RESUMO
BACKGROUND: Alterations in protein glycosylation have been related to malignant transformation and tumour progression. We recently showed that low mRNA levels of Golgi alpha-mannosidase MAN1A1 correlate with poor prognosis in breast cancer patients. METHODS: We analysed the role of MAN1A1 on a protein level using western blot analysis (n=105) and studied the impact of MAN1A1-related glycosylation on the prognostic relevance of adhesion molecules involved in breast cancer using microarray data (n=194). Functional consequences of mannosidase inhibition using the inhibitor kifunensine or MAN1A1 silencing were investigated in breast cancer cells in vitro. RESULTS: Patients with low/moderate MAN1A1 expression in tumours showed significantly shorter disease-free intervals than those with high MAN1A1 levels (P=0.005). Moreover, low MAN1A1 expression correlated significantly with nodal status, grading and brain metastasis. At an mRNA level, membrane proteins ALCAM and CD24 were only significantly prognostic in tumours with high MAN1A1 expression. In vitro, reduced MAN1A1 expression or mannosidase inhibition led to a significantly increased adhesion of breast cancer cells to endothelial cells. CONCLUSIONS: Our study demonstrates the prognostic role of MAN1A1 in breast cancer by affecting the adhesive properties of tumour cells and the strong influence of this glycosylation enzyme on the prognostic impact of some adhesion proteins.
Assuntos
Neoplasias da Mama/metabolismo , Manosidases/metabolismo , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose/fisiologia , Western Blotting , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/biossíntese , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Intervalo Livre de Doença , Feminino , Proteínas Fetais/biossíntese , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Glicosilação , Humanos , Estimativa de Kaplan-Meier , Manosidases/biossíntese , Manosidases/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Taxa de SobrevidaRESUMO
Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.
Assuntos
Actinobacteria , Clonagem Molecular , Expressão Gênica , Manosidases , Actinobacteria/enzimologia , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Catálise , Manosidases/biossíntese , Manosidases/química , Manosidases/genética , Manosidases/isolamento & purificação , Polissacarídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
Expression of pRSETA manb-1601 construct in Hi-Control Escherichia coli BL21 (DE3) cells improved recombinant endo-mannanase (ManB-1601) production by 2.73-fold (1821±100U/ml). A low-cost, agro-industrial residue supplemented industrial medium for enhanced and economical production of ManB-1601 was developed in two mutual phases. Phase-I revealed the potential of various pre- (induction time: 5h, induction mode: lactose 0.5mM) and post-induction [peptone supplementation: 0.94%(w/v), glycerol 0.123%(v/v)] parameters for enhanced production of ManB-1601 and resulted in 4.61-fold (8406±400U/ml) and 2.53-fold (3.30g/l) higher ManB-1601 and biomass production, respectively. Under phase-II, economization of phase-I medium was carried out by reducing/replacing costly ingredients with solubilized-defatted flax seed meal (S-DFSM), which resulted in 3.25-fold (5926U/ml) higher ManB-1601 production. Industrial potential of ManB-1601 was shown in oil extraction from copra as enzyme treatment led to cracks, peeling, fracturing and smoothening of copra, which facilitated higher (18.75%) oil yield.
Assuntos
Cocos/química , Escherichia coli/metabolismo , Manosidases/biossíntese , Óleos de Plantas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Biomassa , Óleo de Coco , Cocos/efeitos dos fármacos , Meios de Cultura , Manosidases/genética , Manosidases/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
Golgi α-mannosidase II (GMII), a key glycosyl hydrolase in the N-linked glycosylation pathway, has been demonstrated to be closely associated with the genesis and development of cancer. In this study, we cloned cDNA-encoding Capra hircus GMII (chGMII) and expressed it in Pichia pastoris expression system. The chGMII cDNA contains an open reading frame of 3432 bp encoding a polypeptide of 1144 amino acids. The deduced molecular mass and pI of chGMII was 130.5 kDa and 8.04, respectively. The gene expression profile analysis showed GMII was the highest expressed gene in the spleen. The recombinant chGMII showed maximum activity at pH 5.4 and 42 °C and was activated by Fe(2+), Zn(2+), Ca(2+), and Mn(2+) and strongly inhibited by Co(2+), Cu(2+), and EDTA. By homology modeling and molecular docking, we obtained the predicted 3D structure of chGMII and the probable binding modes of chGMII-GnMan5Gn, chGMII-SW. A small cavity containing Tyr355 and zinc ion fixed by residues Asp290, His176, Asp178, and His570 was identified as the active center of chGMII. These results not only provide a clue for clarifying the catalytic mechanism of chGMII but also lay a theoretical foundation for subsequent investigations in the field of anticancer therapy for mammals.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Cabras , Manosidases , Baço/enzimologia , Animais , Clonagem Molecular , Expressão Gênica , Cabras/genética , Cabras/metabolismo , Manosidases/biossíntese , Manosidases/genética , Especificidade de Órgãos/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Cytosolic α-mannosidase (Man2C1) trims free oligosaccharides in mammalian cells, and its down-regulation reportedly delays cancer growth by inducing mitotic arrest or apoptosis. However, the mechanism by which Man2C1 down-regulation induces apoptosis is unknown. Here, we demonstrated that silencing of Man2C1 via small hairpin RNAs induced mitochondria-dependent apoptosis in HeLa cells. Expression of CHOP (C/EBP homologous protein), a transcription factor critical to endoplasmic reticulum stress-induced apoptosis, was significantly up-regulated in Man2C1 knockdown cells. However, this enhanced CHOP expression was not caused by endoplasmic reticulum stress. Interestingly, Man2C1 catalytic activity was not required for this regulation of apoptosis; introduction of mutant, enzymatically inactive Man2C1 rescued apoptotic phenotypes of Man2C1 knockdown cells. These results show that Man2C1 has dual functions: one in glycan catabolism and another in apoptotic signaling.
Assuntos
Apoptose/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Manosidases/biossíntese , Mitocôndrias/enzimologia , Transdução de Sinais/fisiologia , Metabolismo dos Carboidratos/fisiologia , Regulação para Baixo/fisiologia , Técnicas de Silenciamento de Genes , Células HeLa , Células Hep G2 , Humanos , Manosidases/genética , Mitocôndrias/genética , Fator de Transcrição CHOP/biossíntese , Fator de Transcrição CHOP/genética , alfa-ManosidaseRESUMO
OBJECTIVE: To find the nature of the protein encoded by 6A8 cDNA. METHODS: In the light of that the ligand of Con A is mannoses and that the function of alpha-mannosidase is trimming of mannoses in the oligosaccharide chains of glycoproteins, COS-7 cells and CNE-2L2 cells, a cloned cell line of human nasopharyngeal carcinoma, were transfected with pRc/CMV-sense 6A8 cDNA or pRc/CMV-antisense 6A8 cDNA and the Con A binding to the cells was examined upon a lazer scan confocal microscope. RESULTS: The data demonstrated that with transfection of pRc/CMV-sense 6A8 cDNA the binding of Con A to the cells was decreased, but the staining of the cells with mAb 6A8 was enhanced, and vise versa for the cells with transfection of pRc/CMV-antisense 6A8 cDNA. CONCLUSIONS: The protein encoded by 6A8 cDNA might be a type of alpha-mannosidase recognized by mAb 6A8.
Assuntos
DNA Complementar/genética , Manosidases/genética , Animais , Anticorpos Monoclonais/farmacologia , Células COS , DNA Antissenso/farmacologia , Humanos , Células Jurkat , Macaca mulatta , Manosidases/biossíntese , Microscopia Confocal , Neoplasias Nasofaríngeas/patologia , Transfecção , Células Tumorais Cultivadas , alfa-ManosidaseRESUMO
The cDNA encoding a mouse homologue of porcine epididymis-specific 135kDa alpha-D-mannosidase (MAN2B2, D28521) was cloned from the mouse testis cDNA library. It was found that 1018 amino acids were coded in its open reading frame, and 62% of the amino acid sequence was identical to that of porcine MAN2B2. In the adult mouse, testis contained higher amounts of mRNA encoding the MAN2B2 homologue than the epididymis, though porcine MAN2B2 was mainly expressed in the narrow region between the caput and corpus epididymis. mRNA of the mouse MAN2B2 homologue was localized exclusively in spermatogonia in the testis. It was specifically expressed in type A spermatogonia at stages IX-XI of spermatogenesis and was detected there until the cell developed into type B spermatogonia. We conclude that the expression of the MAN2B2 homologue can serve as a good marker for the late stages of type A spermatogonia and may have an important role to play in the early step of spermatogenesis in mice.
Assuntos
Manosidases/biossíntese , Espermatogênese/fisiologia , Espermatogônias/enzimologia , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Hibridização In Situ , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/isolamento & purificação , Homologia de Sequência de Aminoácidos , Suínos , Testículo/enzimologiaRESUMO
The v-Ki-Ras oncoprotein dedifferentiates thyroid cells and inhibits nuclear accumulation of the catalytic subunit of cAMP-dependent protein kinase. After activation of v-Ras or protein kinase C, the regulatory subunit of type II protein kinase A, RIIbeta, translocates from the membranes to the cytosol. RIIbeta mRNA and protein were eventually depleted. These effects were mimicked by expressing AKAP45, a truncated version of the RII anchor protein, AKAP75. Because AKAP45 lacks membrane targeting domains, it induces the translocation of PKAII to the cytoplasm. Expression of AKAP45 markedly decreased thyroglobulin mRNA levels and inhibited accumulation of C-PKA in the nucleus. Our results suggest that: 1) The localization of PKAII influences cAMP signaling to the nucleus; 2) Ras alters the localization and the expression of PKAII; 3) Translocation of PKAII to the cytoplasm reduces nuclear C-PKA accumulation, resulting in decreased expression of cAMP-dependent genes, including RIIbeta, TSH receptor, and thyroglobulin. The loss of RIIbeta permanently down-regulates thyroid-specific gene expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Genes ras , Transdução de Sinais , Proteínas de Ancoragem à Quinase A , Animais , Western Blotting , Proteínas de Transporte , Linhagem Celular , Transformação Celular Neoplásica , AMP Cíclico/farmacologia , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Primers do DNA , Regulação Enzimológica da Expressão Gênica , Manosidases/biossíntese , Proteína Oncogênica p21(ras)/biossíntese , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Proteínas/metabolismo , Ratos , Receptores da Tireotropina/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Tireoglobulina/biossíntese , Transfecção , alfa-ManosidaseRESUMO
Golgi alpha-mannosidase II (alpha-MII) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human alpha-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding alpha-MII were isolated. One such gene was found to encode an isozyme, designated alpha-MIIx. A 5-kb cDNA clone encoding alpha-MIIx was then isolated from a human melanoma cDNA library. However, comparison between alpha-MIIx and alpha-MII cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while alpha-MII consists of 1144 amino acid residues. To reevaluate the sequence of alpha-MIIx cDNA, polymerase chain reaction (PCR) was performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the alpha-MIIx genomic sequence revealed that alternative splicing of the alpha-MIIx transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of alpha-MIIx in various tissues, suggesting that the alpha-MIIx gene is a housekeeping gene. COS cells transfected with alpha-MIIx cDNA containing the full-length open reading frame showed an increase of alpha-mannosidase activity. The alpha-MIIx gene was mapped to human chromosome 15q25, whereas the alpha-MII gene was mapped to 5q21-22.
Assuntos
Isoenzimas/genética , Manosidases/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Genoma Humano , Humanos , Isoenzimas/biossíntese , Linfócitos/enzimologia , Manosidases/biossíntese , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Seleção Genética , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Although previous studies have indicated that N-linked oligosaccharides on lysosomal enzymes in Dictyostelium discoideum are extensively phosphorylated and sulfated, the role of these modifications in the sorting and function of these enzymes remains to be determined. We have used radiolabel pulse-chase, subcellular fractionation, and immunofluorescence microscopy to analyze the transport, processing, secretion, and sorting of two lysosomal enzymes in a mutant, HL244, which is almost completely defective in sulfation. [3H]Mannose-labeled N-linked oligosaccharides were released from immunoprecipitated alpha-mannosidase and beta-glucosidase of HL244 by digestion with peptide: N-glycosidase. The size, Man9-10GlcNAc2, and processing of the neutral species were similar to that found in the wild type, but the anionic oligosaccharides were less charged than those from the wild-type enzymes. All of the negative charges on the oligosaccharides for HL244 were due to the presence of 1, 2, or 3 phosphodiesters and not to sulfate esters. The rate of proteolytic processing of precursor forms of alpha-mannosidase and beta-glucosidase to mature forms in HL244 was identical to wild type. The precursor polypeptides in the mutant and the wild type were membrane associated until being processed to mature forms; therefore, sulfated sugars are not essential for this association. Furthermore, the rate of transport of alpha-mannosidase and beta-glucosidase from the endoplasmic reticulum to the Golgi complex was normal in the mutant as determined by the rate at which the newly synthesized proteins became resistant to the enzyme, endo-beta-N-acetylglucosaminidase H. There was no increase in the percentage of newly synthesized mutant precursors which escaped sorting and were secreted, and the intracellularly retained lysosomal enzymes were properly localized to lysosomes as determined by fractionation of cell organelles on Percoll gradients and immunofluorescence microscopy. However, the mutant secreted lysosomally localized mature forms of the enzymes at 2-fold lower rates than wild-type cells during both growth and during starvation conditions that stimulate secretion. Furthermore, the mutant was more resistant to the effects of chloroquine treatment which results in the missorting and oversecretion of lysosomal enzymes. Together, these results suggest that sulfation of N-linked oligosaccharides is not essential for the transport, processing, or sorting of lysosomal enzymes in D. discoideum, but these modified oligosaccharides may function in the secretion of mature forms of the enzymes from lysosomes.
Assuntos
Dictyostelium/enzimologia , Glucosidases/biossíntese , Lisossomos/enzimologia , Manosidases/biossíntese , Oligossacarídeos , Processamento de Proteína Pós-Traducional , Ácidos Sulfúricos , beta-Glucosidase/biossíntese , Acetilglucosaminidase , Transporte Biológico , Cloroquina/farmacologia , Cromatografia por Troca Iônica , Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Cinética , Manosidases/genética , Manosidases/isolamento & purificação , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Oligossacarídeos/isolamento & purificação , alfa-Manosidase , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificaçãoRESUMO
The nucleotide sequence was determined for the alkalophilic Bacillus sp. strain AM-001 beta-mannanase gene which produced two beta-mannanases (A and B) in Escherichia coli transformants. The putative beta-mannanase gene was 1,539 base pairs long and encoded a mature beta-mannanase protein of 487 amino acids and a signal peptide of 26 amino acids. The COOH-terminal amino acid of beta-mannanase A is an arginine residue located at amino acid 513 of the deduced amino acid sequence, and that of beta-mannanase B is a valine residue located at amino acid 365. Deletion derivatives having 1,098 base pairs from the ATG start codon maintained the beta-mannanase activity of the encoded polypeptide. However, clones harboring DNA fragments (1,051 base pairs) shorter than the gene which encoded beta-mannanase B (1,095 base pairs) did not exhibit the beta-mannanase activity. The simultaneous production of both beta-mannanases A and B in an E. coli transformant was demonstrated by the maxicell procedure.
Assuntos
Bacillus/genética , DNA Bacteriano/genética , Escherichia coli/genética , Manosidases/genética , Sequência de Aminoácidos , Aminoácidos/análise , Autorradiografia , Bacillus/enzimologia , Proteínas de Bactérias/análise , Sequência de Bases , Western Blotting , Códon/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Manosidases/biossíntese , Dados de Sequência Molecular , Plasmídeos , beta-ManosidaseRESUMO
A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha-mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.
Assuntos
Dictyostelium/genética , Endocitose , Precursores Enzimáticos/genética , Glucosidases/genética , Lisossomos/enzimologia , Manosidases/genética , Mutação , beta-Glucosidase/genética , Fracionamento Celular , Dictyostelium/enzimologia , Dictyostelium/fisiologia , Precursores Enzimáticos/biossíntese , Cinética , Manosidases/biossíntese , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , alfa-Manosidase , beta-Glucosidase/biossínteseRESUMO
The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development.
Assuntos
Dictyostelium/crescimento & desenvolvimento , Lisossomos/enzimologia , Manosidases/biossíntese , Dictyostelium/enzimologia , Dictyostelium/genética , Dictyostelium/fisiologia , Precursores Enzimáticos/metabolismo , RNA Mensageiro/metabolismo , alfa-ManosidaseRESUMO
The biosynthesis and post-translational modification of mannosidase II, an enzyme required in the maturation of asparagine-linked oligosaccharides in the Golgi complex, has been investigated. Antibody raised against this enzyme purified from rat liver Golgi membranes was used to immunoprecipitate mannosidase II from rat liver, 3T3 cells, or HeLa cells. Mannosidase II immunoprecipitated from rat liver Golgi membranes, when analyzed by polyacrylamide gel electrophoresis, migrated with an apparent molecular weight of approximately 124,000. In contrast, the enzyme purified from rat liver Golgi membranes was shown to contain both the 124,000-dalton component and a 110,000-dalton polypeptide believed to result from degradation of intact mannosidase II during purification. Mannosidase II from 3T3 and HeLa cells migrated on polyacrylamide gels with apparent molecular weights of approximately 124,000 and 134,000-136,000, respectively. When immunoprecipitated from radiolabeled cultures, mannosidase II from both cell types was similar in the following respects: (a) the initial synthesis product had an apparent molecular weight of approximately 124,000; (b) in cultures treated with tunicamycin the initial synthesis product had an apparent molecular weight of approximately 117,000; (c) endoglycosidase H digestion of the initial synthesis product gave an apparent molecular weight similar to the tunicamycin-induced polypeptide; (d) the mature enzyme was mostly (HeLa) or entirely (3T3) resistant to digestion by endoglycosidase H. Loss of [35S]methionine from intracellular mannosidase II occurred with a half-life of approximately 20 h; there was no appreciable accumulation of labeled immuno-reactive material in the medium. HeLa mannosidase II, but not the 3T3 enzyme, was additionally modified 1-3 h after synthesis, the initial synthesis product being converted to a doublet with an apparent molecular weight of approximately 134,000-136,000. Evidence is presented that this mobility shift may result from O-glycosylation. Mannosidase II from both cell types could be labeled with [32P]phosphate or [35S]sulfate. The latter is apparently attached to oligosaccharide as indicated by inhibition of labeling by tunicamycin; the former was shown with the HeLa enzyme to be present as serine phosphate moieties. In addition, [3H]palmitate could be incorporated into the enzyme in 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Complexo de Golgi/enzimologia , Manosidases/biossíntese , Animais , Fibroblastos/ultraestrutura , Glicosídeo Hidrolases/metabolismo , Células HeLa/ultraestrutura , Humanos , Técnicas de Imunoadsorção , Fígado/enzimologia , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Camundongos , Camundongos Endogâmicos BALB C , Monensin/farmacologia , Processamento de Proteína Pós-Traducional , Ratos , Tunicamicina/farmacologiaRESUMO
alpha-Mannosidase and acid phosphatase were induced in alveolar macrophages from the lungs of rabbits following exposure to morpholine. The induction was observed after inhalation of morpholine vapor or when macrophages were cultured in the presence of morpholine. Lysosomal hydrolases were not induced uniformly. In vivo, maximum induction of alpha-mannosidase and acid phosphatase in female rabbits was 1.7-fold and 2-fold, respectively, and in male rabbits, 3-fold and unchanged, respectively. In vitro, maximum hydrolase induction of alpha-mannosidase and acid phosphatase in macrophages from male rabbits was 1.4-fold and 1.3-fold, respectively, and from female rabbits, 1.3-fold and 1.5-fold, respectively. The induction of acid hydrolases in vitro was rapid, reaching a maximum within 4 hr in alveolar macrophages from male rabbits and within 8 hr in those from females. The induction was dose dependent. This study indicates that the lysosomal enzyme system of the alveolar macrophage may be stimulated by a low molecular weight xenobiotic.
Assuntos
Fosfatase Ácida/biossíntese , Lisossomos/enzimologia , Macrófagos/enzimologia , Manosidases/biossíntese , Morfolinas/farmacologia , Alvéolos Pulmonares/enzimologia , Animais , Indução Enzimática/efeitos dos fármacos , Técnicas In Vitro , Fagocitose , Coelhos , alfa-ManosidaseRESUMO
Two types of alpha-D-mannosidase isozymes are present in human white blood cells, human diploid fibroblasts, and HeLa cells. One of these (the S isozyme) constitutes the major alpha-D-mannosidase of the human cells, has a pH optimum of 4.4, and is associated with lysosomes. The other (the F isozyme) is most active at pH 6, is acid labile, and is located in the soluble portion of the cytoplasm. The expression of human lysosomal alpha-D-mannosidase was examined in man-hamster hybrid clones, and was found to be concordant with that of phosphohexose isomerase in 54 of 55 primary clones. A locus specifying human lysosomal alpha-D-mannosidase has therfore been assigned to chromosome 19.