RESUMEN
For the first time Helix lucorum hemocyanin (HlH) has been feruloylated. Two HlH conjugates with 40- and 120- ferulic acid residues were prepared, denoted as FA-HlH-1 and FA-HlH-2. Expectedly, the feruloylation of HlH induced a rearrangement of the protein molecule, a decrease in the ?-helical structure at the expense of ß-structures was observed. Besides, the FA-HlH conjugates were more prone to aggregation, which is probably due to the stabilization of the partially unfolded protein molecules by non-covalent bonding. Interestingly, the thermal stability of HlH was not affected by the modification. The native and feruloylated HlH were not toxic to normal fibroblasts (BJ cells). We observed a decrease in cell viability of breast cancer MCF-7 cells to about 66% after a 48h exposure to 70 µg/well of FA-HlH-2.
Asunto(s)
Ácidos Cumáricos/farmacología , Caracoles Helix/química , Hemocianinas/farmacología , Acilación , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ácidos Cumáricos/síntesis química , Ácidos Cumáricos/toxicidad , Hemocianinas/síntesis química , Hemocianinas/toxicidad , HumanosRESUMEN
Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qß carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qß-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qß-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.
Asunto(s)
Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/uso terapéutico , Epítopos/inmunología , Mucina-1/inmunología , Allolevivirus/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/inmunología , Femenino , Gastrópodos/química , Hemocianinas/síntesis química , Hemocianinas/química , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucina-1/química , Mucina-1/genética , Metástasis de la Neoplasia/inmunología , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas Virales/síntesis química , Proteínas Virales/químicaRESUMEN
Glycoprotein MUC1 is an attractive target for anti-tumor vaccine development. However, the weak immunogenicity of MUC1 remains a significant problem. To solve this problem, several STn derivatives with N-acetyl modifications were synthesized and incorporated into a 20-amino acid MUC1 tandem repeat sequence. The modified STn-MUC1 glycopeptides were further connected to a carrier protein keyhole limpet hemocyanin (KLH). The immunological effects of these synthetic vaccine conjugates were evaluated using the BALB/c mouse model. The results showed that vaccine V2 elicited higher titers of antibodies which cross-reacted with the native STn-MUC1 antigen. Moreover, the elicited antisera reacted with the STn-MUC1 antigen-positive tumor cells, indicating that the carbohydrate antigen modification strategy may hold potential to overcome the weak immunogenicity of natural MUC1 glycopeptides.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/inmunología , Glicopéptidos/síntesis química , Glicopéptidos/farmacología , Hemocianinas/síntesis química , Hemocianinas/farmacología , Animales , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Vacunas contra el Cáncer/farmacología , Línea Celular Tumoral , Femenino , Glicopéptidos/inmunología , Hemocianinas/inmunología , Humanos , Sueros Inmunes/química , Sueros Inmunes/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Mucina-1/química , Neoplasias/prevención & controlRESUMEN
GM1 epitope tetrasaccharide was synthesized by a condensation of sialyl-alpha(2-3)-gal acceptor and gal-beta(1-3)-GalN donor in a highly efficient manner. After introduction of mercaptohexanol to the tetrasaccharide, it was coupled to maleimide-activated KLH carrier protein to give the desired GM1 epitope-KLH conjugate.
Asunto(s)
Epítopos/química , Hemocianinas/química , Lipopolisacáridos/química , Oligosacáridos/química , Epítopos/inmunología , Hemocianinas/síntesis química , Hemocianinas/inmunología , Lipopolisacáridos/síntesis química , Lipopolisacáridos/inmunología , Estructura Molecular , Oligosacáridos/síntesis química , Oligosacáridos/inmunologíaRESUMEN
Vaccines derived from totally synthetic carbohydrate antigens have been shown to elicit an immune response in both preclinical and clinical settings. The vaccines have been proven safe when administered in human clinical trials and are also competent at inducing antibodies that react with aberrant cells expressing the corresponding carbohydrate antigen. The most well studied vaccines have hitherto focused on single carbohydrate antigens, notwithstanding the known heterogeneity of transformed cells. Advances in synthetic organic chemistry have enabled the preparation and subsequent investigation of vaccines that contain several different tumor-associated carbohydrate antigens in a single molecule. These unimolecular constructs could, in principle, serve as superior mimics of cell surface antigens and hence, as multifaceted cancer vaccines. We report here the synthesis of a pentameric vaccine targeting a specific cancer. The new vaccine contains prostate tumor-associated antigens, Tn, TF, STn, Lewis(y), and Globo-H. To reach our goal, antigen-containing amino acid monomers were assembled in a linear fashion to form a glycopeptide containing the five distinct carbohydrate antigen units. The attachment of a linker to the glycopeptide followed by an extraordinary global deprotection and subsequent conjugation to two different immunogenic carriers, keyhole limpet hemocyanin and N-alpha-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, resulted in the vaccine constructs. The results described herein indicate that complex unimolecular multivalent vaccines can be efficiently produced in the laboratory. These fully synthetic vaccines have the potential to stimulate a multifaceted immune response against prostate cancer.
Asunto(s)
Vacunas contra el Cáncer/síntesis química , Cisteína/análogos & derivados , Neoplasias Glandulares y Epiteliales/terapia , Antígenos de Carbohidratos Asociados a Tumores/química , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Secuencia de Carbohidratos , Cisteína/síntesis química , Cisteína/química , Cisteína/inmunología , Hemocianinas/síntesis química , Hemocianinas/química , Hemocianinas/inmunología , Humanos , Lipoproteínas/síntesis química , Lipoproteínas/química , Lipoproteínas/inmunología , Masculino , Datos de Secuencia Molecular , Estructura Molecular , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunologíaRESUMEN
For the purpose of applying the particular antibodies as a new diagnostic procedure for atherosclerosis and related diseases, we successfully achieved the synthesis of the fatty sterol with a linker, then linked the target protein to this sterol. Synthesis was started from pregnenolone and achieved by the Grignard reaction with pentenyl magnesium bromide, regioselective photoaddition of thiolacetic acid toward the 25-double bond, esterification of 3-OH with linoleic anhydride, in situ conjunction of the cross-linker (MBS) to the thiol group after selective deprotection from its acetyl ester, and finally by the reaction with protein such as KLH or albumin through this linker.
Asunto(s)
Pregnenolona/análogos & derivados , Proteínas/inmunología , Esteroles/inmunología , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/metabolismo , Albúminas/síntesis química , Albúminas/inmunología , Albúminas/metabolismo , Animales , Anticuerpos/inmunología , Formación de Anticuerpos , Antígenos/inmunología , Reactivos de Enlaces Cruzados/síntesis química , Reactivos de Enlaces Cruzados/metabolismo , Hemocianinas/síntesis química , Hemocianinas/inmunología , Hemocianinas/metabolismo , Ratones , Pregnenolona/síntesis química , Pregnenolona/inmunología , Pregnenolona/metabolismo , Proteínas/síntesis química , Esteroles/síntesis química , Esteroles/metabolismoRESUMEN
The kinetics of the H2O2 oxidation of separated a, b and c subunits (75 kDa, 657 amino acids) of the arthropod Panulirus interruptus hemocyanin (Hc), in the deoxy Cu(I)2 state, have been studied at 25 degrees C, I = 0.100 M (NaCl). Solutions of oxyHc provide small equilibrium amounts of the deoxy reactant deoxyHc + O2<-->oxyHc (K approx. 10(5) M-1). The reaction is of interest because H2O2 is one of the few molecules which is able to access the active site and oxidise deoxyHc to the metHc Cu(II)2 state. The reaction was studied at pH values in the range 6.8-9.6. Traces of Ca2+ (and other 2+ ions) are controlled by addition of EDTA (5 mM). With or without EDTA hexamer forms are present at the lower pH values. At pH > 8.3 and with EDTA added, a and b (3% sequence difference) give monomer forms. The hexamers are however retained at the higher pH values if Ca2+ (10 mM) is added. As in the corresponding studies of deoxyHc with O2, rate constants for subunit c (42% sequence differences) show no variation with pH and the hexamer is retained over the whole pH-range explored. Rate constants for the reactions of H2O2 with the different monomer and hexamer deoxyHc forms of a, b, and c are in the range 1-75 M-1 s-1. A mild pseudo-catalase activity of metHc leading to reformation of oxyHc also contributes to the reaction.
Asunto(s)
Hemocianinas/análogos & derivados , Peróxido de Hidrógeno/química , Animales , Sitios de Unión , Catalasa/química , Hemocianinas/síntesis química , Hemocianinas/química , Cinética , Nephropidae , Oxidación-ReducciónRESUMEN
Limulus polyphemus hemocyanin is a 3.3 x 10(6)-Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I--V) by DEAE-Sephadex chromatography. Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight-hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight-hexamer molecules from four-hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter-subunit contacts in the native Limulus hemocyanin molecule.