RESUMEN
Surface plasmon resonance technique was investigated for the first time to study the apparent hydrophobicity and association properties of the major bovine caseins: alpha(s)-(alpha(s1)- and alpha(s2)-caseins in a 4:1 proportion), beta-, and kappa-caseins. The apparent hydrophobicities of the caseins were evaluated by a new method based on the binding level of casein on a hydrophobic sensor chip, and kinetic and equilibrium affinity constants were determined for the following casein interactions: alpha(s)/alpha(s), alpha(s)/beta, alpha(s)/kappa, beta/beta, and beta/kappa, using a sensor chip modified with covalent immobilized caseins. The study by surface plasmon resonance technology of these casein interactions under different conditions (pH, ionic strength, calcium concentration, chemical modification) demonstrated that, at neutral pH, electrostatic repulsive forces play an important role since an increase in ionic strength of the medium resulted in a stronger interaction. When charge repulsions were reduced by either acidification, increase in ionic strength, or dephosphorylation, casein interactions were reinforced, presumably due to weak attractive forces. Moreover, in this molecular model, we showed that addition of calcium greatly increased the binding response between the most phosphorylated caseins and that the added calcium (2 mM) participated directly in the formation of bridges between the phosphate groups of the casein molecules.
Asunto(s)
Calcio/química , Caseínas/química , Resonancia por Plasmón de Superficie/métodos , Adsorción , Calcio/farmacología , Caseínas/metabolismo , Cromatografía en Gel , Concentración de Iones de Hidrógeno , Cinética , Concentración Osmolar , Fosforilación , Electricidad EstáticaRESUMEN
The anti-CD4 mAb 13B8.2, directed against the CDR3-like loop of the D1 domain of CD4, inhibits signal transduction pathways leading to both T cell activation and HIV replication. VH9/DSP2/JH2 and Vkappa12-13/Jkappa2 rearrangements, corresponding to genes encoding the heavy and light chain variable regions of the 13B8.2 mAb, were inserted into baculovirus cassettes upstream from pre-installed human Fdgamma1 and Ckappa genes, respectively. After expression in insect cells, a complete correctly-processed Fab was secreted into the culture medium; it was protein-G immunopurified with a yield of 5 mg/L. The chimeric Fab 13B8.2 showed anti-CD4 binding activity with an affinity value of 3.3 nM and recognized the same region on the CDR3-like loop as the parental mAb. The mouse-human Fab inhibited IL2 secretion following antigen presentation and displayed a strong capacity to prevent HIV-1 promoter activation. Taken together, these results indicate that the chimeric Fab retained a major part of the parental 13B8.2 mAb properties and suggest that it might be a valuable therapeutic tool.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Antígenos VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/inmunología , Activación Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Baculoviridae/genética , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Three combinatorial libraries were constructed from unpurified, CD19(+), and antithyroid peroxidase (anti-TPO) B cells extracted from thyroid tissue of Graves' disease patients. Fifteen of the 41 randomly derived anti-TPO single chain variable region fragments (scFvs), showed VH1-3/V lambda 1-51 or VH1-69/V lambda 1-40 heavy/light chain pairing similar to that obtained with TPO-specific scFv derived from an in-cell library. One VH1-3/V lambda 1-51 scFv, A16, showed exactly the same nucleotide sequence as in-cell scFv ICB7, demonstrating that in vivo rearrangement can be obtained from a random combinatorial library. The majority of the scFvs used a heavy chain gene derived from the VH1-3 gene segment, whereas the light chain gene segments used were more heterogeneous, with dominance of the V kappa 1-39 and V lambda 1-51 gene segments. The anti-TPO scFvs showed high affinities to TPO, with values between 0.77 and 12.3 nM, and defined seven antigenic regions on the TPO molecule. The anti-TPO fragments, particularly VH1-3/V lambda 1-51 randomly associated scFv B4, which mimic natural H/L pairing, and VH1-3/V lambda 1-40 in-cell-derived scFv ICA5, efficiently displaced the TPO binding of serum autoantibodies from 20 Graves' disease patients. Our study directly demonstrates that antibodies derived from combinatorial libraries are likely to represent in vivo pairing, leading to high affinity antibody fragments mimicking the binding of serum autoantibodies to TPO.
Asunto(s)
Autoanticuerpos/genética , Yoduro Peroxidasa/inmunología , Adulto , Secuencia de Aminoácidos/genética , Autoanticuerpos/química , Autoanticuerpos/inmunología , Unión Competitiva , Femenino , Biblioteca de Genes , Humanos , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunologíaRESUMEN
The amino-acid sequence of the very high-affinity anti-angiotensin II monoclonal antibody 4D8 was predicted from the nucleotide sequence of the heavy and light chain variable genes. The single-chain variable fragment (scFv) was constructed and expressed in Escherichia coli as a soluble protein and at the surface of the filamentous M13 phage and was compared with the full-length antibody (Ab). The scFv showed the same specificity profile and affinity constant as the intact antibody (5.0x10(10) and 8.0x10(10) M(-1), respectively, by Scatchard analysis). Several peptides from the set of overlapping dodecapeptides covering the variable domains of 4D8 mAb were found to specifically bind biotinylated angiotensin II: peptides from the L1, L2, L3 and H1 regions had the strongest capacity to bind the antigen.
Asunto(s)
Angiotensina II/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Secuencia de Bases , Escherichia coli/metabolismo , Expresión Génica , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Datos de Secuencia Molecular , Oligopéptidos/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , SolubilidadRESUMEN
Intracellular proteins of eukaryotic cells are frequently covalently modified by the addition of long chain fatty acids. These modifications are thought to allow otherwise soluble proteins to associate with membranes by lipid-lipid based hydrophobic interactions. The purpose of this work was to quantify the effect of acyl chain length on hydrophobic interactions between acylated proteins and phospholipid monolayers. The binding of an artificially acylated model protein to electrically neutral phospholipids was studied by surface plasmon resonance, using BIACORE. Kinetic rates for the binding of bovine pancreatic ribonuclease A (RNase A), monoacylated on its N-terminal lysine with fatty acids of 10, 12, 14, 16 or 18 carbon atoms, to phospholipids on hydrophobic sensor chips, were measured. Unlike unmodified ribonuclease, acylated RNase A bound to the phospholipids, and the association level increased with the acyl chain length to reach a maximum for C16. Reproducible kinetics were obtained which did not fit a 1:1 Langmuir model but rather a two-step binding profile.
Asunto(s)
Ácidos Grasos/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Ribonucleasa Pancreática/química , Acilación , Animales , Bovinos , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.
Asunto(s)
Enfermedad de Graves/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/inmunología , Yoduro Peroxidasa/inmunología , Tiroiditis Autoinmune/inmunología , Secuencia de Aminoácidos , Técnicas Químicas Combinatorias , Enfermedad de Graves/etiología , Humanos , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Tiroiditis Autoinmune/etiologíaRESUMEN
The aim of this work was to study the reactivity of antibodies directed against the N-terminus of p53 protein. First, we analysed the cross-reactivity of anti-p53 antibodies from human, mouse and rabbit sera with peptides derived from human, mouse and Xenopus p53. Next, we characterized more precisely a series of monoclonal antibodies directed against the N-terminal part of p53 and produced by immunizing mice with either full length human or Xenopus p53. For each of these mAbs we localized the epitope recognized on human p53 by the Spot method of multiple peptide synthesis, defined critical residues on p53 involved in the interaction by alanine scanning replacement experiments and determined kinetic parameters using real-time interaction analysis. These antibodies could be divided into two groups according to their epitopic and kinetic characteristics and their cross-reactivity with murine p53. Our results indicate that critical residues involved in the interaction of some of these mAbs with p53 correspond to key residues on p53 involved in its interaction with the mdm2 protein. These antibodies could, therefore, represent powerful tools for the study of p53 regulation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos Inmunodominantes/inmunología , Proteínas Nucleares , Proteínas Proto-Oncogénicas/inmunología , Proteína p53 Supresora de Tumor/inmunología , Alanina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión , Secuencia de Consenso , Reacciones Cruzadas , Mapeo Epitopo , Humanos , Epítopos Inmunodominantes/análisis , Epítopos Inmunodominantes/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunología , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Conejos , XenopusRESUMEN
The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety-six 20-mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125I]cTnC, in the presence of 3 mM Ca2+, enabled the assignment of six sites of interaction (residues 19-32, 45-54, 129-138, 145-164, 161-178 and 191-210). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10(-6)-10(-7) M, except for hcTnI [39-58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19-32 from the N-terminal cardio-specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19-32 may correspond to the minimal sequence of the extension which could switch between the N- and C-terminal TnC domains, depending on its phosphorylation state. Finally, two Ca(2+)-dependent cTnC binding domains within the C-terminal part of hcTnI (residues 164-178 and 191-210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium.
Asunto(s)
Miocardio/metabolismo , Troponina C/metabolismo , Troponina I/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biotinilación , Bovinos , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Mutación del Sistema de Lectura , Humanos , Datos de Secuencia Molecular , Aturdimiento Miocárdico/metabolismo , Miocardio/química , Biosíntesis de Péptidos , Péptidos/química , Unión Proteica , Resonancia por Plasmón de Superficie , Factores de Tiempo , Troponina C/química , Troponina C/genética , Troponina I/química , Troponina I/genéticaRESUMEN
gp17, a secretory CD4-binding factor isolated from the human seminal plasma, is identical to the gross cystic disease fluid protein-15, a specific marker for primary and metastatic breast tumors. We previously demonstrated that gp17 binds to CD4 with high affinity and strongly inhibits T lymphocyte apoptosis induced by sequential cross-linking of CD4 and T cell receptor (TCR). To further characterize the gp17/CD4 interaction and map the gp17 binding site, we produced a secreted form of recombinant gp17 fused to human IgG1 Fc, gp17-Ig. We showed that gp17-Ig exhibits a binding affinity for CD4 similar to that of native gp17. As no information about gp17 structure is presently available, 99 overlapping gp17 peptides were synthesized by the Spot method, which allowed the mapping of two CD4 binding regions. Alanine scanning of CD4-reactive peptides identified critical residues, selected for site-directed mutagenesis. Nine gp17-Ig mutants were generated and characterized. Three residues within the carboxy-terminal region were identified as the major binding domain to CD4. The Spot method combined with mutagenesis represents a refined approach to distinguish the contact residues from the ones contributing to the conformation of the CD4-binding domain.
Asunto(s)
Apolipoproteínas , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/química , Glicoproteínas/química , Proteínas de Transporte de Membrana , Vesículas Seminales/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteínas D , Antígenos CD4/metabolismo , Células COS , Proteínas Portadoras/genética , Técnica del Anticuerpo Fluorescente , Glicoproteínas/genética , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Proteínas Recombinantes de Fusión , TransfecciónRESUMEN
beta(2)-Microglobulin, a 12 kDa protein forming part of the class I HLA (histocompatibility locus antigen) major histocompatibility complex, has been used as a prognosis factor for multiple myeloma and as a marker of renal function, and has been shown to be involved in the pathogenesis of dialysis-related amyloidosis. alpha(2)-Macroglobulin has the ability to bind a wide range of physiologically important molecules, thereby influencing their metabolic impact. In this study we show by Western blotting analysis that beta(2)-microglobulin binds to alpha(2)-macroglobulin in vitro. This binding was confirmed by BIAcore analysis, and was shown by ELISA to be concentration-dependent. The sequences of the binding peptides in the mature beta(2)-microglobulin molecule were identified by Spot multiple peptide synthesis and alpha(2)-macroglobulin binding studies. In conclusion, beta(2)-microglobulin interacts specifically with the universal antiprotease a(2)-macroglobulin. The identification of this interaction brings into question some of the axioms on the metabolism of beta(2)-microglobulin, and may help to explain the clinical findings observed in b(2)-microglobulin-related diseases.
Asunto(s)
alfa-Macroglobulinas/química , Microglobulina beta-2/química , Amiloidosis/metabolismo , Sitios de Unión , Western Blotting , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Sitios de Unión , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/aislamiento & purificación , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
In DNA, the deamination of dAMP generates 2'-deoxy-inosine 5'-monophosphate (dIMP). Hypoxanthine (HX) residues are mutagenic since they give rise to A.T-->G.C transition. They are excised, although with different efficiencies, by an activity of the 3-methyl-adenine (3-meAde)-DNA glycosylases from Escherichia coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and yeast (MAG protein). Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E.coli and yeast enzymes are quite inefficient, whereas for the ANPG and the APDG proteins they repair the HX residues with an efficiency comparable to that of alkylated bases, which are believed to be the primary substrates of these DNA glycosylases. Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates. Moreover, using oligo-nucleotides containing a single dIMP residue, we investigated a putative sequence specificity of the enzymes involving the bases next to the HX residue. We found up to 2-5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied. When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dI:T over dI:dG, dI:dC and dI:dA mismatches was observed for both human (ANPG) and E.coli (AlkA) proteins. At variance, the yeast MAG protein removed more efficiently HX from a dI:dG over dI:dC, dI:T and dI:dA mismatches.
Asunto(s)
Disparidad de Par Base/genética , ADN Glicosilasas , ADN/metabolismo , Escherichia coli/enzimología , Inosina Monofosfato/análogos & derivados , N-Glicosil Hidrolasas/metabolismo , Saccharomyces cerevisiae/enzimología , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ADN/química , ADN/genética , Proteínas Fúngicas/metabolismo , Humanos , Hipoxantina/metabolismo , Inosina Monofosfato/genética , Inosina Monofosfato/metabolismo , Cinética , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Piperidinas/metabolismo , Ratas , Especificidad por Sustrato , TermodinámicaRESUMEN
Thyroglobulin (TG) is secreted by the thyrocytes into the follicular lumen of the thyroid. After maturation and hormone formation, TG is endocytosed and delivered to lysosomes. Quality control mechanisms may occur during this bidirectional traffic since 1) several molecular chaperones are cosecreted with TG in vivo, and 2) lysosomal targeting of immature TG is thought to be prevented via the interaction, in acidic conditions, between the Ser(789)-Met(1172) TG hormonogenic domain (BD) and an unidentified membrane receptor. We investigated the secretion and cell surface expression of PDI and other chaperones in the FRTL5 thyroid cell line, and then studied the characteristics of the interaction between TG and PDI. We demonstrated that PDI, but also other chaperones such as calnexin and KDEL-containing proteins are exposed at the cell surface. We observed on living cells or membrane preparations that PDI specifically binds TG in acidic conditions, and that only BD is involved in binding. Surface plasmon resonance analysis of TG/PDI interactions indicated: 1) that PDI bound TG but only in acidic conditions, and that it preferentially recognized immature molecules, and 2) BD is involved in binding even if cysteine-rich modules are deleted. The notion that PDI acts as an "escort" for immature TG in acidic post-endoplasmic reticulum compartments is discussed.
Asunto(s)
Proteínas de Choque Térmico , Proteína Disulfuro Isomerasas/metabolismo , Glándula Tiroides/enzimología , Animales , Unión Competitiva , Proteínas Portadoras/metabolismo , Línea Celular , Cricetinae , Chaperón BiP del Retículo Endoplásmico , Fluoresceína-5-Isotiocianato/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Concentración de Iones de Hidrógeno , Membranas Intracelulares/enzimología , Cinética , Chaperonas Moleculares/metabolismo , Péptidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/aislamiento & purificación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de Superficie , Tiroglobulina/metabolismo , Factores de TiempoRESUMEN
Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.
Asunto(s)
Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Biotina , Dimerización , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , EstreptavidinaRESUMEN
In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves' disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vlambda1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves' disease patients tested were able to strongly inhibit (60-100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.
Asunto(s)
Linfocitos B/enzimología , Linfocitos B/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Yoduro Peroxidasa/inmunología , Biblioteca de Péptidos , Glándula Tiroides/enzimología , Adulto , Secuencia de Aminoácidos , Especificidad de Anticuerpos/genética , Autoanticuerpos/sangre , Linfocitos B/química , Secuencia de Bases , Sitios de Unión de Anticuerpos , Unión Competitiva , Técnicas Químicas Combinatorias/métodos , Femenino , Genes de Inmunoglobulinas , Enfermedad de Graves/sangre , Enfermedad de Graves/genética , Enfermedad de Graves/inmunología , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Yoduro Peroxidasa/sangre , Datos de Secuencia Molecular , Glándula Tiroides/inmunología , Glándula Tiroides/patologíaRESUMEN
The gp17 factor is a secretory product of human seminal vesicle cells which binds to CD4 and acts as a potent inhibitor of T lymphocyte apoptosis induced by CD4 crosslinking and subsequent T-cell receptor (TCR) activation. The protein is identical to gross cystic disease fluid protein-15 (GCDFP-15), a breast tumor secretory marker PIP (prolactin inducible protein), a prolactin-controlled and androgen-controlled protein; secretory actin binding protein (SABP), a seminal plasma actin binding protein and extra-parotid glycoprotein (EP-GP), a secretory protein from the salivary gland. The structure of this protein has not yet been elucidated and no biological function has been clearly attributed to date. Expression of recombinant gp17/GCDFP-15 cDNA in bacteria and insect cells leads to the production of a misfolded insoluble protein. In this study, we describe the production of gp17/GCDFP-15 in two different eukaryotic systems, namely HeLa cells and the Pichia pastoris yeast. Using constructs in which gp17/GCDFP-15 was tagged with enhanced green fluorescent protein (EGFP) in various combinations, we observed expression only when the fusion protein was directed to the secretory compartment by the correct signal peptide. The resulting fluorescent protein was inefficiently secreted, thus suggesting that gp17/GCDFP-15 is not appropriately post-translationally processed and/or transported in HeLa cells. The use of the P. pastoris secretory pathway allowed instead the accumulation in the culture medium of a GCDFP-15/gp17 species which retained the ability to bind to CD4 and also most of the biochemical and immunological properties of the native protein. The production of an active recombinant molecule opens the way to correlate the structural properties of this peculiar factor to its ability to bind several proteins, including CD4, and to block CD4-mediated T cell programmed death.
Asunto(s)
Apolipoproteínas , Antígenos CD4/química , Proteínas Portadoras/química , Glicoproteínas/química , Proteínas de Transporte de Membrana , Vesículas Seminales/química , Apolipoproteínas D , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/inmunología , Glicoproteínas/biosíntesis , Glicoproteínas/inmunología , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes , Masculino , Pichia , Unión Proteica , Proteínas Recombinantes , TransfecciónRESUMEN
It is widely accepted that the serum of healthy individuals contains natural antibodies only against those blood group A or B antigens that are not expressed on the individual's red blood cells. The mechanisms involved in tolerance to autologous blood group antigens remain unclear. In the present study, we show that IgM and IgG antibodies reactive with autologous blood group antigens are present in the immunoglobulin fraction of normal human serum. Natural IgG anti-A antibodies purified by affinity chromatography from IgG of individuals of blood group A exhibited an affinity for A trisaccharide antigen in the micromolar range and agglutinated A red cells at sixfold higher concentrations than those required for agglutination with affinity-purified anti-A IgG of individuals of blood group B. Whereas autoantibodies reactive with self A and B antigens are readily detected in purified IgG and IgM fractions, their expression is restricted in whole serum as a result of complementary interactions between variable regions of antibodies. These observations suggest that tolerance to autologous ABO blood group antigens is dependent on peripheral control of antibody autoreactivity.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/inmunología , Autoanticuerpos/sangre , Adulto , Complejo Antígeno-Anticuerpo/análisis , Autoantígenos/inmunología , Ensayo de Inmunoadsorción Enzimática , Hemaglutinación , Humanos , Tolerancia Inmunológica , Inmunoglobulina G/sangre , Inmunoglobulina M/sangreRESUMEN
Intravenous immunoglobulin (IVIg) therapy is associated with a broad range of immunomodulatory activities. Several of the postulated mechanisms of IVIg action relate to the presence of antibodies to molecules relevant for regulation of the immune response. This article reports that IVIg contains antibodies to the Arg-Gly-Asp (RGD) sequence, and the attachment site of a number of adhesive extracellular matrix proteins, including ligands for beta1, beta3, and beta5 integrins. Anti-RGD antibodies were identified in IVIg by enzyme-linked immunosorbent assay and by using the BIAcore (BIAcore, Uppsala, Sweden) technology. The affinity of anti-RGD antibodies to a synthetic RGD-containing peptide and to fibronectin (Fn) was found to be in the micromolar range. F(ab')2 fragments specific for RGD were purified from IVIg by affinity chromatography. Anti-RGD F(ab')2 antibodies inhibited adenosine diphosphate induced alphaIIb/beta3 integrin-mediated platelet aggregation and the adhesion of activated alpha4beta1 integrin-expressing B cells to Fn. Adhesion of unstimulated platelets to fibrinogen (Fg) involving both the gamma-chain dodecapeptide sequence and the RGD sequence was inhibited by anti-RGD antibodies. In addition, adhesion of thrombin-stimulated platelets to von Willebrand factor or Fg was completely inhibited by affinity-purified anti-RGD antibodies. Our results suggest that the presence of natural IgG antibodies to the RGD motif may contribute to the immunomodulatory and anti-inflammatory effects of therapeutic preparations of normal IgG.
Asunto(s)
Anticuerpos/farmacología , Linfocitos B/patología , Inmunoglobulinas Intravenosas/farmacología , Oligopéptidos/inmunología , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Anticuerpos/inmunología , Adhesión Celular/efectos de los fármacos , Fibronectinas , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Adhesividad Plaquetaria/inmunología , Agregación Plaquetaria/inmunología , Receptores Inmunológicos/inmunologíaRESUMEN
Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.
Asunto(s)
Antígenos CD/fisiología , Interleucina-6/fisiología , Glicoproteínas de Membrana/fisiología , Paraproteinemias/inmunología , Receptores de Interleucina-6/fisiología , Anticuerpos Monoclonales , Antígenos CD/sangre , Antígenos CD/aislamiento & purificación , Receptor gp130 de Citocinas , Electroforesis en Gel de Poliacrilamida , Epítopos/análisis , Femenino , Humanos , Interleucina-6/farmacología , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/aislamiento & purificación , Persona de Mediana Edad , Paraproteinemias/sangre , Unión Proteica , Receptores de Interleucina-6/sangre , Receptores de Interleucina-6/aislamiento & purificación , Transducción de Señal/inmunologíaRESUMEN
In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non-overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab' fragments from 4 different specific anti-CEA MAbs recognizing 4 CEA epitopes (Gold 1-4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross-inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) M(-1) for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab')2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5- and 8.1-fold increase, respectively, as compared with the parental F(ab')2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab')2 by injection of 131I-BpAb/125I-F(ab')2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post-injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab')2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab')2, one of the best parental F(ab')2 fragments used in our study.