Assuntos
Lectinas/metabolismo , Análise em Microsséries , Polissacarídeos/metabolismo , Animais , Sítios de Ligação , Glicoconjugados/química , Glicoconjugados/metabolismo , Humanos , Lectinas/química , Análise em Microsséries/métodos , Nanotecnologia/métodos , Polissacarídeos/química , Ligação ProteicaAssuntos
Aldeído Liases/química , Anticorpos Catalíticos/química , Evolução Molecular Direcionada , Proteínas/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Anticorpos Catalíticos/genética , Anticorpos Catalíticos/metabolismo , Catálise , Regiões Determinantes de Complementaridade , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção EnzimáticaRESUMO
We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.
Assuntos
Anticorpos Antineoplásicos/sangue , Antígenos Glicosídicos Associados a Tumores/imunologia , Vacinas Anticâncer/imunologia , Carcinoma/imunologia , Mucina-1/imunologia , Animais , Antígenos Glicosídicos Associados a Tumores/química , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hemocianinas/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Mucina-1/química , Vacinas Conjugadas/imunologiaRESUMO
The design and evaluation of a new genetic selection system for evolving catalytic antibodies with aldolase activity are described. Through a series of model selections, we have identified selection conditions where expression of a catalytically active antibody confers a growth advantage to Escherichia coli. In addition, we provide evidence that the growth advantage is a direct result of catalytic activity.
Assuntos
Anticorpos Catalíticos/genética , Frutose-Bifosfato Aldolase/imunologia , Seleção Genética , Sequência de Bases , Primers do DNA , Escherichia coli/crescimento & desenvolvimentoRESUMO
Posttranslational modifications of proteins regulate many biological processes, including metabolism, signal transduction, and gene expression. The synthetic challenges associated with generating homogeneous populations of selectively modified proteins, however, have hindered detailed studies of the effects of these modifications on protein structure and function. Here, we report an approach to the cotranslational synthesis of selectively glycosylated proteins in which the modified amino acid is genetically encoded. We show that myoglobin containing beta-N-acetylglucosamine (GlcNAc)-serine at a defined position can be expressed in Escherichia coli in good yield and with high fidelity. The beta-GlcNAc moiety can be recognized by a saccharide-binding protein, or subsequently modified with a galactosyltransferase to build more complex carbohydrates. This approach should be generally applicable to other posttranslational modifications such as protein phosphorylation, acetylation, and methylation.