RESUMEN
U3 small nucleolar RNA (snoRNA) is a member of the Box C/D family of snoRNAs which functions in ribosomal RNA processing. U3-55k is a protein that has been found to interact with U3 but not other members of the Box C/D snoRNA family. We have found that interaction of the U3-55k protein with U3 RNA in vivo is mediated by the conserved Box B/C motif which is unique to U3 snoRNA. Mutation of Box B and Box C, but not of other conserved sequence elements, disrupted interaction of U3-55k with U3 RNA. Furthermore, a fragment of U3 containing only these two conserved elements was bound by U3-55k in vivo. RNA binding assays performed in vitro indicate that Box C may be the primary determinant of the interaction. We have cloned the cDNA encoding the Xenopus laevis U3-55k protein and find strong homology to the human sequence, including six WD repeats. Deletion of WD repeats or sequences near the C-terminus of U3-55k resulted in loss of association with U3 RNA and also loss of localization of U3-55k to the nucleolus, suggesting that protein-protein interactions contribute to the localization and RNA binding of U3-55k in vivo.
Asunto(s)
ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Unión Proteica , ARN Nucleolar Pequeño/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Ribonucleoproteínas Nucleolares Pequeñas/química , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.
Asunto(s)
Aequorina/sangre , Galactosiltransferasas/sangre , Lectinas de Plantas , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Animales , Biotina/metabolismo , Secuencia de Carbohidratos , Bovinos , Humanos , Lectinas/metabolismo , Microquímica/métodos , Leche/química , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Proteínas Recombinantes/metabolismo , Albúmina Sérica Bovina/metabolismoRESUMEN
We report here that both the mouse teratocarcinoma F9 cells and F9 cells induced to differentiate by treatment with retinoic acid contain cell surface glycoconjugates with terminal alpha-linked galactose residues, as shown by agglutination of cells with antisera to blood type B, but not to type A. In addition, both cell types contain high numbers of binding sites for Griffonia simplicifolia-I, a lectin which binds to terminal alpha-linked galactose residues, although differentiated F9 cells contain approximately 50% more binding sites/cell for this lectin. We have also confirmed that differentiation is accompanied by a decrease in the expression of the fucose-containing stage-specific embryonic antigen (SSEA)-1, as evidenced by the fact that F9 cells, but not differentiated F9 cells, are agglutinated by monoclonal antibody to this antigen. Since these results indicate that surface glycoconjugates contain terminal alpha-linked galactose residues, we assayed cell extracts for the enzyme UDP-Gal:beta-D-Gal-alpha 1,3-galactosyltransferase. We have found that F9 cell extracts contain this activity, and differentiation results in a significant increase in the specific activity of the enzyme, from approximately 2 nmol/mg h in F9 extracts to 7 nmol/mg h in RA/F9 extracts. It has been suggested that the loss of the SSEA-1 antigen upon differentiation of F9 cells is due to decreased activity of the enzyme GDP-Fuc:beta-D-GlcNAc-alpha 1, 3-fucosyltransferase. We therefore determined the activities of this fucosyltransferase and several other glycosyltransferases, which included UDP-GlcNAc:beta-D-Gal-beta 1,3-N-acetylglucosaminyltransferase, UDP-Gal:beta-D-GlcNAc-beta 1,4-galactosyltransferase, and GDP-Fuc:beta-D-GlcNAc-alpha 1,6-fucosyltransferase. We have found that extracts from both cell types contain these enzyme activities; differentiation, however, does not result in substantial changes in any of these activities.