RESUMEN
T cell responses are generally regarded as specific for protein-derived peptide antigens. This is based on the molecular paradigm dictated by the T cell receptor (TCR) recognition of peptide-major histocompatibility complexs, which provides the molecular bases of the specificity and restriction of the T cell responses. An increasing number of findings in the last 20 years have challenged this paradigm, by showing the existence of T cells specific for lipid antigens presented by CD1 molecules. CD1-restricted T cells have been proven to be frequent components of the immune system and to recognize exogenous lipids, derived from pathogenic bacteria, as well as cell-endogenous self-lipids. This represents a young and exciting area of research in immunology with intriguing biological bases and a potential direct impact on human health.
Asunto(s)
Presentación de Antígeno , Antígenos CD1/inmunología , Autoantígenos/inmunología , Lípidos/inmunología , Autotolerancia/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Animales , Antígenos Bacterianos/inmunología , Antígenos CD1/genética , Antígenos de Neoplasias/inmunología , Antígenos de Plantas/inmunología , Autoinmunidad , Escherichia coli/inmunología , Reordenamiento Génico de Linfocito T , Humanos , Leucemia/inmunología , Ratones , Mycobacterium tuberculosis/inmunología , Polen/inmunología , Conformación Proteica , Estructura Terciaria de Proteína , Especificidad de la EspecieRESUMEN
Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.
Asunto(s)
Alérgenos/genética , Proteínas de Plantas/genética , Polen/genética , Alérgenos/química , Alérgenos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antígenos de Plantas , Secuencia de Bases , Cartilla de ADN/genética , Expresión Génica , Humanos , Lolium/genética , Lolium/inmunología , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Polen/química , Conformación Proteica , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Rinitis Alérgica Estacional/diagnóstico , Rinitis Alérgica Estacional/inmunología , Pruebas CutáneasRESUMEN
The tailpiece of secretory Ig-mu-chains (mu(s)tp) is highly conserved throughout evolution: in particular, a carboxy-terminal cysteine residue (Cys575) and a glycan linked to Asn563 are found in all species sequenced so far. Here we show that the mu(s)tp oligosaccharide moieties are important for the binding of J-chains and for the process of IgM polymerization. In the absence of the mu(s)tp glycans, pentamers cannot be assembled and polymers containing six or more subunits are secreted. Despite their increased valency, these molecules have a lower association rate with antigen than wild-type polymers. Unexpectedly, the C-terminal oligosaccharides also affect kinetic parameters on unpolymerized subunits. Thus, monomers lacking the C-terminal sugars because of either site-directed mutagenesis or selective enzymatic deglycosylation with endoglycosidase H, have a lower k(on) for the antigen. Taken together, our results indicate that the C-terminal mu-chain glycans can shape the structure of mu(s2)L2 subunits and their further assembly into polymers.
Asunto(s)
Inmunoglobulina M/química , Inmunoglobulina M/fisiología , Cadenas mu de Inmunoglobulina/química , Cadenas mu de Inmunoglobulina/fisiología , Polisacáridos/química , Animales , Sitios de Unión , Línea Celular , Secuencia Conservada , Evolución Molecular , Glicosilación , Hemólisis , Inmunoglobulina M/genética , Cadenas mu de Inmunoglobulina/genética , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.
Asunto(s)
Alérgenos/inmunología , Inmunoglobulina E/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Lolium/inmunología , Proteínas de Plantas/inmunología , Polen/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Antígenos de Plantas , Unión Competitiva , Mapeo Epitopo , Epítopos , Histamina/metabolismo , Humanos , Inmunoglobulina E/genética , Fragmentos Fab de Inmunoglobulinas/genética , Mastocitos/inmunología , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Recombinantes/inmunologíaRESUMEN
The authors describe the production, via recombinant DNA technology, of bifunctional polypeptides for immunoenzymatic assays. These molecules contain an apolipoprotein moiety fused to an active beta-galactosidase enzyme, and are used as tracers in competition assays with specific monoclonal antibodies. The final colorimetric result is inversely correlated with the apolipoproteins plasma values. This technology, named RIECA (Recombinant Immunoenzymatic Competition Assay) is very accurate and flexible and may be applied to a wide range of diagnostic interest.
Asunto(s)
Apolipoproteínas A/sangre , Apolipoproteínas B/sangre , Técnicas para Inmunoenzimas/instrumentación , Anticuerpos Monoclonales , Humanos , beta-GalactosidasaRESUMEN
Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner. We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments. Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose). The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis. Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.
Asunto(s)
Anticuerpos Monoclonales/genética , Calmodulina/genética , Fragmentos de Inmunoglobulinas/genética , Secuencia de Aminoácidos , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Secuencia de Bases , Calmodulina/química , Clonación Molecular , Simulación por Computador , Escherichia coli/genética , Colorantes Fluorescentes , Expresión Génica , Fragmentos de Inmunoglobulinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/inmunología , Proteínas Recombinantes de FusiónAsunto(s)
Alérgenos/inmunología , Lolium/inmunología , Proteínas de Plantas/inmunología , Polen/inmunología , Proteínas Recombinantes de Fusión/inmunología , Alérgenos/genética , Anticuerpos/inmunología , Antígenos de Plantas , Mapeo Epitopo , Humanos , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The possibility of obtaining immobilized horseradish peroxidase (HRP) materials with K'(m) values close to that of the native enzyme, but with good thermal stability, was investigated. The photochemical reaction was used as the immobilization methodology. Temperature and catalyst concentration were found to be the main parameters able to control the immobilization reaction mechanism more than type of functional monomer, polymer-matrix, and enzyme-polymer ratios. By carrying out the immobilization reaction at 35 degrees C and using either bisacryloylpiperazine (BAP) or hexhydro-1,3,5-triacryloyl-s-triazine (HTsT) as the functional monomer, materials with a good thermal stabilization (the retained activity after 240 min at 60 degrees C was between 65-25%) as well as kinetic constants (0.6-0.8 x 10(-4)M) similar to that of the free enzyme (0.57 x 10(-4)M) were obtained. Since low K'(m) values were obtained also using a high polymer content (pBAP copolymers, 25%; pHTsT copolymers, 30%) and neither limitation to substrate diffusion nor a reduction of the enzyme mobility was found, the enzyme should be linked to the matrix during the last steps of monomer polymerization, and it should have an external disposition with respect to the support.
RESUMEN
A human monoclonal IgG1(kappa) antibody (hmAb) specific for a sequential epitope comprised within the 5-1-1 fragment of the NS4 region of hepatitis C virus (HCV) has been recently generated (1). In this study, B-cell clone supernatant containing the hmAb was purified by passage over a protein A affinity column. Preincubation with synthetic oligopeptides containing the epitope recognized by the hmAb resulted in complete inhibition of binding to the whole recombinant protein, attesting to its specificity. Calculation of the dissociation constant (Kd) using a synthetic 20-mer as antigen gave a value of 3.3372 x 10(-8) M, consistent with that of a human IgG. We predict that this hmAb will be helpful in characterizing the as yet unidentified native NS4 protein of HCV.
Asunto(s)
Anticuerpos Monoclonales , Especificidad de Anticuerpos , Hepacivirus/inmunología , Unión Competitiva/fisiología , Epítopos , HumanosRESUMEN
IgM are glycoproteins secreted by plasma cells as (mu2L2)5+J or (mu2L2)6 polymers. In most species, mu- and J-chains bear five and one N -glycans, respectively. Here we compare the terminal glycosylation patterns of 4-hydroxy-3-nitrophenylacetyl (NP)-specific IgM secreted by transfectants of the J558L mouse myeloma deficient in the alpha2,6 sialyltransferase [alpha2,6ST(N)] or by a hybridoma expressing this enzyme (B1.8 cells). The absence of alpha2,6-sialylation results in an increased addition of alpha1, 3-galactosyl residues to mu- and J-chain N-glycans. Since alpha1, 3-galactosyltransferase (alpha1,3Gal-T) is similarly expressed in the two cell lines, these results indicate that a competition reaction occurs in vivo between alpha2,6ST(N) and alpha1,3Gal-T. In the alpha2,6ST(N) deficient transfectants, mu-chains lacking the C-subterminal Cys575 residue, which are secreted mainly in the form of mu2L2 monomers, are more efficiently capped by alpha1, 3-galactosyl residues, confirming that polymerization significantly reduces the accessibility of mu-chain glycans to the Golgi processing enzymes involved in the biogenesis of antennary sugars. Functional assays indicate that IgM sialylation affects antigen-binding and complement-dependent hemolysis of haptenated red blood cells.
Asunto(s)
Disacáridos/metabolismo , Epítopos/metabolismo , Mieloma Múltiple/metabolismo , Animales , Biopolímeros , Proteínas del Sistema Complemento/metabolismo , Glicosiltransferasas/metabolismo , Inmunoglobulina M/metabolismo , Ratones , Mieloma Múltiple/patología , Polisacáridos/metabolismo , Células Tumorales CultivadasRESUMEN
Although atopic allergy affects =20% of the total population, the relationship between the protein structure and immunogenic activity of the allergens is still largely unknown. We observed that group 5 grass allergens are characterized by repeated structural motifs. Using a new algorithm, TEPITOPE, we predicted promiscuous HLA-DR ligands within the repeated motifs of the Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.
Asunto(s)
Alérgenos/química , Biología Computacional , Epítopos de Linfocito T/aislamiento & purificación , Lolium/inmunología , Proteínas de Plantas/química , Alérgenos/inmunología , Alérgenos/metabolismo , Secuencia de Aminoácidos , Antígenos de Plantas , Células Clonales , Biología Computacional/métodos , Citocinas/metabolismo , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Polen , Unión Proteica/inmunología , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Programas Informáticos , Linfocitos T/inmunologíaRESUMEN
The cDNA for streptavidin (residues 15-159) was subcloned into an expression vector in fusion at the N-terminus with the T7-tag (12 residues). Conditions were found to express the protein in Escherichia coli in a soluble, assembled, and active form. The protein was purified in two simple steps which involved heating at 75 degreesC and affinity chromatography on iminobiotin agarose. The purified protein was obtained in yields of 70 mg per liter of bacterial culture. Electron spray mass spectrometry analysis showed that the recombinant streptavidin had the expected molecular mass without covalent modifications. ELISA and surface plasmon resonance analyses showed it to be functionally analogous to the natural streptavidin. This appears to be an improvement over the reported methods of recombinant streptavidin production which involve protein renaturation or the use of eukaryotic expression systems.
Asunto(s)
Escherichia coli/química , Proteínas Recombinantes/aislamiento & purificación , Estreptavidina/química , Streptomyces/química , Biotina/metabolismo , Cromatografía de Afinidad , Escherichia coli/genética , Proteínas Fúngicas/aislamiento & purificación , Expresión Génica/genética , Espectrometría de Masas , Oligopéptidos/genética , Unión Proteica/fisiologíaRESUMEN
NKT cells are a small subset of T lymphocytes which express an invariant V(alpha24JalphaQ TCR and recognize glycolipids presented by CD1d. In adults, NKT cells have a memory phenotype, frequently associated with oligoclonal expansion, express NK cell markers, and produce TO cytokines upon primary stimulation. Because of these features, NKT cells are regarded as lymphocytes of innate immunity. We investigated NKT cells from cord blood to see how these cells appear in the absence of exogenous stimuli. We found that NKT cells are present at comparable frequencies in cord blood and adult peripheral blood mononuclear cells and in both cases display a memory (CD45RO+CD62L-) phenotype. However, neonatal NKT cells differ from their adult counterparts by the following characteristics: (1) they express markers of activation, such as CD25; (2) they are polyclonal; (3) they do not produce cytokines in response to primary stimulation. Together, our data show that human NKT cells arise in the newborn with an activated memory phenotype, probably due to recognition of an endogenous ligand(s). The absence of oligoclonal expansion and primary effector functions also suggest that neonatal NKT cells, despite their activated memory phenotype, require a further priming/differentiation event to behave as fully functional cells of innate immunity.
Asunto(s)
Memoria Inmunológica/inmunología , Células Asesinas Naturales/inmunología , Antígenos Comunes de Leucocito/biosíntesis , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Adulto , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Biomarcadores , Sangre Fetal , Antígenos HLA-DR/biosíntesis , Humanos , Inmunofenotipificación , Recién Nacido , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Selectina L/biosíntesis , Lectinas Tipo C , Leucocitos Mononucleares/citología , Ratones , Receptores de Interleucina-2/biosíntesisRESUMEN
The molecular cloning of the cDNA encoding for an isoallergenic form of Lol p II, a major rye grass (Lolium perenne) pollen allergen, was performed by polymerase chain reaction amplification on mRNA extracted from pollen. The amino acid sequence derived from the cDNA was truncated by 4 and 5 residues at the NH2- and COOH-terminal ends, respectively, and differed only in one position from that previously reported. This cDNA was expressed in Escherichia coli by fusion to the carboxyl terminus of the human ferritin H-chain. The molecule was produced in high yields as a soluble protein and was easily purified. The protein retains the multimeric quaternary structure of ferritin, and it exposes on the surface the allergenic moiety, which can be recognized in Western blotting and in enzyme-linked immunosorbent assay experiments by specific IgE from allergic patients. The recombinant allergen was used to analyze the sera of 26 patients allergic to L. perenne compared with control sera. The results were in good agreement with the values obtained with the radioallergosorbent test assay. In addition, histamine release experiments in whole blood from an allergic patient and skin prick tests showed that the recombinant allergen retains some of the biological properties of the natural compound. These findings indicate that the availability of homogeneous recombinant allergens may be useful for the development of more specific diagnostic and therapeutic procedures. Moreover, this expression system may be of more general interest for producing large amounts of soluble protein domains in E. coli.
Asunto(s)
Alérgenos/genética , Proteínas de Plantas/genética , Alérgenos/biosíntesis , Alérgenos/inmunología , Secuencia de Aminoácidos , Antígenos de Plantas , Secuencia de Bases , Clonación Molecular , ADN Complementario , Escherichia coli , Liberación de Histamina , Humanos , Lolium/inmunología , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Homología de Secuencia de Aminoácido , Pruebas CutáneasRESUMEN
Specific targeting of radioactive agents to tumour cells has been the main objective of the in vivo use of monoclonal antibodies and their fragments. In particular, specific antibodies to carcinoembryonic antigen (CEA)-expressing tumours can be used either for diagnosis or therapy, if targeting could be improved. The expression of antibody fragments in bacteria allows the preparation of engineered molecules with antigen-binding properties and a better penetration into the tumour. A specific anti-CEA single-chain Fv fragment was produced in bacteria and purified. Its binding activity has been demonstrated in ELISA, immunocytochemistry, immunohistochemistry, fluorescence-activated cell sorting and the kinetic parameters determined by the plasmon surface resonance.
Asunto(s)
Anticuerpos Antineoplásicos/biosíntesis , Antígeno Carcinoembrionario/inmunología , Fragmentos de Inmunoglobulinas/biosíntesis , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/aislamiento & purificación , Secuencia de Bases , Biotecnología , Línea Celular , Cartilla de ADN/genética , Escherichia coli/genética , Vectores Genéticos , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Inmunohistoquímica , Cinética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the HER-2 extracellular domain that was shared by the scFv and the parental MAb. BlAcore analysis indicated a Koff of 9.3 x 10(-4) s(-1), similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean t1/2beta of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different human carcinomas overexpressing HER-2.