RESUMEN
The complete coding sequence of a BDIX rat gene homologous to the human ABO gene was determined. Identification of the exon-intron boundaries, obtained by comparison of the coding sequence with rat genomic sequences from data banks, revealed that the rat gene structure is identical to that of the human ABO gene. It localizes to rat chromosome 3 (q11-q12), a region homologous to human 9q34. Phylogenetic analysis of a set of sequences available for the various members of the same gene family confirmed that the rat sequence belongs to the ABO gene cluster. The cDNA was transfected in CHO cells already stably transfected with an alpha1,2fucosyltransferase in order to express H oligosaccharide acceptors. Analysis of the transfectants by flow cytometry indicated that A but not B epitopes were synthesized. Direct assay of the enzyme activity using 2' fucosyllactose as acceptor confirmed the strong UDP-GalNAc:Fucalpha1,2GalalphaGalNAc transferase (Atransferase) activity of the enzyme product and allowed detection of a small UDP-Gal:Fucalpha1,2GalalphaGal transferase (B transferase) activity. The presence of the mRNA and of the A and B antigens was searched in various BDIX rat tissues. There was a general good concordance between the presence of the mRNA and that of the A antigen. Tissue distributions of the A and B antigens in the homozygous BDIX rat strain were largely different, indicating that these antigens cannot be synthesized by alleles of the same gene in this rat inbred strain.
Asunto(s)
N-Acetilgalactosaminiltransferasas/genética , Sistema del Grupo Sanguíneo ABO/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Mapeo Cromosómico , Clonación Molecular , Cricetinae , ADN Complementario/genética , Evolución Molecular , Humanos , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/biosíntesis , N-Acetilgalactosaminiltransferasas/metabolismo , Oligosacáridos/biosíntesis , Oligosacáridos de Cadena Ramificada , Especificidad de Órganos , Filogenia , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Trisacáridos/biosíntesis , Trisacáridos/metabolismoRESUMEN
The aim of the study was to assess the isolation of HDL by fast protein liquid chromatography (FPLC) to perform kinetics studies of apolipoprotein (apo)A-I-HDL labelled with a stable isotope. Comparison between FPLC and ultracentrifugation has been made. ApoA-I-HDL kinetics were studied by infusion of [5.5.5-(2)H(3)]leucine for 14 h in five subjects. Using FPLC, prebeta(1) HDL and alphaHDL (HDL(2) and HDL(3)) were separated from 200 microl of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in prebeta(1) HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to alphaHDL suggested the existence of a mixture of apoA-I-HDL sub-classes. From this difference in enrichments, the turnover rate of total HDL-UC, usually assumed to be alphaHDL, was probably overestimated in previous studies. To our knowledge, this study is the first report which provides a convenient tool to distinguish enrichments of apoA-I in prebeta(1) HDL and alphaHDL from total HDL previously used for kinetic measurements. This original and new method should help to understand the kinetics of HDL in humans and the reverse cholesterol transport dynamics.
Asunto(s)
Apolipoproteína A-I/sangre , Cromatografía Líquida de Alta Presión/métodos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/aislamiento & purificación , Apolipoproteína A-I/aislamiento & purificación , Humanos , Cinética , Lipoproteínas HDL/clasificación , UltracentrifugaciónRESUMEN
Neutral mucin oligosaccharides from the small intestine of control rats and rats infected with the parasite Nippostrongylus brasiliensis were released and analyzed by gas chromatography-mass spectrometry. Infected animals expressed seven blood group A-like structures that were all absent in the control animals. The blood group A nature of these epitopes was confirmed by blood group A reactivity of the prepared mucins, of which Muc2 was one. Transferase assays and Northern blotting on small intestines from infected animals showed that an alpha-N-acetylgalactosaminyltransferase similar to the human blood group A glycosyltransferase had been induced. The expression was a transient event, with a maximum at day 6 of the 13-day-long infection. The rat blood group A glycosyltransferase was cloned, revealing two forms with an amino acid similarity of 95%. Both types had blood group A transferase activity and were probably allelic because none of 12 analyzed inbred strains carried both types. The second type was found in outbred rats and in one inbred strain. First generation offspring of inbred rats of each type were heterozygous, further supporting the allelic hypothesis. The transient induction and the large allelic variation could suggest that glycosyltransferases are part of a dynamic system altering mucins and other glycoconjugates as a protecting mechanism against microbial challenges.