RESUMEN
BACKGROUND: The anion exchanger, Band 3, carries antigens in the Diego blood group system, and can carry the Band 3-Memphis phenotype. Although Di(b) is of high prevalence and Band 3-Memphis is of low prevalence in humans, it has been suggested that both are on the ancestral gene. We determined the orthologue nucleotide sequences corresponding to these two polymorphic sites, Di(a)/Di(b) (2561T > C; Leu854Pro) and Band 3-Memphis(166A > G; Lys56Glu) in several nonhuman primates. METHODS: Genomic DNA was extracted from blood samples of great apes, lesser apes, old world monkeys, new world monkeys and prosimians. PCR amplifications were done with primer pairs that were located in the flanking intronic regions of Exon 4 and Exon 19; and the amplified products were sequenced. RESULTS: Amino acid sequence alignment of nonhuman primates band 3 with that of human showed extensive homologies. In exon 4, Glu56Lys polymorphic site showed Glu similar to Band 3-Memphis type and in exon 19, Leu854Pro polymorphic site showed Pro indicating Di(b) phenotype. CONCLUSIONS: The nonhuman primates have nucleotide sequences of Di(b)(2561C) in cis to Band 3-Memphis (166G), which is consistent with the assertion that the Di(b) and Band 3-Memphis phenotype represents the ancestral Band 3 gene.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/genética , Antígenos de Grupos Sanguíneos/genética , Primates/sangre , Primates/genética , Secuencia de Aminoácidos , Animales , ADN/química , ADN/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.
Asunto(s)
ADP Ribosa Transferasas/inmunología , Proteínas de Ciclo Celular/inmunología , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/inmunología , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reacciones Cruzadas , Endotelio Vascular/inmunología , Eritrocitos/inmunología , Proteínas Ligadas a GPI , Humanos , Macrófagos/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Monocitos/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Tonsila Palatina/metabolismo , Ratas , Alineación de Secuencia , Bazo/inmunología , Venas Umbilicales/inmunologíaRESUMEN
BACKGROUND: The Gerbich blood group system antigens are carried on glycophorin C (GPC) and glycophorin D (GPD) and variants thereof. These glycoproteins have been expressed in a heterologous system to study the individual antigens and to determine whether Ana is antithetical to Ge2. STUDY DESIGN AND METHODS: cDNAs encoding GPC, GPD, GPC.Yus, GPC.Ge, GPC.Lsa, and GPD.Lsa were transfected and stably expressed in a human embryonic kidney cell line (293T). Individual Gerbich antigens were analyzed with MoAbs and human polyclonal antibodies by flow cytometry and immunoblotting. Recombinant GPD and GPD.Ana were expressed transiently and analyzed for expression of Ge2 and Ana antigens. RESULTS: All recombinant variants were detected with sialidase-resistant and -sensitive anti-Ge2, anti-Ge3, and anti-Ge4. Ge4 antigen expression was depressed in GPC.Ls(a) transfectants as well as on Ls(a+) RBCs. GPD.An(a) recombinant protein expressed Ana and Ge2 antigens. CONCLUSION: Cell lines stably expressing glycosylated Gerbich proteins were developed in a heterologous system by transfecting individual variant forms of GPC and GPD. Unexpectedly, it was found that Ge4 antigen is reduced in both the GPC.Ls(a) recombinant and the Ls(a+) RBCs. It was also shown that Ana and Ge2 antigens were expressed on a single GPD.An(a) protein and, therefore, they cannot be antithetical.