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1.
J Med Chem ; 63(22): 13913-13950, 2020 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-33155811

RESUMEN

A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine-citrulline dipeptide linkers via heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02 µmol/kg).


Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Benzodiazepinas/química , Diseño de Fármacos , Inmunoconjugados/farmacología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Neoplasias Gástricas/tratamiento farmacológico , Tetrahidroisoquinolinas/química , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antineoplásicos/química , Apoptosis , Benzodiazepinas/metabolismo , Proliferación Celular , Femenino , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/inmunología , Proteínas Ligadas a GPI/inmunología , Humanos , Inmunoconjugados/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Mesotelina , Ratones , Ratones SCID , Carcinoma Pulmonar de Células Pequeñas/patología , Neoplasias Gástricas/patología , Relación Estructura-Actividad , Tetrahidroisoquinolinas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Nanoscale ; 12(5): 2946-2960, 2020 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-31994576

RESUMEN

Effective treatment of glioma and other central nervous system (CNS) diseases is hindered by the presence of the blood-brain barrier (BBB). A novel nano-delivery vehicle system composed of PLGA-lysoGM1/DOX micelles was developed to cross the BBB for CNS treatment. We have shown that doxorubicin (DOX) as a model drug encapsulated in PLGA-lysoGM1 micelles can achieve up to 3.8% loading efficiency and 61.6% encapsulation efficiency by the orthogonal test design. Our in vitro experiments demonstrated that PLGA-lysoGM1/DOX micelles had a slow and sustainable drug release under physiological conditions and exhibited a high cellular uptake through the macropinocytosis and the autophagy/lysosomal pathways. In vivo experimental studies in zebrafish and mice confirmed that PLGA-lysoGM1/DOX micelles could cross the BBB and be specifically accumulated in the brain. Moreover, an excellent anti-glioma effect was observed in intracranial glioma-bearing rats. Therefore, PLGA-lysoGM1/DOX micelles not only effectively can cross the BBB, but our results also suggest that they have great potential for anti-glioma therapy and other central nervous system diseases.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Doxorrubicina , Sistemas de Liberación de Medicamentos , Gangliósido G(M1)/análogos & derivados , Glioma/tratamiento farmacológico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Animales , Barrera Hematoencefálica/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Gangliósido G(M1)/farmacocinética , Gangliósido G(M1)/farmacología , Glioma/metabolismo , Glioma/patología , Humanos , Masculino , Ratones , Micelas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacología , Pez Cebra
3.
FEBS J ; 287(12): 2612-2630, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-31769916

RESUMEN

A 15-kDa lectin, termed SeviL, was isolated from Mytilisepta virgata (purplish bifurcate mussel). SeviL forms a noncovalent dimer that binds strongly to ganglio-series GM1b oligosaccharide (Neu5Acɑ2-3Galß1-3GalNAcß1-4Galß1-4Glc) and its precursor, asialo-GM1 (Galß1-3GalNAcß1-4Galß1-4Glc). SeviL also interacts weakly with the glycan moiety of SSEA-4 hexaose (Neu5Acα2-3Galß1-3GalNAcß1-3Galα1-4Galß1-4Glc). A partial protein sequence of the lectin was determined by mass spectrometry, and the complete sequence was identified from transcriptomic analysis. SeviL, consisting of 129 amino acids, was classified as an R(icin B)-type lectin, based on the presence of the QxW motif characteristic of this fold. SeviL mRNA is highly expressed in gills and, in particular, mantle rim tissues. Orthologue sequences were identified in other species of the family Mytilidae, including Mytilus galloprovincialis, from which lectin MytiLec-1 was isolated and characterized in our previous studies. Thus, mytilid species contain lectins belonging to at least two distinct families (R-type lectins and mytilectins) that have a common ß-trefoil fold structure but differing glycan-binding specificities. SeviL displayed notable cytotoxic (apoptotic) effects against various cultured cell lines (human breast, ovarian, and colonic cancer; dog kidney) that possess asialo-GM1 oligosaccharide at the cell surface. This cytotoxic effect was inhibited by the presence of anti-asialo-GM1 oligosaccharide antibodies. With HeLa ovarian cancer cells, SeviL showed dose- and time-dependent activation of kinase MKK3/6, p38 MAPK, and caspase-3/9. The transduction pathways activated by SeviL via the glycosphingolipid oligosaccharide were triggered apoptosis. DATABASE: Nucleotide sequence data have been deposited in the GenBank database under accession numbers MK434191, MK434192, MK434193, MK434194, MK434195, MK434196, MK434197, MK434198, MK434199, MK434200, and MK434201.


Asunto(s)
Gangliósido G(M1)/análogos & derivados , Lectinas/química , Lectinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mytilidae/química , Oligosacáridos/metabolismo , Animales , Sitios de Unión , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Células HeLa , Humanos , Lectinas/aislamiento & purificación , Proteínas Quinasas Activadas por Mitógenos/química , Mytilidae/metabolismo , Oligosacáridos/química , Especificidad de la Especie
4.
Sci Rep ; 9(1): 12243, 2019 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-31439922

RESUMEN

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor - the cholera toxin (CT). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewisx (Lex), however, where and how the CT binds to Lex remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunits of the CT bound to the Lex trisaccharide, and complementary quantitative binding data for CT holotoxins. Lex, and also L-fucose alone, bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. Lex is the first histo-blood group antigen of non-secretor phenotype structurally investigated in complex with CT. Together with the quantitative binding data, this allows unique insight into why individuals with non-secretor phenotype are more prone to severe cholera than so-called 'secretors'.


Asunto(s)
Antígenos de Grupos Sanguíneos/metabolismo , Toxina del Cólera/química , Cólera/metabolismo , Gangliósido G(M1)/análogos & derivados , Vibrio cholerae/metabolismo , Sitios de Unión , Antígenos de Grupos Sanguíneos/química , Cólera/microbiología , Toxina del Cólera/metabolismo , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Glicosilación , Humanos , Unión Proteica , Vibrio cholerae/química , Vibrio cholerae/genética
5.
Nano Lett ; 19(3): 1875-1882, 2019 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-30719917

RESUMEN

Viruses, such as influenza A, typically bind to the plasma membrane of their host by engaging multiple membrane receptors in parallel, thereby forming so-called multivalent interactions that are created by the collective action of multiple weak ligand-receptor bonds. The overall interaction strength can be modulated by changing the number of engaged receptors. This feature is used by viruses to achieve a sufficiently firm attachment to the host's plasma membrane but also allows progeny viruses to leave the plasma membrane after completing the virus replication cycle. Design of strategies to prevent infection, for example, by disturbing these attachment and detachment processes upon application of antivirals, requires quantification of the underlying multivalent interaction in absence and presence of antivirals. This is still an unresolved problem, as there is currently no approach available that allows for determining the valency (i.e., of the number of receptors bound to a particular virus) on the level of single viruses under equilibrium conditions. Herein, we track the motion of single influenza A/X31 viruses (IAVs; interacting with the ganglioside GD1a incorporated in a supported lipid bilayer) using total internal reflection fluorescence microscopy and show that IAV residence time distributions can be deconvoluted from valency effects by taking the IAV mobility into account. The so-derived off-rate distributions, expressed in dependence of an average, apparent valency, show the expected decrease in off-rate with increasing valency but also show an unexpected peak structure, which can be linked to a competition in the opposing functionalities of the two influenza A virus spike proteins, hemagglutinin (HA), and neuraminidase (NA). By application of the antiviral zanamivir that inhibits the activity of NA, we provide direct evidence, how the HA/NA balance modulates this virus-receptor interaction, allowing us to assess the inhibition concentration of zanamivir based on its effect on the multivalent interaction.


Asunto(s)
Hemaglutininas/química , Gripe Humana/virología , Neuraminidasa/química , Receptores Virales/química , Membrana Celular/química , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/química , Hemaglutininas/genética , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/genética , Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Membrana Dobles de Lípidos/química , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Receptores Virales/genética , Zanamivir
6.
Curr Eye Res ; 44(6): 664-670, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30688114

RESUMEN

Purpose: Most complex gangliosides in vertebrates are formed from ganglioside GM3. GM3 deficiency in humans can result in epilepsy and visual impairment. To investigate whether a deficiency of GM3 is involved in visual function, ST3GAL5-/- mice with mutations in the ST3GAL5 gene-coded GM3 synthase were employed. Materials and Methods: Sixty mice were employed in this study. The glycosphingolipids of mice retinas were analyzed through high performance thin layer chromatography. The morphology of the optic nerves and retinas were evaluated by hematoxylin and eosin staining and immunohistochemical analysis using an anti-glial fibrillary acidic protein (GFAP) antibody. An electroretinogram (ERG) was applied on the eyes of 4, 9, 12, and 14-month-old mice. Also, visual evoked potential (VEP) was applied on 13-month-old mice. Results: The GM3 in the retinas was detected in ST3GAL5+/+ mice but not ST3GAL5-/- mice. Also, GM1b and GD1α expressions and lactosylceramide accumulation were found in the ST3GAL5-/- mouse retinas. There was no significant difference in GFAP expression in the retinas or optic discs between ST3GAL5+/+ and ST3GAL5-/- mice. Furthermore, the outcome of ERG and VEP analysis showed no disparity between the two strains in 13 and 14-month-old mice. Conclusion: In the eye, neither histopathological abnormalities nor abnormal functions of the retina were found in GM3-deficient mice. Differing from the situation in patients with GM3 deficiency, the lack of GM3 in mice did not lead to optic nerve atrophy.


Asunto(s)
Retina/enzimología , Sialiltransferasas/deficiencia , Agudeza Visual/fisiología , Animales , Antígenos CD/metabolismo , Combinación de Medicamentos , Electrorretinografía , Potenciales Evocados Visuales/fisiología , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Lactosilceramidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Midriáticos/farmacología , Fenilefrina/farmacología , Proteína Quinasa C-alfa/metabolismo , Pupila/efectos de los fármacos , Tropicamida/farmacología
7.
Glycobiology ; 29(3): 242-259, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30535277

RESUMEN

The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N/O-glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N/O- glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N/O-glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g., m/z 1905.634, 2158.777 and 2905.036) and 15 O-glycan peaks (e.g., 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m/z 2205.766) was unique in HC, and 9 O-glycan peaks (e.g., m/z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N/O-glycans in saliva.


Asunto(s)
Biomarcadores de Tumor/genética , Virus de la Hepatitis B/genética , Polisacáridos/genética , Análisis por Matrices de Proteínas , Biomarcadores de Tumor/química , Biomarcadores de Tumor/aislamiento & purificación , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Femenino , Fibrosis/genética , Fibrosis/virología , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/química , Gangliósido G(M1)/genética , Virus de la Hepatitis B/aislamiento & purificación , Virus de la Hepatitis B/patogenicidad , Hepatitis Crónica/genética , Hepatitis Crónica/virología , Humanos , Lectinas/química , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Masculino , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Saliva/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
8.
Clin Cancer Res ; 24(20): 5178-5189, 2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-30021910

RESUMEN

Purpose: The ganglioside fucosyl-GM1 (FucGM1) is a tumor-associated antigen expressed in a large percentage of human small cell lung cancer (SCLC) tumors, but absent in most normal adult tissues, making it a promising target in immuno-oncology. This study was undertaken to evaluate the preclinical efficacy of BMS-986012, a novel, nonfucosylated, fully human IgG1 antibody that binds specifically to FucGM1.Experimental Design: The antitumor activity of BMS-986012 was evaluated in in vitro assays using SCLC cells and in mouse xenograft and syngeneic tumor models, with and without chemotherapeutic agents and checkpoint inhibitors.Results: BMS-986012 showed a high binding affinity for FcγRIIIa (CD16), which resulted in enhanced antibody-dependent cellular cytotoxicity (ADCC) against FucGM1-expressing tumor cell lines. BMS-986012-mediated tumor cell killing was also observed in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) assays. In several mouse SCLC models, BMS-986012 demonstrated efficacy and was well tolerated. In the DMS79 xenograft model, tumor regression was achieved with BMS-986012 doses of 0.3 mg/kg and greater; antitumor activity was enhanced when BMS-986012 was combined with standard-of-care cisplatin or etoposide. In a syngeneic model, tumors derived from a genetically engineered model of SCLC were treated with BMS-986012 or anti-FucGM1 with a mouse IgG2a Fc and their responses evaluated; when BMS-986012 was combined with anti-PD-1 or anti-CD137 antibody, therapeutic responses significantly improved.Conclusions: Single-agent BMS-986012 demonstrated robust antitumor activity, with the addition of chemotherapeutic or immunomodulatory agents further inhibiting SCLC growth in the same models. These preclinical data supported evaluation of BMS-986012 in a phase I clinical trial of patients with relapsed, refractory SCLC. Clin Cancer Res; 24(20); 5178-89. ©2018 AACR.


Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Gangliósido G(M1)/análogos & derivados , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos de Neoplasias/inmunología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Gangliósido G(M1)/antagonistas & inhibidores , Gangliósido G(M1)/inmunología , Gangliósido G(M1)/metabolismo , Humanos , Inmunohistoquímica , Inmunomodulación/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Unión Proteica , Receptores de IgG/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Rapid Commun Mass Spectrom ; 32(17): 1481-1490, 2018 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-29876976

RESUMEN

RATIONALE: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc-GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc-GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc-GM1 biomarkers present in various biological matrices has not been reported to date. METHODS: Individual Fuc-GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high-resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre-concentrate the soluble and drug-bound forms of Fuc-GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc-GM1 analogs in biological matrices. RESULTS: The concentration of individual Fuc-GM1 analogs in the standard mixture was estimated to be 7-34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 µL), the lower limit of quantification (LLOQ) of each analog was 0.2-0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE-HPLC/MS/MS method was demonstrated by detecting endogenous Fuc-GM1 analogs present in rat blood and in several lung cancer cell lines. CONCLUSIONS: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc-GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug-bound Fuc-GM1.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Gangliósido G(M1)/análogos & derivados , Neoplasias Pulmonares/sangre , Espectrometría de Masas en Tándem/métodos , Animales , Anticuerpos Monoclonales/análisis , Biomarcadores/sangre , Biomarcadores/química , Gangliósido G(M1)/sangre , Gangliósido G(M1)/química , Gangliósido G(M1)/aislamiento & purificación , Humanos , Ratas
10.
Org Biomol Chem ; 16(22): 4076-4080, 2018 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-29789847

RESUMEN

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacßSph) was chemically synthesized (on a 13 g scale), subjected to sequential one-pot multienzyme (OPME) glycosylation reactions with facile C18-cartridge purification, followed by improved acylation conditions to form target gangliosides, including fucosyl GM1 which has never been synthesized before.


Asunto(s)
Antígenos de Neoplasias/química , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M3)/síntesis química , Gangliósido G(M1)/síntesis química , Glicosilación , Lactosa/química , Esfingosina/análogos & derivados , Esfingosina/química
11.
Methods Enzymol ; 597: 239-263, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28935104

RESUMEN

Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes. The functional evaluations of the synthesized fluorescent gangliosides demonstrated the great influence of fluorescent dye on the physical properties of gangliosides in PMs and revealed the fluorescent ganglioside analogs which show similar behaviors to the native gangliosides.


Asunto(s)
Bioquímica/métodos , Gangliósido G(M1)/química , Gangliósido G(M2)/química , Gangliósido G(M3)/química , Gangliósidos/química , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/síntesis química , Gangliósido G(M2)/análogos & derivados , Gangliósido G(M2)/síntesis química , Gangliósido G(M3)/análogos & derivados , Gangliósido G(M3)/síntesis química , Gangliósidos/síntesis química , Glicoesfingolípidos/síntesis química , Glicoesfingolípidos/química , Microdominios de Membrana , Ácidos Siálicos/química
12.
Protein Expr Purif ; 138: 1-6, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28673733

RESUMEN

The cholera toxin B subunit (CTB) is a nontoxic portion of the cholera toxin that retains mucosal adjuvant properties. Expression of CTB in Escherichia coli is difficult as CTB aggregates and accumulates as insoluble inclusion bodies. To remedy this problem, the periplasmic chaperone, SKP, was investigated as possible co-expression partner to increase the solubility of recombinant CTB (rCTB) in E. coli. The result showed co-expression of SKP enhanced the soluble expression of rCTB in E. coli. Moreover, soluble rCTB was successfully expressed and secreted into the periplasmic space through the direction of the LTB leader signal. rCTB in periplasm was purified using an immobilized d-galactose resin; GM1-ELISA experiments showed that rCTB retains strong GM1 ganglioside-binding activity. Intranasal administration of ovalbumin (OVA) with rCTB significantly induced both mucosal and humoral immune responses specific to OVA. These data indicate that co-expression of the molecular chaperone SKP with CTB increased the solubility of rCTB while maintaining its function.


Asunto(s)
Adyuvantes Inmunológicos/genética , Toxina del Cólera/genética , Escherichia coli/genética , Inmunidad Mucosa/efectos de los fármacos , Proteínas Quinasas Asociadas a Fase-S/genética , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Animales , Toxina del Cólera/administración & dosificación , Toxina del Cólera/inmunología , Clonación Molecular , Escherichia coli/metabolismo , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Expresión Génica , Inmunización , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Periplasma/química , Periplasma/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Solubilidad
13.
Biochim Biophys Acta Gen Subj ; 1861(10): 2494-2506, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28734966

RESUMEN

Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40ms and 12ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.


Asunto(s)
Gangliósido G(M1)/síntesis química , Gangliósido G(M2)/síntesis química , Gangliósido G(M3)/síntesis química , Gangliósidos/síntesis química , Microdominios de Membrana/metabolismo , Sondas Moleculares/síntesis química , Antígenos CD59/química , Antígenos CD59/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/metabolismo , Gangliósido G(M2)/análogos & derivados , Gangliósido G(M2)/metabolismo , Gangliósido G(M3)/análogos & derivados , Gangliósido G(M3)/metabolismo , Gangliósidos/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microdominios de Membrana/ultraestructura , Sondas Moleculares/metabolismo , Imagen Individual de Molécula
14.
Int J Mol Sci ; 17(8)2016 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-27529215

RESUMEN

The ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of GD1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34⁺ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Neoplasias de la Mama/metabolismo , Sialiltransferasas/metabolismo , Animales , Antígenos CD34/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/metabolismo , Humanos , Ratones , Pericitos/metabolismo , Sialiltransferasas/genética
15.
ACS Chem Neurosci ; 7(8): 1107-11, 2016 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-27276519

RESUMEN

Ganglioside Hp-s1 is isolated from the ovary of sea urchin Diadema setosum. It exhibited better neuritogenic activity than GM1 in pheochromocytoma 12 cells. To explore the roles of glucosyl moiety of Hp-s1 in contributing to the neurogenic activity, we developed feasible procedures for synthesis of Hp-s1 analogues (2a-2f). The glucosyl moiety of Hp-s1 was replaced with α-glucose, α-galactose, ß-galactose, α-mannose, and ß-mannose, and their biological activities on SH-SY5Y cells and natural killer T (NKT) cells were evaluated. We found that the orientation of C-2 hydroxyl group at glucosyl moiety of Hp-s1 plays an important role to induce neurite outgrowth of SH-SY5Y cells. Surprisingly, compound 2d could activate NKT cells to produce interleukin 2, although it did not show great activity on neurite outgrowth of SH-SY5Y cells. In general, the Hp-s1 might be considered as a lead compound for the development of novel drugs aimed at modulating the activity of neuronal cells.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Gangliósido G(M1) , Neurogénesis/efectos de los fármacos , Animales , Conformación de Carbohidratos , Línea Celular Tumoral , Citocinas/metabolismo , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/química , Gangliósido G(M1)/farmacología , Glicosilación/efectos de los fármacos , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Modelos Químicos , Estructura Molecular , Neuritas/efectos de los fármacos , Neuroblastoma
16.
Glycobiology ; 26(9): 984-998, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27102283

RESUMEN

Some gangliosides, sialic acid-containing glycosphingolipids, have been considered as tumor-associated antigens. GD1α or a GD1α synthase gene ST6GalNAc5 was reported to be involved in the metastasis of murine lymphomas or human breast cancers, respectively. But expression patterns of 0-series gangliosides GD1α and its precursor GM1b in human cancers have not yet been investigated mainly due to lack of specific antibodies. We established specific monoclonal antibodies (mAbs) reactive with GD1α or GM1b using gangliosides from brain tissues of GM3 synthase (St3gal5)-deficient mice as immunogens. We used GM2/GD2 synthase (B4galnt1)-deficient mice to immunize by liposomes embedded with GD1α or acidic glycolipid fractions from brain of St3gal5-deficient mice. Specificities of established mAbs as analyzed by enzyme-linked immunosorbent assay and thin-layer chromatography-immunostaining were very high among various gangliosides. Increased expression of GD1α and reduced GM1b in the St6galnac5 cDNA-transfected RAW117 cell line also substantiated the specificities of two mAbs. Then, we analyzed expression of GD1α and GM1b, and of relevant glycosyltransferase genes in various human cancer cell lines using generated anti-GD1α mAb 122 or anti-GM1b mAb MR155A-7. A few human cancer cell lines showed significant expression of these gangliosides with reasonable expression of relevant glycosyltransferase genes.


Asunto(s)
Gangliósido G(M1)/análogos & derivados , N-Acetilgalactosaminiltransferasas/genética , Sialiltransferasas/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Gangliósido G(M1)/biosíntesis , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Gangliósidos/genética , Gangliósidos/metabolismo , Regulación Enzimológica de la Expresión Génica , Glicoesfingolípidos/genética , Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Humanos , Ratones , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/metabolismo , Metástasis de la Neoplasia , Sialiltransferasas/metabolismo
17.
Biochim Biophys Acta ; 1861(7): 606-16, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27102612

RESUMEN

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.


Asunto(s)
Colestanos/farmacología , Colestenonas/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Toxina del Cólera/química , Endocitosis/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Exocitosis/efectos de los fármacos , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/química , Microdominios de Membrana/efectos de los fármacos , Microelectrodos , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Unión Neuromuscular/fisiología , Rana ridibunda , Coloración y Etiquetado , Vesículas Sinápticas/efectos de los fármacos , Técnicas de Cultivo de Tejidos
18.
PLoS One ; 11(4): e0153265, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27070150

RESUMEN

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM1 and GM1-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM1 has approximately 7 times higher CTB binding capacity than GM1. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM1 caused the increased binding capacity. This was unintuitive, as GM1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM1 and GM2, we observed the mild binding avidity fucosyl-GM1 activated GM2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM2 receptors have negligible binding avidity in pure GM2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.


Asunto(s)
Toxina del Cólera/metabolismo , Gangliósido G(M1)/análogos & derivados , Membrana Dobles de Lípidos/metabolismo , Toxina del Cólera/química , Microscopía por Crioelectrón , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Gangliósido G(M2)/química , Gangliósido G(M2)/metabolismo , Técnicas In Vitro , Cinética , Membrana Dobles de Lípidos/química , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Modelos Biológicos , Nanotecnología , Unión Proteica , Dióxido de Silicio/química , Plata/química , Resonancia por Plasmón de Superficie
19.
J Reprod Dev ; 62(3): 249-55, 2016 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-26860251

RESUMEN

Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs.


Asunto(s)
Blastocisto/citología , Gangliósido G(M1)/análogos & derivados , Oocitos/citología , Animales , Apoptosis , Núcleo Celular/metabolismo , Células Cultivadas , Fase de Segmentación del Huevo , Células del Cúmulo/citología , Desarrollo Embrionario , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Femenino , Fertilización , Gangliósido G(M1)/fisiología , Meiosis , Metafase , Ovario/metabolismo , Sialiltransferasas/metabolismo , Porcinos , beta-Galactosida alfa-2,3-Sialiltransferasa
20.
Glycobiology ; 26(5): 532-9, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26715202

RESUMEN

Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 µs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures.


Asunto(s)
Cápside/química , Factor H de Complemento/química , Gangliósido G(M1)/análogos & derivados , Simulación de Dinámica Molecular , Virus 40 de los Simios/química , Cápside/metabolismo , Factor H de Complemento/metabolismo , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Humanos , Virus 40 de los Simios/metabolismo
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