RESUMEN
HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.
Asunto(s)
Antígenos HLA/química , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana , Estructura Secundaria de Proteína , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Membrana Celular/metabolismo , Cristalografía por Rayos X/métodos , Antígenos HLA/genética , Hemocromatosis/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Receptores de Transferrina/químicaRESUMEN
In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A. A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments. As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes. Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein. Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2. The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC. The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC. Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos Par 6 , Hemocromatosis/genética , Proteínas de la Membrana , ARN Citoplasmático Pequeño , Simportadores , Secuencia de Aminoácidos , Autoantígenos/genética , Bacterias/genética , Sitios de Unión , Northern Blotting , Butirofilinas , Proteínas Portadoras/genética , Clonación Molecular , Secuencia Conservada , ADN Complementario , Antígenos HLA/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Histonas/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares , Proteínas/genética , Proteínas/metabolismo , Ribonucleoproteínas/genética , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de Aminoácido , Lugares Marcados de Secuencia , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Distribución Tisular , Factores de Transcripción , Transcripción Genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.