RESUMEN
We describe a novel gene, Blimp-1 (for B lymphocyte-induced maturation protein), transcripts of which are rapidly induced during the differentiation of B lymphocytes into immunoglobulin secretory cells and whose expression is characteristic of late B and plasma cell lines. The 856 amino acid open reading frame contains five Krüppel-type zinc finger motifs and proline-rich and acidic regions similar to those of known transcription factors. Serological studies show an approximately 100 kd protein that localizes to the nucleus. Stable or transient transfection of Blimp-1 into B cell lymphoma lines leads to the expression of many of the phenotypic changes associated with B cell differentiation into an early plasma cell stage, including induction of J chain message and immunoglobulin secretion, up-regulation of Syndecan-1, and increased cell size and granularity. Thus, Blimp-1 appears to be a pleiotropic regulatory factor capable of at least partially driving the terminal differentiation of B cells.
Asunto(s)
Linfocitos B/fisiología , Diferenciación Celular/genética , Genes de Inmunoglobulinas/genética , Células Plasmáticas/fisiología , Proteínas Represoras , Factores de Transcripción/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario/aislamiento & purificación , Cadenas J de Inmunoglobulina/análisis , Cadenas J de Inmunoglobulina/biosíntesis , Inmunoglobulina M/análisis , Inmunoglobulina M/biosíntesis , Ratones , Datos de Secuencia Molecular , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Reacción en Cadena de la Polimerasa , Factor 1 de Unión al Dominio 1 de Regulación Positiva , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Análisis de Secuencia de ADN , Factores de Transcripción/análisis , Factores de Transcripción/fisiología , TransfecciónRESUMEN
All intra J chain disulfide bridges of human sIgA, the disulfide bonds between the J chain and the two IgA monomers, and one inter IgA monomer disulfide bridge were determined. sIgA was isolated from colostrum of healthy women and digested with IgA1-specific protease followed by cyanogen bromide cleavage. This procedure generated fragments of 140 kDa, 60 kDa, and 28 kDa. The 28-kDa polypeptide comprised the complete J chain covalently bound to two alpha 1 chain octapeptides derived from the C-termini of two alpha 1 chains. The 28-kDa fragment was digested with trypsin. The resulting peptides were purified by RP-HPLC, and subsequently characterized by amino-acid analysis, mass spectrometry, and gas phase sequencing. These data unequivocally show that the J chain cysteines C1-C6, C4-C5, and C7-C8 form intra chain disulfide bridges. The second (C2) and the third (C3) J chain cysteines are disulfide linked to two alpha chain cysteines (C17) joining the two IgA monomers of sIgA tail to tail. The remaining two alpha chains of the two monomers are directly bound to each other via their ultimate cysteines (C17-C17). A new model for the J chain in sIgA is presented.
Asunto(s)
Disulfuros/análisis , Inmunoglobulina A Secretora/análisis , Cadenas J de Inmunoglobulina/análisis , Serina Endopeptidasas , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Calostro/química , Bromuro de Cianógeno , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Hidrólisis , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Pepsina A , Péptido Hidrolasas , Estructura Terciaria de Proteína , TripsinaAsunto(s)
Mama/inmunología , Inmunoglobulina A/biosíntesis , Lactancia , Adulto , Células Productoras de Anticuerpos , Mama/anatomía & histología , Mama/metabolismo , Recuento de Células , Calostro/inmunología , Citoplasma/inmunología , Femenino , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Cadenas J de Inmunoglobulina/análisis , Inmunoglobulina M/análisis , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Aparato Lagrimal/inmunología , Aparato Lagrimal/metabolismo , Tamaño de los Órganos , Glándula Parótida/anatomía & histología , Glándula Parótida/inmunología , Glándula Parótida/metabolismo , EmbarazoRESUMEN
Purified bovine colostral intact immunoglobulin M (IgM) exhibited the presence of an anodal, single, fast moving band (noncovalently bound form) when subjected to analytical polyacrylamide gel electrophoresis at an alkaline pH in urea. Reduced and alkylated or sulfitolysed bovine colostral IgM (devoid of the noncovalently bound form) also showed the presence of a similar band (covalently bound form). The molecular weight of both the covalently bound and noncavalently bound forms of the fast component was determined to be 16,500 by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. In addition, the non-cavalently bound form of the fast-moving component was found to be antigenically identical to the covalently bound form. The noncovalently bound form sedimented as a single peak at 1.56 S. Antiserum against the fast-moving component precipitated neither bovine colostral IgG nor mu-chains and bovine serum albumin, but precipitated native or denatured intact IgM (devoid of the non-covalently bound form) and human J-chains and vice versa, thus permitting the fast-moving components to be classified as J-chains. Radioalkylation experiments revealed the presence of 9.7 sulfhydryl groups per mole, for both the covalently and non-covalently bound forms of bovine J-chain. The stoichiometry of J-chain, determined from the densitometric tracing of the reduced and alkylated bovine colostral IgM (devoid of the noncovalently bound J-chain) in stained analytical polyacrylamide gels, revealed the presence of one J-chain per IgM molecule. On the other hand the amount of non-covalently bound form of J-chain was determined to be 1.2 per molecule of IgM.