RESUMEN
Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Anticuerpos Monoclonales/inmunología , Antígenos de Grupos Sanguíneos/inmunología , Proteínas Portadoras/inmunología , Proteínas de la Membrana/inmunología , Espectrina/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/aislamiento & purificación , Western Blotting , Cromatografía de Afinidad , Detergentes/farmacología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Membrana Eritrocítica/química , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/inmunología , Humanos , Isoantígenos/química , Isoantígenos/inmunología , Isoantígenos/aislamiento & purificación , Sistema del Grupo Sanguíneo de Kell/química , Sistema del Grupo Sanguíneo de Kell/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
The human Kx blood group antigen is carried by a 37,000 M(r) apparent molecular mass membrane polypeptide which is deficient in rare individuals with the McLeod syndrome. The X-linked human XK gene is transcribed in many tissues including adult skeletal muscle and brain, sieges of disorders observed in McLeod syndrome. We report here the cloning of the orthologous mouse XK mRNA. Comparison of XK from human and mouse revealed 80% sequence similarity at the amino acid level. The mouse XK gene is organized in two exons and is expressed in many tissues, but its expression pattern is slightly different from that of the human gene. The presence in mouse erythrocyte membrane of a 43,000 M(r) Kx-related protein was demonstrated by immunoblotting with a rabbit antiserum directed against the human protein. With non-reduced samples, a 140,000 M(r) species was detected instead of the 43,000 M(r) protein, suggesting that, as demonstrated in the Kx polypeptide might be complexed with another protein in mouse red cells, presumably the homologue of the human Kell protein of 93,000 M(r).
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Antígenos de Grupos Sanguíneos/genética , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Membrana Eritrocítica/química , Biblioteca de Genes , Sistema del Grupo Sanguíneo de Kell , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Músculo Esquelético , Unión Proteica , Distribución TisularRESUMEN
Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Eritrocitos/metabolismo , Sistema del Grupo Sanguíneo de Kell/metabolismo , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Proteínas Sanguíneas/química , Proteínas Portadoras/química , Humanos , Sistema del Grupo Sanguíneo de Kell/química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Ácido PalmíticoRESUMEN
Kell and Kx are two quantitatively minor proteins from the human erythrocyte membrane which carry blood groups antigens and are thought to be a metalloprotease and a membrane transporter, respectively. In the red cell membrane, these proteins form a complex stabilized by disulfide bond(s). Phosphorylation status of these proteins was studied, in the presence or absence of effectors of several kinases, either on intact cells incubated with [32P]-orthophosphate or on ghosts incubated with [gamma-32P]ATP. Purification of Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity allowed to establish that (i) neither protein is phosphorylated on tyrosine; (ii) the Kell protein is a putative substrate for Casein Kinase II (CKII) and Casein Kinase I (CKI) but not for protein kinase C (PKC), whereas Kx protein is phosphorylated by CKII and PKC but not by CKI; (iii) Protein Kinase A neither phosphorylates the Kell nor the Kx proteins.
Asunto(s)
Antígenos de Superficie/química , Eritrocitos/química , Sistema del Grupo Sanguíneo de Kell/química , Proteínas de la Membrana/química , Quinasa de la Caseína II , Caseína Quinasas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Disulfuros/química , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Membrana Eritrocítica/química , Humanos , Fosforilación , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The Kx protein is an erythrocyte membrane polypeptide which is deficient in rare individuals suffering from the McLeod syndrome. The gene encoding this protein has been recently cloned and the Kx protein independently purified as a covalent complex with the Kell blood group protein. To further study the Kx membrane protein, antisera raised in rabbits against six synthetic peptides derived from the primary sequence of this protein were characterized. All antisera but two precipitated the recombinant Kx protein synthesized in coupled transcription-translation in vitro. Three antisera reacted on immunoblots with the 37 kD Kx protein present in the purified Kell-Kx complex and in SDS red cell membrane lysates from variants with different Kell blood group phenotypes, including Ko, which lack the Kell protein of 93 kD. However, no reactivity was found with McLeod preparations lacking Kx protein, thus clearly indicating that these antibodies have a Kx specificity. Unexpectedly, the relative amount of Kx protein in Ko cells was found to be lower than in red cells with common Kell phenotypes, suggesting that the absence of the Kell protein may alter the amount of Kx in the membrane.