RESUMEN
Bovine beta-1,4-galactosyltransferase (beta-1,4-GT; EC 2.4.1.90) belongs to the glycosyltransferase family and as such shares a general topology: an N-terminal cytoplasmic tail, a signal anchor followed by a stem region and a catalytic domain at the C-terminal end of the protein. cDNA constructs of the N-terminal deleted forms of beta-1,4-GT were prepared in pGEX-2T vector and expressed in E. coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins accumulated within inclusion bodies as insoluble aggregates that were solubilized in 5 M guanidine HCl and required an 'oxido-shuffling' reagent for regeneration of the enzyme activity. The recombinant beta-1,4-GT, devoid of the GST domain, has 30-85% of the sp. act. of bovine milk beta-1,4-GT with apparent Kms for N-acetylglucosamine and UDP-galactose similar to those of milk enzyme. Deletion analyses show that both beta-1,4-GT and lactose synthetase activities remain intact even in the absence of the first 129 residues (pGT-d129). The activities are lost when either deletions extend up to residue 142 (pGT-d142) or Cys134 is mutated to Ser (pGT-d129C134S). These results suggest that the formation of a disulfide bond involving Cys134 holds the protein in a conformation that is required for enzymatic activity.
Asunto(s)
Cisteína , Escherichia coli/genética , Eliminación de Gen , Expresión Génica , N-Acetil-Lactosamina Sintasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Glutatión/metabolismo , Glutatión Transferasa/genética , Cinética , Datos de Secuencia Molecular , Mutagénesis , N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/metabolismo , Reacción en Cadena de la Polimerasa , Desnaturalización Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
To examine the role of the NH2-terminal region of the 402-residue-long beta-1,4-galactosyltransferase (beta-1,4-GT), a series of mutants and chimeric cDNA were constructed by polymerase chain reaction and transiently expressed in COS-7 cells, the enzyme activities were measured, and the protein was localized in the cells by subcellular fractionation or indirect immunofluorescence microscopy. We showed earlier that the deletion of the amino-terminal cytoplasmic tail and transmembrane domain from GT abolishes the stable expression of this protein in mammalian cells (Masibay, A.S., Boeggeman, E., and Qasba, P.K. (1992) Mol. Biol. Rep. 16, 99-104). Further deletion analyses of the amino-terminal region show that the first 21 amino acids of beta-1,4-GT are not essential for the stable production of the protein and are consistently localized in the Golgi apparatus. In addition, analysis of hybrid constructs showed that residues 1-25 of alpha-1,3-galactosyltransferase can functionally replace the beta-1,4-GT amino-terminal domain (residues 1-43). This fusion protein also showed Golgi localization. On the other hand, the alpha-2,6-sialyltransferase/beta-1,4-GT fusion protein (alpha-2,6-ST/beta-1,4-GT) needed additional COOH-terminal sequences flanking the transmembrane domain of the alpha-2,6-ST for stability and Golgi localization. Substitution of Arg-24, Leu-25, Leu-26, and His-33 of the beta-1,4-GT transmembrane by Ile (pLFM) or substitution of Tyr by Ile at positions 40 and 41 coupled with the insertion of 4 Ile residues at position 43 (pLB) released the mutant proteins from the Golgi and was detected on the cell surface. Our results show that (a) the transmembrane domains of beta-1,4-GT, alpha-1,3-galactosyltransferase, and alpha-2,6-ST, along with its stem region, all play a role in Golgi targeting and participate in a common mechanism that allows the protein to be processed properly and not be degraded in vivo; (b) increasing the length of the transmembrane domain overrides the Golgi retention signal and directs the enzyme to the plasma membrane; and (c) the length of the hydrophobic region of the transmembrane domain of beta-1,4-GT is an important parameter but is not sufficient by itself for Golgi retention.
Asunto(s)
Aparato de Golgi/metabolismo , N-Acetil-Lactosamina Sintasa/genética , N-Acetil-Lactosamina Sintasa/metabolismo , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Membrana Celular/enzimología , ADN Recombinante/metabolismo , Immunoblotting , Datos de Secuencia Molecular , Mutagénesis , N-Acetil-Lactosamina Sintasa/análisis , Oligodesoxirribonucleótidos , Conformación Proteica , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , TransfecciónRESUMEN
To determine the biological role, if any, of the NH2-terminal region of beta-1,4-galactosyltransferase (GT; EC 2.4.1.90), we constructed deletion mutants and expressed them in COS-7 cells. Each deletion construct was analyzed for enzymatic activity, protein production and mRNA transcription. All of the deletion mutants were transcribed to produce GT mRNA, but the GT protein was not detected in those constructs whose transmembrane (aa 14-42) domain was deleted. The results suggest that the transmembrane region is essential for the stability of the protein and perhaps contain sequences critical for the proper targeting of the molecule. The possible role of the NH2-terminal signal anchor domain in the in vivo regulation of GT is discussed.
Asunto(s)
N-Acetil-Lactosamina Sintasa/genética , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular , Clonación Molecular , ADN de Cadena Simple , Vectores Genéticos , Datos de Secuencia Molecular , Mutación , N-Acetil-Lactosamina Sintasa/química , Reacción en Cadena de la PolimerasaRESUMEN
The beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) is localized in the trans-cisternae of the Golgi apparatus where it catalyzes the transfer of galactose from UDP-galactose to the N-acetylglucosamine residue of secretory and membrane-bound glycoproteins. Given the potential role of GT in cell-cell interaction and the fact that numerous cell surface events occur during cell growth we studied the possible relationship between GT expression and 3T3 cell growth. The level of GT mRNA increases 3--4-fold 2 h after serum-stimulation of quiescent 3T3 cells. Protein biosynthesis inhibitors like cycloheximide and anisomycin superinduce GT mRNA expression. Concomitant with this increase is an observed rise in the level of GT protein as well as an increase in overall GT enzymatic activity. Antibody-binding studies and direct enzyme assays of intact cells, along with subcellular fractionation experiments indicate that there is an increase in both Golgi and cell surface-associated GT pools upon serum-stimulation of resting cells. We conclude that GT is a member of the cell-cycle dependent genes whose expression is growth regulated.
Asunto(s)
División Celular , N-Acetil-Lactosamina Sintasa/genética , ARN Mensajero/análisis , Animales , Regulación Enzimológica de la Expresión Génica , Aparato de Golgi/enzimología , Interfase , RatonesRESUMEN
We have identified four cDNA clones, cl-1, cl-5, cl-15, and cl-16, that represent genes induced by serum in resting mouse 3T3 cells. Partial sequence analysis of the four cDNAs indicated that cl-15 corresponds to the mouse beta-actin gene. Comparison of the DNA sequences of the other three clones with the sequence data bank (Genbank) showed little homology to other known DNA sequences and thus represent novel genes. The level of the mRNAs corresponding to the four genes began to increase in resting cells following serum stimulation, reached a peak between 5 h and 8 h and then started to decline. Inhibitors of transcription diminished the induction of the mRNAs corresponding to the four genes. Cycloheximide and anisomycin had little effect on the induction of beta actin mRNA while the induction of the other three genes was suppressed by the same inhibitors. 12-O-Tetradecanoylphorbol-13-acetate and the calcium ionophore A23187 enhanced the expression of the cl-16 mRNA while epidermal growth factor, fibroblast growth factor, or insulin enhanced the expression of cl-1- and cl-5-specific transcripts. The level of beta-actin mRNA was elevated in resting cells by epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate and to a lesser extent by fibroblast growth factor, insulin, and dibutyryl cyclic AMP-elevating agents. Pertussis toxin, an inhibitor of the action of G proteins, did not significantly suppress the activation of the four genes by serum. However, 2-aminopurine, a protein kinase inhibitor, suppressed the induction of the four transcripts in serum-stimulated cells. The possible pathways involved in the activation of these genes in resting cells are discussed.
Asunto(s)
Ciclo Celular/genética , Activación Transcripcional , Actinas/genética , Animales , Células Cultivadas , Clonación Molecular , Regulación de la Expresión Génica , Biblioteca de Genes , Sustancias de Crecimiento/fisiología , Ratones , Poli A/biosíntesis , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/biosíntesis , Transcripción Genética/efectos de los fármacosRESUMEN
The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.
Asunto(s)
Regulación de la Expresión Génica , Lipoproteína Lipasa/genética , Mutación , Animales , Northern Blotting , Southern Blotting , ADN/análisis , Lipoproteína Lipasa/deficiencia , Ratones , Polimorfismo de Longitud del Fragmento de Restricción , ARN/análisisRESUMEN
A bovine beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by alpha-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.
Asunto(s)
Genes , Lactosa Sintasa/genética , N-Acetil-Lactosamina Sintasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , ADN/genética , Femenino , Cinética , Datos de Secuencia Molecular , N-Acetil-Lactosamina Sintasa/metabolismo , Placenta/enzimología , Plásmidos , Embarazo , Mapeo Restrictivo , TransfecciónRESUMEN
We isolated cDNA clones that represent genes whose expression is enhanced when resting Swiss mouse 3T3 cells are stimulated to proliferate with serum. Two clones (designated pME1 and pMR6) were analyzed further. A partial sequence analysis of the pME1 insert DNA indicated that it contained a 104-base-pair stretch with extensive homology to the 3' untranslated region of gamma actin. Similar analysis of the insert DNA from the pMR6 clone indicated that it did not correspond to any previously reported gene sequence. We used the pME1 clone as a probe to determine the level of gamma actin-specific transcript in 3T3 cells under a variety of conditions. The level of gamma actin-specific mRNA began to increase in resting cells upon serum stimulation and reached a peak at 6 h. Thereafter its level declined, and by 24 h it was hardly detectable. In contrast, pMR6-specific transcript was detectable in resting cells but remained elevated even at 24 h poststimulation. The level of gamma-actin mRNA was elevated in resting cells by 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and bombesin and to a lesser extent by cholera toxin, fibroblast-derived growth factor, and dibutyryl cyclic AMP. However, insulin, vasopressin, or epidermal growth factor failed to enhance gamma-actin mRNA levels in resting cells. Inhibitors of transcription diminished the induction of gamma-actin mRNA. Gamma-actin gene was superinduced in serum-stimulated cells by cycloheximide, an inhibitor of translation. Analysis of proteins from serum-stimulated cells by two-dimensional gel electrophoresis indicated that enhanced transcription of gamma-actin mRNA resulted in a concomitant increase in the corresponding actin protein. The possible role of gamma actin, a component of the cytoskeleton, in the regulation of cell growth is discussed.
Asunto(s)
Actinas/genética , Regulación de la Expresión Génica , ARN Mensajero/biosíntesis , Animales , Sangre/metabolismo , Ciclo Celular , División Celular , Línea Celular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Sustancias de Crecimiento/farmacología , Ratones , Hibridación de Ácido NucleicoRESUMEN
The detection of p30 by means of an indirect thin-layer immunoassay (TIA) is described. Extracts from 20 samples can be analyzed in approximately 2 h with a detection limit of approximately 50 ng. The p30 protein was detected in seminal stains which had been stored at room temperature for six months and at 130 degrees C for 4 h. Blood, saliva, urine, perspiration, and tears did not interfere with the method. The reliability of the method was demonstrated in a blind study.