neoplasias do Colo do Útero/genética , Carcinoma/tratamento farmacológico , Carcinoma/terapia , Divisão Celular/efeitos dos fármacos , Citomegalovirus/genética , Feminino , Ganciclovir/farmacologia , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Humanos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Proteínas Secretadas Inibidoras de Proteinases , Inibidor Secretado de Peptidases Leucocitárias , Simplexvirus/genética , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/terapiaRESUMO
Originally conceived and applied for the treatment of inherited monogenetic defects such as adenosine deaminase deficiency and cystic fibrosis, gene therapy was later applied to the treatment of cancer. Such a genetic strategy seemed rational given the recognition that cancer typically develops in a multistep process involving alterations of a number of different genes as demonstrated in familial polyposis and colorectal cancer through the work of Vogelstein et al. Because of the numerous alterations that may result in the eventual development of cancer, there is no obvious single choice for a therapeutic gene. Although one may view this as an obstacle, it also allows for a variety of possible therapeutic interventions. This review focuses on the known genetic defects that occur in ovarian cancer, the gene therapy strategies suggested by such defects, and the approaches under current development for the treatment of this disease. As such, this work also describes some of the approved human gene therapy protocols. Finally, an overview of the problems and directions for future growth and research is presented.
Assuntos
Terapia Genética , Neoplasias Ovarianas/terapia , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Terapia Genética/tendências , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Mutagênese , Neoplasias Ovarianas/genética , Pró-FármacosRESUMO
Human umbilical vein endothelial cells (HUVECs) were evaluated for utility as a vector to achieve a bystander effect and killing of ovarian carcinoma cell lines. After demonstrating that HUVECs could be transduced with the reporter gene LacZ encoded by an adenoviral vector, appropriate cell killing of the AdCMVHSV-TK-transduced HUVECs was exhibited after treatment with 20 microM ganciclovir. Mixing experiments were then performed to determine whether the transduced HUVECs would demonstrate a bystander effect with the ovarian cancer cell lines. When 50% AdCMVHSV-TK-transduced HUVECs were mixed with untransduced ovarian cancer cells, > 70% of all cells were killed. Finally, s.c. and i.p. injections of herpes simplex-thymidine kinase-expressing HUVECs and SKOV3ip1 tumor cells were performed to evaluate the effects of HUVECs in in vivo models. These studies showed a decrease in tumor growth s.c. as well as a statistically significant survival prolongation (P < 0.05) in the i.p. model. These findings suggest that endothelial cells may be used as a vehicle for the delivery of cytotoxicity (bystander effect) in molecular chemotherapy.
Assuntos
Endotélio Vascular/fisiologia , Terapia Genética/métodos , Neoplasias Ovarianas/terapia , Animais , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos , Humanos , Camundongos , Camundongos SCID , Simplexvirus/enzimologia , Timidina Quinase/biossíntese , Timidina Quinase/genética , Transdução Genética , Células Tumorais CultivadasRESUMO
OBJECTIVE: The aim of this study was to evaluate the response to salvage treatment in recurrent ovarian cancer treated initially with paclitaxel-based chemotherapy. METHODS: A retrospective review of patients with recurrent ovarian cancer treated with surgical debulking and paclitaxel-based chemotherapy was performed. All cases received second-line treatment with a response evaluated by clinical or surgical means. Data analysis was conducted using the SAS statistical package. RESULTS: Fifty cases of advanced stage disease were available for review. Patients received paclitaxel and cisplatin or carboplatin with a 72.0% response rate. The median time to recurrence after primary treatment was 6 months. Second-line treatment included cisplatin or carboplatin (50%), Taxol (10%), or lutetium (22%), an intraperitoneal radiolabeled monoclonal antibody targeted to TAG-72. A 52.0% clinical response to salvage treatment was detected. With a median follow-up of 7 months, 68.0% of patients had experienced recurrence or progression of their disease. The median time to second recurrence was 5 months. Cases sensitive to initial paclitaxel-containing chemotherapy responded to any of the salvage treatments more frequently than chemotherapy-resistant tumors (88.5% versus 11.5%, P < 0.05). CONCLUSIONS: Recurrent ovarian cancer patients initially treated with paclitaxel-based chemotherapy frequently responded to salvage treatment. However, the duration of response was brief, and hospitalization for treatment-related side-effects was common. Tumor response to initial paclitaxel/platinum treatment was predictive of future response to second-line agents. Current salvage therapies appear to provide little benefit in cases of tumors resistant to primary chemotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Terapia de Salvação , Adulto , Idoso , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Carboplatina/administração & dosagem , Cisplatino/administração & dosagem , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Paclitaxel/administração & dosagem , Recidiva , Indução de Remissão , Estudos Retrospectivos , Resultado do TratamentoRESUMO
OBJECTIVE: Our aim was to determine whether histopathological variables predict persistent high-grade squamous intraepithelial lesions (HGSIL) after large-loop excision of the transformation zone (LLETZ). MATERIALS AND METHODS: All patients with cervical intraepithelial neoplasia (CIN) grade 2 or 3 on a LLETZ specimen with at least one follow-up Papanicolaou (Pap) test were identified. Histopathological variables were evaluated for the potential to predict HGSIL on a follow-up Pap test. Variables examined included endocervical margin status, ectocervical margin status, endocervical curettage (ECC) result, presence or absence of endocervical glandular involvement, and presence or absence of koilocytosis. RESULTS: Two hundred and nineteen cases were identified. A follow-up Pap test showed HGSIL in 16 patients (7.3%). Of the histopathological variables studied, only a positive ECC at the time of LLETZ conization predicted HGSIL on follow-up cytology (p =.0002). Endocervical margin status, ectocervical margin status, presence or absence of glandular involvement, and presence or absence of koilocytosis were not associated significantly with HGSIL at follow-up. CONCLUSION: Most histopathological factors from LLETZ conization do not predict reliably the presence of HGSIL at the time of follow-up Pap test. A positive ECC at the time of LLETZ, however, may predict those patients destined to have persistence or recurrence. These findings suggest that conservative follow-up is warranted after LLETZ conization.
RESUMO
Binding of the Fc region of IgG antibodies to low affinity Fc gamma receptors (Fc gammaR) triggers important effector functions in the immune system. The type IIIb Fc gammaR (Fc gammaRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of Fc gammaRIIIb (sFc gammaRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab')2) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K(A)) of 1.3 +/- 0.6 x 10(6) M(-1) and 2.6 +/- 0.4 x 10(5) M(-1), respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 +/- 0.2 x 10(6) M(-1)), whereas that for human IgG3 was twofold higher (4.2 +/- 0.4 x 10(5) M(-1)). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 >> IgG4 >>> IgG2. Thus, the extracellular polypeptide of Fc gammaRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.
Assuntos
Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Animais , Cricetinae , Escherichia coli/genética , Células Eucarióticas , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/classificação , Técnicas In Vitro , Cinética , Células Procarióticas , Ligação Proteica , Receptores de IgG/química , Receptores de IgG/classificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex. The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t. All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t. Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t. In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t. Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells.
Assuntos
Retículo Endoplasmático/metabolismo , Dobramento de Proteína , Receptores de IgE/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Transporte Biológico , Células CHO , Linhagem Celular , Cricetinae , Glicosilação , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/imunologia , Receptores de IgE/genética , Tunicamicina/farmacologiaRESUMO
epsilon BP (IgE-binding protein) is a 31,000 M(r) protein originally identified in rat basophilic leukemia (RBL) cells. The protein is composed of two domains with the amino-terminal domain containing a highly conserved repetitive sequence and the carboxyl-terminal domain containing consensus sequences shared by other beta-galactoside-binding soluble lectins. The protein has wide tissue distribution, is found on cell surfaces and in extracellular milieu. By combined efforts from several research groups including ours a multifunctional nature of this lectin began to emerge. This review emphasizes the following characteristics of epsilon BP: (i) epsilon BP is secreted by cells such as macrophages; (ii) like many other lectins, epsilon BP functions at least bivalently; (iii) epsilon BP has specificity for distinct oligosaccharide structures that have a terminal galactose not masked by sialic acids; and (iv) in addition to binding IgE, epsilon BP binds to surfaces of various cell types via lectin-carbohydrate interaction. Importantly, epsilon BP binds to the IgE receptor on mast cells. We propose that epsilon BP can function as a modulatory protein on various cells by cross-linking critical cell surface glycoproteins. The proposed action of epsilon BP on mast cells is presented as a model.
Assuntos
Antígenos de Diferenciação/fisiologia , Lectinas/fisiologia , Mastócitos/imunologia , Receptores de IgE/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/química , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Galectina 3 , Lectinas/biossíntese , Lectinas/química , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peso MolecularRESUMO
IgE is highly glycosylated, but the function of the oligosaccharide side chains is largely unknown. The previous discovery of an animal lectin, IgE-binding protein (epsilon BP), affords an opportunity to study potential carbohydrate-dependent effector functions of IgE. epsilon BP is a beta-galactoside-specific lectin with binding affinity for IgE and is now known to be equivalent to carbohydrate-binding protein 35 and the Mac-2 Ag; thus, it may have multiple functions in addition to IgE binding. We have previously shown that rat r epsilon BP recognizes sialidase-treated human myeloma IgE to a much greater extent than the untreated IgE. In contrast, human epsilon BP binds essentially equivalently to a monoclonal murine IgE with or without sialidase pretreatment. To validate a possible role for epsilon BP in the IgE system, we investigated the pattern of recognition of epsilon BP for various polyclonal human IgE samples. We show that polyclonal IgE derived from four individuals with hyper-IgE syndrome or atopic dermatitis recognizes epsilon BP and that there is individual variation in the proportion of IgE recognized by epsilon BP, ranging from greater than 60% for one sample to almost undetectable levels in another. We conclude that epsilon BP does indeed recognize polyclonal IgE and that this recognition is modulated by sialylation of IgE oligosaccharides. Furthermore, there exist different IgE glycoforms, varying in the degree of sialylation, and these are distributed in a distinct manner in different individuals.
Assuntos
Antígenos de Diferenciação/metabolismo , Imunoglobulina E/química , Galectina 3 , Humanos , Hipergamaglobulinemia/metabolismo , Hipersensibilidade/imunologia , Imunoglobulina E/metabolismo , Lectinas/metabolismo , Ligação Proteica , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismoRESUMO
Multiple mRNA species encoding several predicted forms of the high-affinity IgE receptor alpha subunit (Fc epsilon RI-alpha) have been previously characterized from rat basophilic leukemia cells. Using the polymerase chain reaction procedure, we have extended these findings to show that one Fc epsilon RI-alpha mRNA variant, characterized by a 163-bp deletion within the coding sequence, exists in normal rat connective tissue mast cells as well as in both transformed and non transformed murine mast cell lines. In addition, a partial murine Fc epsilon RI-alpha genomic clone, spanning the internal-deletion sequence, has been identified, and from analysis of this sequence a mechanism of alternative pre-mRNA splicing is proposed. Finally, mRNA variants have been translated in a cell-free system and the protein products partially characterized.
Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Transformação Celular Neoplásica/genética , Mastócitos/ultraestrutura , RNA Mensageiro/genética , Receptores Fc/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada/química , Eletroforese em Gel de Poliacrilamida , Variação Genética , Masculino , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos , Receptores de IgE , Dodecilsulfato de Sódio , Transcrição GênicaRESUMO
IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.
Assuntos
Escherichia coli/genética , Imunoglobulina E/metabolismo , Linfocinas/metabolismo , Proteínas Secretadas pela Próstata , Animais , Linhagem Celular , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Leucemia Basofílica Aguda/imunologia , Linfocinas/genética , Linfocinas/isolamento & purificação , Peso Molecular , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
IgE-binding protein (epsilon BP) refers to a protein originally identified in rat basophilic leukemia cells by virtue of its affinity for IgE. It is now known to be a beta-galactoside-binding lectin equivalent to carbohydrate-binding protein 35 (CBP 35). More recently, its identity to Mac-2, a macrophage cell-surface protein, has been established. cDNA coding for human epsilon BP has been cloned from a human HeLa cell cDNA library and contains an open reading frame of 750 base pairs encoding a 250 amino acid protein. Like the rat and murine counterparts, the human epsilon BP amino acid sequence can be divided into two domains with the amino-terminal domain consisting of a highly conserved repetitive sequence (YPGXXXPGA) and the carboxyl-terminal domain containing sequences shared by other S-type lectins. The human epsilon BP sequence exhibits extensive homology to murine and rat epsilon BP with 84% and 82% identity, respectively. The homology is particularly striking in the carboxyl-terminal domain where 95% identity is found between human and murine sequences in a stretch of over 70 amino acids. A survey of epsilon BP mRNA expression from several lymphocyte cell lines revealed that the level of epsilon BP transcription may reflect a relationship between cell differentiation and epsilon BP expression. Finally, human epsilon BP was purified from several human cell lines and shown to possess lactose-binding characteristics and cross-species reactivity to murine IgE. Surprisingly, three different human myeloma IgE proteins did not show reactivity to human epsilon BP. However, after neuraminidase treatment of each human IgE, pronounced binding to epsilon BP was observed, thereby indicating that only specific IgE glycoforms can be recognized by epsilon BP.
Assuntos
Antígenos de Diferenciação/genética , Proteínas de Transporte/genética , Hemaglutininas/genética , Imunoglobulina E/metabolismo , Lectinas/genética , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , DNA/genética , Galactose/metabolismo , Galectina 3 , Células HeLa/química , Hemaglutininas/metabolismo , Humanos , Lectinas/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas do Mieloma/metabolismo , Especificidade de Órgãos , Ratos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The predicted amino acid sequence of carbohydrate-binding protein 35 (CBP35; Mr approximately 35,000), a galactose-specific lectin in many mouse and human cells, has been compared to the predicted sequence of an IgE-binding protein (epsilon BP) originally identified in rat basophilic leukemia cells. The sequences of the two proteins showed that: (a) 85% of the amino acid residues are identical; (b) the polypeptide chains are comprised of two distinct domains; and (c) highly conserved internal repetitive sequences are present. Consistent with these observations, antisera raised against CBP35 or against a synthetic peptide derived from the epsilon BP sequence cross-reacted with both proteins. Moreover, fractionation of extracts of mouse 3T3 fibroblasts over an IgE-Sepharose affinity column showed that CBP35 bound to IgE; this binding was reversed by addition of lactose. Conversely, fractionation of extracts of rat basophilic leukemia cells over an affinity column of Sepharose derivatized with N-(epsilon-amino-caproyl)-D-galactosamine showed that epsilon BP was a galactose-binding protein. Furthermore, epsilon BP bound to IgE-Sepharose could be eluted by lactose. Finally, transcription and translation in vitro of the cDNA corresponding to epsilon BP yielded a polypeptide containing carbohydrate-binding activity. All of these results strongly support the conclusion that CBP35 and epsilon BP are mouse and rat homologues, respectively.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Fc/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Proteínas de Transporte/genética , Linhagem Celular , Células Cultivadas , Humanos , Immunoblotting , Imunoglobulina E/metabolismo , Peso Molecular , Biossíntese de Proteínas , Receptores Fc/genética , Receptores de IgE , Transcrição GênicaRESUMO
We show that IgE-binding protein (epsilon BP) is found primarily in the cytoplasm of rat basophilic leukemia (RBL) cells and COS-1 cells transfected with epsilon BP cDNA. Antibodies to a synthetic peptide internal to epsilon BP were generated that specifically recognized epsilon BP by protein immunoblotting. These antibodies also bind the surface of RBL cells. Surprisingly, blot hybridization analysis of RNA from nine various normal rat tissues showed that the epsilon BP gene is transcribed in all the tissues tested as well as in a mouse macrophage-like cell line.
Assuntos
Proteínas de Transporte/isolamento & purificação , Imunoglobulina E/metabolismo , Linfocinas/isolamento & purificação , Proteínas Secretadas pela Próstata , Animais , Reações Antígeno-Anticorpo , Basófilos , Sítios de Ligação de Anticorpos , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular , Clonagem Molecular , Citoplasma/análise , DNA , Haplorrinos , Leucemia/imunologia , Leucemia/metabolismo , Linfocinas/genética , Linfocinas/imunologia , Peptídeos/imunologia , Coelhos , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos B/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.
Assuntos
Imunoglobulina E/imunologia , Receptores Fc/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Basófilos/imunologia , Clonagem Molecular , DNA/genética , Mastócitos/imunologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Receptores Fc/imunologia , Receptores de IgERESUMO
Six peptides, representing contiguous amino acid sequences within the C epsilon 2, C epsilon 3 and C epsilon 4 domains of murine IgE, were selected for synthesis on the basis of overall hydropathy, degree of homology to both human and rat IgE and, where possible, inclusion of a native cysteine residue. Antibodies were produced against each peptide by immunizing rabbits with peptide-KLH conjugates. Each anti-peptide antiserum exhibited good reactivity with the corresponding immunizing peptide (titer: 10(-4) to 10(-5] and four of the six antisera exhibited a distinct preferential murine IgE reactivity compared to four other murine immunoglobulin classes (IgG1, IgG2b, IgM and IgA). In addition, one antiserum (anti-epsilon peptide 5), raised against a peptide with 80% homology to human IgE, reacted comparably with both human and murine IgE. Each IgE-reactive antiserum was screened for the ability to stimulate mediator release from IgE-sensitized rat basophilic leukemia (RBL) cells. Two of the four IgE-reactive antisera strongly stimulated 3H-serotonin release (anti-epsilon peptides 4 and 5), one antiserum showed weak activity (anti-epsilon peptide 3) and the remaining anti-peptide serum (anti-epsilon peptide 6), which exhibited the highest anti-IgE reactivity, exhibited no detectable stimulatory activity. Individual anti-peptide antibodies were subsequently tested for the potential to bind to receptor-bound IgE. Anti-epsilon peptide 3 was shown to exhibit the least binding, anti-epsilon peptide 6 showed the highest magnitude of binding while anti-epsilon peptides 4 and 5 exhibited intermediate values. We conclude from this study that sequences defined by epsilon-peptides 4 and 5 are not significantly involved in the receptor binding mechanism whereas epsilon-peptide 3 is likely to be most proximal to the IgE-receptor recognition site of those sequences studied. Finally, we suggest that the epsilon-peptide 6 sequence is in such an orientation in cell-bound IgE that, while it is accessible to external antibody, effective cross-linking of the IgE-receptor complex cannot be achieved through this determinant.