RESUMEN
Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A.
Asunto(s)
Citocinas , Lípido A , Lipopolisacáridos , Lípido A/química , Lípido A/farmacología , Lípido A/análogos & derivados , Lípido A/síntesis química , Citocinas/metabolismo , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Humanos , Antígeno 96 de los Linfocitos/metabolismo , Antígeno 96 de los Linfocitos/química , Diseño de Fármacos , Relación Estructura-Actividad , Transducción de Señal/efectos de los fármacosRESUMEN
Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.
Asunto(s)
Selectina E , Virus de la Influenza A , Polisacáridos , Antígeno Sialil Lewis X , Polisacáridos/química , Polisacáridos/metabolismo , Virus de la Influenza A/metabolismo , Antígeno Sialil Lewis X/metabolismo , Antígeno Sialil Lewis X/química , Selectina E/metabolismo , Selectina E/química , Humanos , Oligosacáridos/química , Oligosacáridos/síntesis química , Oligosacáridos/metabolismo , Receptores Virales/metabolismo , Receptores Virales/química , Epítopos/química , Epítopos/metabolismo , AnimalesRESUMEN
Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.
Asunto(s)
Células Epiteliales/metabolismo , Liasa de Heparina/metabolismo , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/metabolismo , Sulfatos/metabolismo , Sulfotransferasas/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Antitrombina III/química , Antitrombina III/genética , Antitrombina III/metabolismo , Sitios de Unión , Unión Competitiva , Secuencia de Carbohidratos , Línea Celular , Córnea/citología , Córnea/metabolismo , Células Epiteliales/patología , Células Epiteliales/virología , Factor Xa/química , Factor Xa/genética , Factor Xa/metabolismo , Inhibidores del Factor Xa/química , Inhibidores del Factor Xa/metabolismo , Expresión Génica , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Liasa de Heparina/química , Liasa de Heparina/genética , Heparitina Sulfato/química , Herpesvirus Humano 1/crecimiento & desarrollo , Interacciones Huésped-Patógeno/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Análisis por Micromatrices , Unión Proteica , Proteolisis , Bibliotecas de Moléculas Pequeñas , Especificidad por Sustrato , Sulfatos/química , Sulfotransferasas/química , Sulfotransferasas/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Virus de la Influenza A/genética , Mutación , Neuraminidasa/genética , Infecciones por Orthomyxoviridae/virología , Ácidos Siálicos/metabolismo , Replicación Viral , Sustitución de Aminoácidos , Animales , Sitios de Unión , Perros , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Virus de la Influenza A/aislamiento & purificación , Células de Riñón Canino Madin Darby , Neuraminidasa/química , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/patología , Unión ProteicaRESUMEN
Guillain-Barré syndrome is often caused by Campylobacter jejuni infection that has induced antibodies to the lipo-oligosaccharide (LOS) that cross-react with gangliosides at peripheral nerves causing polyneuropathy. To examine fine specificities of anti-ganglioside antibodies and develop a more robust platform for diagnosis and disease monitoring, we developed a chemoenzymatic approach that provided an unprecedented panel of oligosaccharides composed of the inner-core of the LOS of C. jejuni extended by various ganglioside mimics. The compounds and corresponding ganglio-oligosaccharides were printed as a microarray to examine binding specificities of lectins, anti-ganglioside antibodies, and serum antibodies of GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis, disease monitoring, and immunological research.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Materiales Biomiméticos/química , Campylobacter jejuni/enzimología , Síndrome de Guillain-Barré/diagnóstico , Lipopolisacáridos/química , Oligosacáridos/química , Especificidad de Anticuerpos , Técnicas Biosensibles , Reacciones Cruzadas , Gangliósidos/química , Humanos , Lectinas/química , Análisis por Matrices de Proteínas , Suero/química , Bibliotecas de Moléculas Pequeñas/análisisRESUMEN
The parasitic worm, Schistosoma mansoni, expresses unusual fucosylated glycans in a stage-dependent manner that can be recognized by the human innate immune receptor DC-SIGN, thereby shaping host immune responses. We have developed a synthetic approach for mono- and bis-fucosylated LacdiNAc (LDN-F and LDN-DF, respectively), which are epitopes expressed on glycolipids and glycoproteins of S. mansoni. It is based on the use of monosaccharide building blocks having carefully selected amino-protecting groups, facilitating high yielding and stereoselective glycosylations. The molecular interaction between the synthetic glycans and DC-SIGN was studied by NMR and molecular modeling, which demonstrated that the α1,3-fucoside of LDN-F can coordinate with the Ca2+ -ion of the canonical binding site of DC-SIGN allowing for additional interactions with the underlying LDN backbone. The 1,2-fucoside of LDN-DF can be complexed in a similar manner, however, in this binding mode GlcNAc and GalNAc of the LDN backbone are placed away from the protein surface resulting in a substantially lower binding affinity. Glycan microarray binding studies showed that the avidity and selectivity of binding is greatly enhanced when the glycans are presented multivalently, and in this format Lex and LDN-F gave strong responsiveness, whereas no binding was detected for LDN-DF. The data indicates that S. mansoni has developed a strategy to avoid detection by DC-SIGN in a stage-dependent manner by the addition of a fucoside to a number of its ligands.
Asunto(s)
Moléculas de Adhesión Celular , Lectinas Tipo C , Polisacáridos , Receptores de Superficie Celular , Schistosoma mansoni , Animales , Moléculas de Adhesión Celular/inmunología , Glicosilación , Humanos , Inmunidad Innata , Lectinas Tipo C/inmunología , Polisacáridos/inmunología , Receptores de Superficie Celular/inmunología , Schistosoma mansoni/inmunologíaRESUMEN
Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs, and thus, the reported synthetic principles will find broad applicability.
Asunto(s)
Glicosiltransferasas/química , Leche Humana/química , Oligosacáridos/síntesis química , Femenino , Humanos , Análisis por Micromatrices , Oligosacáridos/químicaRESUMEN
Disialosyl globopentaosylceramide (DSGb5) is often expressed by renal cell carcinomas. To investigate properties of DSGb5, we have prepared its oligosaccharide moiety by chemically synthesizing Gb5 which was enzymatically sialylated using the mammalian sialyltransferases ST3Gal1 and ST6GalNAc5. Glycan microarray binding studies indicate that Siglec-7 does not recognize DSGb5, and preferentially binds Neu5Acα(2,8)Neu5Ac containing glycans.
Asunto(s)
Carcinoma de Células Renales/química , Inhibidores Enzimáticos/farmacología , Globósidos/farmacología , Neoplasias Renales/química , Oligosacáridos/farmacología , Sialiltransferasas/antagonistas & inhibidores , Antígenos de Neoplasias , Conformación de Carbohidratos , Inhibidores Enzimáticos/química , Globósidos/síntesis química , Globósidos/química , Células HEK293 , Humanos , Análisis por Micromatrices , Oligosacáridos/química , Sialiltransferasas/metabolismo , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
We describe a chemoenzymatic strategy that can give a library of differentially fucosylated and sialylated oligosaccharides starting from a single chemically synthesized tri-N-acetyllactosamine derivative. The common precursor could easily be converted into 6 different hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as a free amine (GlcNH2 ) or Boc (GlcNHBoc). Fucosylation of the resulting compounds by a recombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties, providing access to 6 selectively mono- and bis-fucosylated oligosaccharides. Conversion of the GlcNH2 or GlcNHBoc moieties into the natural GlcNAc, followed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds. The oligosaccharides were printed as a microarray that was probed by several glycan-binding proteins, demonstrating that complex patterns of fucosylation can modulate glycan recognition.
RESUMEN
A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC classâ II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Collectively, the immunological data demonstrate engagement of helper T-cells in immune activation. A synthetic methodology was developed for a penta-glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1-based cancer vaccines that elicited potent immune responses employed exogenous helper T-epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T-epitope will be more attractive, because it will activate cognate CD4+ T-cells that will provide critical tumor-specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.
Asunto(s)
Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/inmunología , Glicopéptidos/inmunología , Mucina-1/inmunología , Vacunas Sintéticas/inmunología , Animales , Vacunas contra el Cáncer/química , Glicopéptidos/química , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Conformación Molecular , Mucina-1/química , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/químicaRESUMEN
Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.
Asunto(s)
Factores de Crecimiento de Fibroblastos/química , Heparitina Sulfato/química , Análisis por Micromatrices , Animales , Sitios de Unión , Conformación de Carbohidratos , Línea Celular , Proliferación Celular , Ligandos , Ratones , Resonancia por Plasmón de SuperficieRESUMEN
A major objective of nanomedicine is to combine in a controlled manner multiple functional entities into a single nanoscale device to target particles with great spatial precision, thereby increasing the selectivity and potency of therapeutic drugs. A multifunctional nanoparticle is described for controlled conjugation of a cytotoxic drug, a cancer cell targeting ligand, and an imaging moiety. The approach is based on the chemical synthesis of polyethylene glycol that at one end is modified by a thioctic acid for controlled attachment to a gold core. The other end of the PEG polymers is modified by a hydrazine, amine, or dibenzocyclooctynol moiety for conjugation with functional entities having a ketone, activated ester, or azide moiety, respectively. The conjugation approach allowed the controlled attachment of doxorubicin through an acid-labile hydrazone linkage, an Alexa Fluor dye through an amide bond, and a glycan-based ligand for the cell surface receptor CD22 of B-cells using strain promoted azide-alkyne cycloaddition. The incorporation of the ligand for CD22 led to rapid entry of the nanoparticle by receptor-mediated endocytosis. Covalent attachment of doxorubicin via hydrazone linkage caused pH-responsive intracellular release of doxorubicin and significantly enhanced the cytotoxicity of nanoparticles. A remarkable 60-fold enhancement in cytotoxicity of CD22 (+) lymphoma cells was observed compared to non- targeted nanoparticles.
Asunto(s)
Antineoplásicos/química , Doxorrubicina/química , Sistemas de Liberación de Medicamentos/métodos , Oro/química , Linfoma de Células B/química , Nanopartículas/química , Polietilenglicoles/química , Polímeros/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Ciclooctanos/química , Ciclooctanos/metabolismo , Doxorrubicina/farmacología , Portadores de Fármacos , Endocitosis , Humanos , Concentración de Iones de Hidrógeno , Linfoma de Células B/metabolismoRESUMEN
Niemann-Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Azidas/farmacocinética , Proteínas Portadoras/genética , Línea Celular , Colesterol/metabolismo , Dextranos/farmacocinética , Endosomas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Galactosamina/farmacocinética , Glicoconjugados/genética , Glicoconjugados/metabolismo , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genética , Transporte de Proteínas/fisiología , Sialoglicoproteínas/metabolismo , Proteínas de Transporte Vesicular , beta-Ciclodextrinas/farmacologíaRESUMEN
Major histocompatibility complex (MHC) class I and II are glycoproteins that can present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes. Here, the importance of the modification of protein antigens by glycans on cellular uptake, proteolytic processing, presentation by MHC and subsequent T-cell priming is reviewed. Antigen glycosylation is important for a number of diseases and vaccine design. All of the key proteins involved in antigen recognition and the orchestration of downstream effector functions are glycosylated. The influence of protein glycosylation on immune function and disease is covered.
Asunto(s)
Inmunidad Adaptativa/fisiología , Antígenos/metabolismo , Complejo Mayor de Histocompatibilidad/fisiología , Glicoproteínas , Glicosilación , VacunasRESUMEN
The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam(3)CysSK(4), a peptide T(helper) epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Mucina-1/inmunología , Neoplasias/inmunología , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos de Neoplasias/química , Vacunas contra el Cáncer/química , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Humanos , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Virus del Tumor Mamario del Ratón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Neoplasias/patología , Linfocitos T Citotóxicos/inmunología , Carga Tumoral/inmunología , Vacunas Sintéticas/químicaRESUMEN
The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O-linked saccharides. Although tumor-associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T-lymphocytes (CTLs) and ADCC-mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous Thelper peptide, and a TLR2 (Pam3 CysSK4 ) or TLR9 (CpG-ODN 1826) agonist. It was found that the Pam3 CysSK4 -containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor-associated glycopeptide. The unique adjuvant properties of Pam3 CysSK4 , which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor-specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/uso terapéutico , Glicopéptidos/uso terapéutico , Mucina-1/uso terapéutico , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 9/agonistas , Adyuvantes Inmunológicos/química , Secuencia de Aminoácidos , Animales , Mama/inmunología , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Glicopéptidos/química , Glicopéptidos/inmunología , Glicosilación , Humanos , Inmunidad Celular , Inmunización , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mucina-1/química , Mucina-1/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
A game of tag: N-Glycans on the surface of living cells were selectively tagged by exogenously administering recombinant ST6Galâ I sialyltransferase and azide-modified CMP-Neu5Ac. This modification was followed by a strain-promoted cycloaddition using a biotin-modified dibenzylcyclooctynol (red star=biotin). The methodology will make it possible to dissect the mechanisms that underlie altered glycoconjugate recycling and storage in disease.
Asunto(s)
Polisacáridos/química , Sialiltransferasas/química , Química Clic/métodos , Fibroblastos/química , Fibroblastos/metabolismo , Humanos , Ácido N-Acetilneuramínico , Polisacáridos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/síntesis química , Ácidos Siálicos/química , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Francisella tularensis, which is a Gram negative bacterium that causes tularemia, has been classified by the Center for Disease Control and Prevention (CDC) as a category A bioweapon. The development of vaccines, immunotherapeutics, and diagnostics for F. tularensis requires a detailed knowledge of the saccharide structures that can be recognized by protective antibodies. We have synthesized the inner core region of the lipopolysaccharide (LPS) of F. tularensis to probe antigenic responses elicited by a live and subunit vaccine. The successful preparation of the target compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl ether (All), and levulinoyl (Lev) ester. The ability to remove the protecting groups in different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A comparison of the (1)H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment of the inner core oligosaccharide of F. tularensis . The observation that immunizations with LPS lead to antibody responses to the inner core saccharides provides an impetus to further explore this compound as a vaccine candidate.
Asunto(s)
Vacunas Bacterianas/síntesis química , Vacunas Bacterianas/inmunología , Francisella tularensis/inmunología , Lipopolisacáridos/síntesis química , Lipopolisacáridos/inmunología , Tularemia/inmunología , Tularemia/prevención & control , Animales , Formación de Anticuerpos , Vacunas Bacterianas/química , Femenino , Francisella tularensis/química , Inmunización , Inmunoconjugados/química , Inmunoconjugados/inmunología , Lipopolisacáridos/química , Ratones , Ratones Endogámicos BALB CRESUMEN
Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represent a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location, and in particular it has been shown that DIBO derivative 4 can enter cells, thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.
Asunto(s)
Cicloparafinas/química , Fibroblastos/química , Glicoconjugados/química , Coloración y Etiquetado , Cicloparafinas/síntesis química , Cicloparafinas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Glicoconjugados/síntesis química , Glicoconjugados/metabolismo , Humanos , Células Jurkat , Cinética , Estructura MolecularRESUMEN
Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc-modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.