RESUMEN
The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (mu(0)) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F(1) (DQ52(+/-)) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52(-) allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM(+) B cells of bone marrow and spleen used the DQ52(-) allele less frequently. On DJ joints of the DQ52(-) allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52(-) allele. In splenic B cells of the DQ52(-/-) mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3' JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.
Asunto(s)
Diversidad de Anticuerpos/genética , Eliminación de Gen , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencias Reguladoras de Ácidos Nucleicos/inmunología , Alelos , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/patología , Secuencia de Bases , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , ADN Complementario/aislamiento & purificación , Marcación de Gen , Marcadores Genéticos/inmunología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región de Unión de la Inmunoglobulina/biosíntesis , Región de Unión de la Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/metabolismo , Cadenas mu de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/aislamiento & purificación , Recuento de Linfocitos , Linfopenia/genética , Linfopenia/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/inmunología , Transcripción Genética/inmunologíaRESUMEN
BACKGROUND: A number of allergenic proteins in animal danders have been characterized at the molecular level, but little is known of their biologic functions. We have found that the prevalence of IgE antibodies among patients with cattle-associated asthma is highest against a dander protein referred to as BDA20. OBJECTIVE: The study was performed to characterize the molecular structure of BDA20,* the predominant allergen in bovine dander. METHODS: Clones encoding allergens were identified and isolated from a complementary DNA library by immunoblotting and DNA hybridization and sequenced. Recombinant proteins were produced in Escherichia coli. Immunoreactivity of the recombinant proteins and amino acid sequences of peptides obtained from native BDA20 after Lys-C cleavage were used to identify clones coding for BDA20. RESULTS: In this article we report the cDNA and amino acid sequences of BDA20. Homology comparisons showed that BDA20 belongs to the family of lipocalins. CONCLUSIONS: The results link a dander allergen to a group of functionally important proteins. Lipocalins are present in various body fluids and secretions of several animal species in which they function as carriers of small hydrophobic molecules, such as retinoids and pheromones. If allergenicity proves to be a property shared by lipocalins, our results will have considerable implications for allergen research.
Asunto(s)
Alérgenos/genética , Proteínas , Piel/inmunología , Animales , Antígenos de Plantas , Secuencia de Bases , Bovinos , Secuencia de Consenso , Cartilla de ADN/química , ADN Complementario/genética , Humanos , Hipersensibilidad/inmunología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Piel/químicaRESUMEN
Immunoscreening of a cDNA library from bovine skin led to isolation of clones coding for an allergen named BDA11. Sequence analysis of the clones revealed that they can encode a protein of 11.6 kDa with a predicted pI of 5.19. Allergenicity of BDA11 was verified by the IgE reactivity in cattle-allergic patients' sera with the recombinant protein produced in Escherichia coli. A biochemically purified native allergen of 11 kDa from bovine dander was identified as BDA11 by peptide sequencing. Homology comparisons showed that BDA11 had a 63.4% amino acid identity with human psoriasin. Psoriasin is a calcium-binding protein expressed in keratinocytes, and it is strongly up-regulated in psoriatic skin. BDA11 also had segments homologous with calcium-binding proteins from three other species.