RESUMO
Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.
Assuntos
Colesterol , Esfingomielinas , Esfingomielinas/química , Esfingomielinas/metabolismo , Colesterol/química , Colesterol/metabolismo , Humanos , Animais , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/química , Microscopia/métodos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Membrana Celular/químicaRESUMO
Intestinal mucins play an essential role in the defense against bacterial invasion and the maintenance of gut microbiota, which is instrumental in the regulation of host immune systems; hence, its dysregulation is a hallmark of metabolic disease and intestinal inflammation. However, the mechanism by which intestinal mucins control the gut microbiota as well as disease phenotypes remains nebulous. Herein, we report that N-acetylglucosamine (GlcNAc)-6-O sulfation of O-glycans on intestinal mucins performs a protective role against obesity and intestinal inflammation. Chst4-/- mice, lacking GlcNAc-6-O sulfation of the mucin O-glycans, showed significant weight gain and increased susceptibility to dextran sodium sulfate-induced colitis as well as colitis-associated cancer accompanied by significantly reduced immunoglobulin A (IgA) production caused by an impaired T follicular helper cell-mediated IgA response. Interestingly, the protective effects of GlcNAc-6-O sulfation against obesity and intestinal inflammation depend on the gut microbiota, evidenced by the modulation of the gut microbiota by cohousing or microbiota transplantation reversing disease phenotypes and IgA production. Collectively, our findings provide insight into the significance of host glycosylation, more specifically GlcNAc-6-O sulfation on intestinal mucins, in protecting against obesity and intestinal inflammation via regulation of the gut microbiota.
Assuntos
Microbioma Gastrointestinal , Mucinas , Animais , Camundongos , Mucinas/metabolismo , Acetilglucosamina/metabolismo , Polissacarídeos/metabolismo , Inflamação , ObesidadeRESUMO
Angiogenin 4 bearing ribonuclease activity is an endogenous antimicrobial protein expressed in small and large intestine. However, the crucial amino acid residues responsible for the antibacterial activity of Ang4 and its impact on gut microbiota remain unknown. Here, we report the contribution of critical amino acid residues in the functional regions of Ang4 to its activity against Salmonella typhimurium LT2 and the effect of Ang4 on gut microbiota in mice. We found that Ang4 binds S. typhimurium LT2 through two consecutive basic amino acid residues, K58 and K59, in the cell-binding segment and disrupts the bacterial membrane integrity at the N-terminal α-helix containing residues K7 and K30, as evidenced by the specific mutations of cationic residues of Ang4. We also found that the RNase activity of Ang4 was not involved in its bactericidal activity, as shown by the H12 mutant, which lacks RNase activity. In vivo administration of Ang4 through the mouse rectum and subsequent bacterial 16S rRNA gene sequencing analyses demonstrated that administration of Ang4 not only increased beneficial bacteria such as Lactobacillus, Akkermansia, Dubosiella, Coriobacteriaceae UCG-002, and Adlercreutzia, but also decreased certain pathogenic bacteria, including Alistipes and Enterohabdus, indicating that Ang4 regulates the shape of gut microbiota composition. We conclude that Ang4 kills bacteria by disrupting bacterial membrane integrity through critical basic amino acid residues with different functionalities rather than overall electrostatic interactions and potentially maintains gut microflora in vivo under physiological and pathophysiological conditions.
RESUMO
Low-density lipoprotein (LDL)-cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin-inducible rapid degradation of oxysterol-binding protein-related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL-derived cholesterol from late endosomes to focal adhesion kinase (FAK)-/integrin-positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P2 -containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P2 generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P2 in NPC1-containing late endosomes in a FAK-dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL-cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P2 exchange between late and recycling endosomes.
Assuntos
Transporte Biológico/fisiologia , LDL-Colesterol/metabolismo , Endossomos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/fisiologia , HumanosRESUMO
ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation. Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L. ORP2 showed specific affinity for PI(4,5)P2, PI(3,4,5)P3 and PI(4)P, with suggestive Kd values in the µM range. Also binding of cholesterol by ORP2 was detectable, but a Kd could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions. Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs.
Assuntos
Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Gotículas Lipídicas/metabolismo , Fosfatidilinositóis/metabolismo , Receptores de Esteroides/metabolismo , Colesterol/genética , Retículo Endoplasmático/genética , Endossomos/genética , Células HeLa , Humanos , Fosfatidilinositóis/genética , Receptores de Esteroides/genéticaRESUMO
Sialidases are glycosidases responsible for the removal of α-glycosidically linked sialic acid residues from carbohydrate portions of glycoproteins and glycolipids, this process being the initial step in the degradation of such glycoconjugates. Sialic acids are considered to play important roles in various biological processes largely in two ways, one related to their hydrophilic and acidic properties exerting physicochemical effects on the glycoconjugates to which they are attached, and the other as recognition sites or in an opposing fashion as masking sites. The removal of sialic acids catalyzed by a sialidase, therefore greatly influences many biological processes through changing the conformation of glycoproteins and through recognition and masking of biological sites of functional molecules. Sialidases are found widely distributed in metazoan animals, from echinoderms to mammals, and are also present in viruses and other microorganisms, including fungi, protozoa, and bacteria even mostly lacking sialic acids. In mammals, there are four forms of sialidase (Neu1, Neu2, Neu3, and Neu4), differing in their major subcellular localization and enzymatic properties. They have been implicated in regulation of various cellular activities, such as cell differentiation, cell growth, and cell adhesion and motility, depending on their particular properties. In contrast, in microorganisms the enzymes appear to play roles limited to nutrition and pathogenesis. In this chapter, the focus is on mammalian sialidases preferentially hydrolyzing gangliosides, mostly Neu3 and Neu4, with an attempt to provide a brief overview of their physiological and pathological roles.
Assuntos
Gangliosídeos/metabolismo , Doenças Metabólicas/enzimologia , Doenças Metabólicas/fisiopatologia , Neuraminidase/metabolismo , Animais , HumanosRESUMO
Tay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal ß-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa-/- mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by ß-hexosaminidase B. Therefore, the presence of the sialidase (s) allows the consequences of the Hexa defect to be bypassed. To determine if the sialidase NEU3 contributes to GM2 ganglioside degradation, we generated a mouse model with combined deficiencies of HEXA and NEU3. The Hexa-/-Neu3-/- mice were healthy at birth, but died at 1.5 to 4.5months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa-/-Neu3-/- mice revealed the abnormal accumulation of GM2 ganglioside. Histological and immunohistochemical analysis demonstrated cytoplasmic vacuolation in the neurons. Electron microscopic examination of the brain, kidneys and testes revealed pleomorphic inclusions of many small vesicles and complex lamellar structures. The Hexa-/-Neu3-/- mice exhibited progressive neurodegeneration with neuronal loss, Purkinje cell depletion, and astrogliosis. Slow movement, ataxia, and tremors were the prominent neurological abnormalities observed in these mice. Furthermore, radiographs revealed abnormalities in the skeletal bones of the Hexa-/-Neu3-/- mice. Thus, the Hexa-/-Neu3-/- mice mimic the neuropathological and clinical abnormalities of the classical early-onset Tay-Sachs patients, and provide a suitable model for the future pre-clinical testing of potential treatments for this condition.
Assuntos
Gangliosidoses GM2/genética , Hexosaminidase B/genética , Neuraminidase/genética , Doença de Tay-Sachs/genética , Animais , Química Encefálica/genética , Vesículas Citoplasmáticas/patologia , Gangliosidoses GM2/metabolismo , Gliose/genética , Gliose/patologia , Glicoesfingolipídeos/metabolismo , Coxeadura Animal/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuraminidase/deficiência , Neurônios/patologia , Células de Purkinje/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Doença de Tay-Sachs/patologiaRESUMO
BACKGROUND: Glioblastoma multiforme is one of the most malignant tumors of the human central nervous system characterized by high degree of invasiveness. Focusing on this invasive nature, we investigated whether ganglioside-specific sialidase NEU3 might be involved, because gangliosides are major components of brain tissues, and cell surface sialic acids, as target residues of sialidase catalysis, are thought to be closely related to cell invasion. METHODS: NEU3 mRNA levels of human glioblastoma specimens were evaluated by quantitative RT-PCR. Human glioblastoma cell lines, U251, A172, and T98G were used for cell invasion and migration by transwell and cell scratching assay. The molecules involved in the signaling cascade were investigated by western blot and immunofluorescent microscopy. RESULTS: NEU3 expression was down-regulated in human glioblastoma specimens. In the human glioblastoma cell lines, NEU3 overexpression reduced invasion and migration by promoting the assembly of focal adhesions through reduced calpain-dependent proteolysis, but NEU3 silencing resulted in accelerating cell invasion via disassembly of focal adhesions. In NEU3-silenced cells, elevation of calpain activity and GM3 accumulation were observed, as results of reduced sialidase hydrolysis, localization of calpain and GM3 at the cell lamellipodium being demonstrated by immunofluorescence microscopy. CONCLUSION: Sialidase NEU3 was found to exert a great influence on cell invasion in regulation of calpain activity and focal adhesion disassembly and consequent invasive potential of glioblastoma cells. GENERAL SIGNIFICANCE: This first demonstration of sialidase involvement in invasive potential of gliolastoma cells may point to NEU3 as an attractive treatment target of human gliomas.
Assuntos
Proliferação de Células/genética , Glioblastoma/genética , Invasividade Neoplásica/genética , Neuraminidase/genética , Calpaína/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Adesões Focais/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Humanos , Masculino , ProteóliseRESUMO
Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, GM3 ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro Double-knockout mice also have reduced levels of GM1 ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of GM2 in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of ß-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies.-Pan, X., De Britto Pará De Aragão, C., Velasco-Martin, J. P., Priestman, D. A., Wu, H. Y., Takahashi, K., Yamaguchi, K., Sturiale, L., Garozzo, D., Platt, F. M., Lamarche-Vane, N., Morales, C. R., Miyagi, T., Pshezhetsky, A. V. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides.
Assuntos
Encéfalo/metabolismo , Gangliosídeos/metabolismo , Neuraminidase/metabolismo , Neurônios/fisiologia , Animais , Encéfalo/patologia , Células Cultivadas , Embrião de Mamíferos , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Mucolipidoses/metabolismo , Neuraminidase/genéticaRESUMO
This paper describes the first total synthesis of the proposed structure for aromin, an annonaceous acetogenin possessing an unusual bis-THF ring system, and its 4S,7R-isomer. The key steps involve an oxidative cyclization of a couple of terminal-diene alcohols and an intermolecular metathesis of an alkenyl tetrahydrofuran with an enone carrying a tetrahydrofuranyl lactone. The spectral data of both samples did not match those of aromin. Re-examination of the NMR data using the CAST/CNMR Structure Elucidator and chemical derivations suggested that the real structure of aromin should be revised to be a tetrahydropyran acetogenin, montanacin D. Cytotoxicities in human solid tumor cell lines for synthetic samples were also evaluated.
Assuntos
Acetogeninas/síntese química , Acetogeninas/química , Acetogeninas/farmacologia , Annonaceae/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , Ciclização , Ensaios de Seleção de Medicamentos Antitumorais , Furanos/química , Humanos , Espectrometria de Massas , Estrutura Molecular , Oxirredução , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
The central molecular event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC), which is a sialoglycoprotein, into the disease-associated, transmissible form denoted PrPSc. Recent studies revealed a correlation between the sialylation status of PrPSc and incubation time to disease and introduced a new hypothesis that progression of prion diseases could be controlled or reversed by altering the sialylation level of PrPC. Of the four known mammalian sialidases, the enzymes that cleave off sialic acid residues, only NEU1, NEU3 and NEU4 are expressed in the brain. To test whether cellular sialidases control the steady-state sialylation level of PrPC and to identify the putative sialidase responsible for desialylating PrPC, we analyzed brain-derived PrPC from knockout mice deficient in Neu1, Neu3, Neu4, or from Neu3/Neu4 double knockouts. Surprisingly, no differences in the sialylation of PrPC or its proteolytic product C1 were noticed in any of the knockout mice tested as compared to the age-matched controls. However, significantly higher amounts of the C1 fragment relative to full-length PrPC were detected in the brains of Neu1 knockout mice as compared to WT mice or to the other knockout mice. Additional experiments revealed that in neuroblastoma cell line the sialylation pattern of C1 could be changed by an inhibitor of sialylatransferases. In summary, this study suggests that targeting cellular sialidases is apparently not the correct strategy for altering the sialylation levels of PrPC, whereas modulating the activity of sialylatransferases might offer a more promising approach. Our findings also suggest that catabolism of PrPC involves its α-cleavage followed by desialylation of the resulting C1 fragments by NEU1 and consequent fast degradation of the desialylated products.
Assuntos
Neuraminidase/metabolismo , Fragmentos de Peptídeos/metabolismo , Príons/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Eletroforese em Gel Bidimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/antagonistas & inibidores , Neuraminidase/deficiência , Neuraminidase/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Regional lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) is a crucial event for its progression, associated with a high rate of mortality. Sialidase, a key enzyme for the regulation of cellular sialic acids through catalyzing the initial step of degradation of glycoproteins and glycolipids, has been implicated in cancer progression. To facilitate the development of novel treatments for HNSCC, we have investigated whether sialidase is involved in the progression of this cancer. We found plasma membrane-associated sialidase (NEU3) to be significantly upregulated in tumor compared to non-tumor tissues; particularly, an increase in its mRNA levels was significantly associated with lymph node metastasis. To understand the mechanisms, we analyzed the NEU3-mediated effects on the malignant phenotype using squamous carcinoma HSC-2 and SAS cells. NEU3 promoted cell motility and invasion, accompanied by the increased expression of MMP-9, whereas NEU3 silencing or the activity-null mutant did not. NEU3 enhanced phosphorylation of epidermal growth factor receptor (EGFR), and an EGFR inhibitor, AG1478, abrogated the NEU3-induced MMP9 augmentation. These findings identify NEU3 as a participant in HNSCC progression through the regulation of EGFR signaling and thus as a potential target for inhibiting EGFR-mediated tumor progression.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Neuraminidase/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia em Camada Fina , Progressão da Doença , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neuraminidase/análise , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Carcinoma de Células Escamosas de Cabeça e Pescoço , Transfecção , Regulação para CimaRESUMO
We previously demonstrated that sialidase NEU3, a key glycosidase for ganglioside degradation, is up-regulated in various human cancers, leading to increased cell invasion, motility and survival of cancer cells possibly through activation of EGF signaling. Its up-regulation is also important for promotion of the stage of colorectal carcinogenesis in vivo in human NEU3 transgenic mice treated with azoxymethane for the induction of aberrant crypt foci in the colon mucosa, accompanied by enhanced phosphorylation of EGF receptor (EGFR). To address whether the activation of EGF signaling by the sialidase is associated with oncogenic transformation, we here analyzed the effects of overexpression of NEU3 and EGFR in NIH-3T3 cells. When NEU3 was stably transfected with or without EGFR, it was associated with significant increases in clonogenic growth, clonogenicity on soft agar and in vivo tumor growth in nude mice either with or without the receptor overexpression in the presence of EGF, compared with the levels in their vector controls. Despite the fact that the endogenous level of EGFR is known to be extremely low in these cells, NEU3 significantly enhanced the phosphorylation of Akt and ERK, as well as that of the receptor. The NEU3-mediated activation was largely abrogated by the EGFR inhibitor AG1478 or PD153035, but significant clonogenic growth still remained. NEU3 was then found to activate Src kinase, and the clonogenicity was completely suppressed by an Src inhibitor, PP2. The activity-null mutants failed to activate Src and EGFR, indicating that ganglioside modulation by NEU3 may be necessary for the activation. NEU3 and Src were co-immunoprecipitated with EGFR in NEU3- and EGFR- transfected cells. These findings identify NEU3 as an essential participant in tumorigenesis through the EGFR/Src signaling pathway and a potential target for inhibiting EGFR-mediated tumor progression.
Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Neuraminidase/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Animais , Carcinogênese/genética , Proliferação de Células , Ativação Enzimática , Receptores ErbB/genética , Gangliosídeos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Neuraminidase/genética , Transfecção , Regulação para CimaRESUMO
The plasma membrane-associated sialidase NEU3 is a key enzyme for ganglioside degradation. We previously demonstrated remarkable up-regulation of NEU3 in various human cancers, with augmented malignant properties. Here, we provide evidence of a close link between NEU3 expression and Wnt/ß-catenin signaling in colon cancer cells by analyzing tumorigenic potential and cancer stem-like characteristics. NEU3 silencing in HT-29 and HCT116 colon cancer cells resulted in significant decrease in clonogenicity on soft agar and in vivo tumor growth, along with down-regulation of stemness and Wnt-related genes. Analyses further revealed that NEU3 enhanced phosphorylation of the Wnt receptor LRP6 and consequently ß-catenin activation by accelerating complex formation with LRP6 and recruitment of GSK3ß and Axin, whereas its silencing exerted the opposite effects. NEU3 activity-null mutants failed to demonstrate the activation, indicating the requirement of ganglioside modulation by the sialidase for the effects. Under sphere-forming conditions, when stemness genes are up-regulated, endogenous NEU3 expression was found to be significantly increased, whereas NEU3 silencing suppressed sphere-formation and in vivo tumor incidence in NOD-SCID mice. Increased ability of clonogenicity on soft agar and sphere formation by Wnt stimulation was abrogated by NEU3 silencing. Furthermore, NEU3 was found to regulate phosphorylation of ERK and Akt via EGF receptor and Ras cascades, thought to be additionally required for tumor progression. The results indicate an essential contribution of NEU3 to tumorigenic potential through maintenance of stem-like characteristics of colon cancer cells by regulating Wnt signaling at the receptor level, in addition to tumor progression via Ras/MAPK signaling.
Assuntos
Neoplasias do Colo/metabolismo , Gangliosídeos/metabolismo , Neuraminidase/metabolismo , Animais , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Células HCT116 , Células HEK293 , Células HT29 , Xenoenxertos , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Fatores de Transcrição TCF/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Aberrant sialylation in glycoproteins and glycolipids is a characteristic feature of malignancy. Human sialidases, which catalyze the removal of sialic acid residues from glycoconjugates, have been implicated in cancer progression. They have been detected in a wide variety of human cells and tissues, but few studies have focused on their existence in human serum. Among the four types identified to date, we previously demonstrated that plasma membrane-associated ganglioside sialidase (NEU3) is markedly upregulated in various human cancers, including examples in the colon and prostate. Here, using a sensitive assay method, we found a significant increase of sialidase activity in the serum of patients with prostate cancer compared with that in healthy subjects having low activity, if any. Activity was apparent with gangliosides as substrates, but only to a very limited extent with 4-methylumbelliferyl sialic acid, a good synthetic substrate for sialidases other than human NEU3. The serum sialidase was also almost entirely immunoprecipitated with anti-NEU3 antibody, but not with antibodies for other sialidases. Interestingly, sera additionally contained inhibitory activity against the sialidase and also against recombinant human NEU3. The sialidase and inhibitor activities could be separated by exosome isolation and by hydrophobic column chromatography. The serum sialidase was assessed by a sandwich ELISA method using two anti-NEU3 antibodies. The results provide strong evidence that the serum sialidase is, in fact, NEU3, and this subtype may, therefore, be a potential utility for novel diagnosis of human cancers.
Assuntos
Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/sangue , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/antagonistas & inibidores , Neuraminidase/sangue , Neoplasias da Próstata/sangue , Biomarcadores Tumorais/biossíntese , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Gangliosídeos/metabolismo , Humanos , Masculino , Neuraminidase/biossíntese , Neuraminidase/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
The plasma membrane-associated sialidase NEU3 plays crucial roles in regulation of transmembrane signaling, and its aberrant up-regulation in various cancers contributes to malignancy. However, it remains uncertain how NEU3 is naturally activated and locates to plasma membranes, because of its Triton X-100 requirement for the sialidase activity in vitro and its often changing subcellular location. Among phospholipids examined, we demonstrate that phosphatidic acid (PA) elevates its sialidase activity 4 to 5 times at 50 µM in vitro at neutral pH and promotes translocation to the cell surface and cell migration through Ras-signaling in HeLa and COS-1 cells. NEU3 was found to interact selectively with PA as assessed by phospholipid array, liposome coprecipitation, and ELISA assays and to colocalize with phospholipase D (PLD) 1 in response to epidermal growth factor (EGF) or serum stimulation. Studies using tagged NEU3 fragments with point mutations identified PA- and calmodulin (CaM)-binding sites around the N terminus and confirmed its participation in translocation and catalytic activity. EGF induced PLD1 activation concomitantly with enhanced NEU3 translocation to the cell surface, as assessed by confocal microscopy. These results suggest that interactions of NEU3 with PA produced by PLD1 are important for regulation of transmembrane signaling, this aberrant acceleration probably promoting malignancy in cancers.
Assuntos
Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Neuraminidase/metabolismo , Ácidos Fosfatídicos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Células COS , Proliferação de Células , Células Cultivadas , Chlorocebus aethiops , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Camundongos , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Fosfolipase D/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
Sialic acid-binding lectin (SBL) is a multi-functional protein that is isolated from oocytes of Rana catesbeiana. It has both lectin and ribonuclease (enzyme) properties, and therefore is called leczyme. We examined the anti-tumor effects of SBL and discovered that SBL has potential as a new type of anti-cancer drug. SBL causes a cancer-selective induction of apoptosis by multiple signaling pathways whereby RNA is its target. It is suggested that the mitochondrial pathway and endoplasmic reticulum stress-mediated pathway participate in SBL-induced signaling. The synergistic anti-tumor effects with other molecules, such as tumor necrosis factor-related apoptosis ligand and interferon γ, have been reported. In this study, we summarize the effects of SBL and focus on its cancer-selective apoptotic properties. In addition, we present a possible explanation for its cancer specificity.
RESUMO
Sialic acid-binding lectin (SBL), isolated from oocytes of Rana catesbeiana, is leczyme and has both lectin and ribonuclease (RNase) activities. A remarkable antitumor effect of SBL has also been reported. SBL agglutinates various kinds of tumor cells but not normal cells. SBL agglutination activity is not affected by mono- or oligosaccharides. However, SBL-induced agglutination and antitumor effects are inhibited by sialomucin but not asialomucin. In addition, SBL has very little effect on sialidase-treated cells. SBL causes cancer-selective induction of apoptosis by multiple signaling pathways, which target RNA. Synergistic antitumor effects with other molecules, such as tumor necrosis factor-related apoptosis ligand (TRAIL) and interferon- γ (IFN-γ), have been reported. Thus, SBL may be a novel candidate molecule for anticancer drug development. Sialoglycoconjugates on the tumor cell surface may be associated with lectin activity and antitumor effects of SBL. We review the properties of SBL, particularly its lectin, RNase, and antitumor activities, and comprehensively examine the potential application of SBL for clinical purposes.
Assuntos
Proteínas de Anfíbios/uso terapêutico , Antineoplásicos/uso terapêutico , Lectinas/uso terapêutico , Neoplasias/tratamento farmacológico , Ribonucleases/uso terapêutico , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Lectinas/química , Lectinas/farmacologia , Dados de Sequência Molecular , Rana catesbeiana , Ribonucleases/química , Ribonucleases/farmacologiaRESUMO
Malignant mesothelioma is a highly aggressive tumor with poor prognosis. An effective drug for treatment of malignant mesothelioma is greatly needed. Sialic acid-binding lectin (SBL) isolated from oocytes of Rana catesbeiana is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity, so it could be developed as a new type of anticancer drug. The validity of SBL for treatment of malignant mesothelioma was assessed using three malignant mesotheliomas and a non-malignant mesothlial cell line. Effectiveness of combinatorial treatment of SBL and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was also elucidated and characterized. SBL induced tumor-selective cytotoxicity that was attributed to induction of apoptosis. Combinatorial treatment of SBL and TRAIL showed synergistic apoptosis-inducing effect. Additional experiments revealed that Bid was the mediating molecule for the synergistic effect in SBL and TRAIL. These results suggested that SBL could be a promising candidate for the therapeutics for malignant mesothelioma. Furthermore, the combinatorial treatment of SBL and TRAIL could be an effective regimen against malignant mesothelioma.