RESUMEN
Mucin 1 (MUC1) peptide fused with Q11 (MUC1-Q11) having 35 residues has previously been shown to form amyloid fibrils. Using time-dependent and high-resolution atomic force microscopy (AFM) imaging, it is revealed that the formation of individual MUC1-Q11 fibrils entails nucleation and extension at both ends. This process can be altered by local mechanical perturbations using AFM probes. This work reports two specific perturbations and outcomes. First, by increasing load while maintaining tip-surface contact, the fibrils are cut during the scan due to shearing. Growth of fibrils occurs at the newly exposed termini, following similar mechanism of the MUC1-Q11 nucleation growth. As a result, branched fibrils are seen on the surface whose orientation and length can be controlled by the nuclei orientation and reaction time. In contrast to the "one-time-cut", fibrils can be continuously fragmented by modulation at sufficiently high amplitude. As a result, short and highly branched fibrils accumulate and pile on surfaces. Since the fibril formation and assembly of MUC1-Q11 can be impacted by local mechanical force, this approach offers a nonchemical and label-free means to control the presentation of MUC1 epitopes, and has promising application in MUC1 fibril-based immunotherapy.
Asunto(s)
Péptidos/química , Amiloide/química , Microscopía de Fuerza Atómica , Mucina-1/químicaRESUMEN
Oligo(ethylene glycol)-linked light fluorous tags have been found to be optimal for conjugating to glycans for both high-yield enzymatic glycosylation reactions using one-pot multienzyme (OPME) systems and quick product purification using fluorous solid-phase extraction (FSPE) cartridges. The combination of OPME glycosylation systems and the FSPE cartridge purification scheme provides a highly effective strategy for facile synthesis and purification of glycans.
Asunto(s)
Flúor/química , Complejos Multienzimáticos/metabolismo , Polisacáridos/biosíntesis , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicosilación , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Extracción en Fase SólidaRESUMEN
A series of STn-MUC1 and ST-MUC1 glycopeptides containing naturally occurring and non-natural sialic acids have been chemoenzymatically synthesized from Tn-MUC1 glycopeptide using one-pot multienzyme (OPME) approaches. In situ generation of the sialyltransferase donor cytidine 5'-monophosphate-sialic acid (CMP-Sia) using a CMP-sialic acid synthetase in the presence of an extra amount of cytidine 5'-triphosphate (CTP) and removal of CMP from the reaction mixture by flash C18 cartridge purification allow the complete consumption of Tn-MUC1 glycopeptide for quantitative synthesis of STn-MUC1. A Campylobacter jejuni ß1-3GalT (CjCgtBΔ30-His6) mutant has been found to catalyze the transfer of one or more galactose residues to Tn-MUC1 for the synthesis of T-MUC1 and galactosylated T-MUC1. Sialylation of T-MUC1 using Pasteurella multocida α2-3-sialyltransferase 3 (PmST3) with Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) and Escherichia coli sialic acid aldolase in one pot produced ST-MUC1 efficiently. These glycopeptides are potential cancer vaccine candidates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glicopéptidos/biosíntesis , Ácidos Siálicos/química , Antígenos Virales de Tumores/química , Proteínas Bacterianas/genética , Secuencia de Bases , Campylobacter jejuni/enzimología , Clonación Molecular , Escherichia coli/enzimología , Glicopéptidos/química , Datos de Secuencia Molecular , Mutación , Neisseria meningitidis/enzimología , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Pasteurella multocida/enzimología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned from Bifidobacterium longum strain ATCC55813 and used efficiently with a Pasteurella multocida inorganic pyrophosphatase (PmPpA) with or without a monosaccharide 1-kinase for one-pot multienzyme synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their derivatives. Further chemical diversification of a UDP-mannose derivative resulted in the formation of UDP-N-acetylmannosamine.
Asunto(s)
Bifidobacterium/enzimología , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Azúcares de Uridina Difosfato/biosíntesis , Galactoquinasa/metabolismo , Pirofosfatasa Inorgánica/metabolismo , Pasteurella multocida/enzimología , Uridina Difosfato Galactosa/biosíntesis , Uridina Difosfato Galactosa/química , Uridina Difosfato Glucosa/biosíntesis , Uridina Difosfato Glucosa/química , Azúcares de Uridina Difosfato/químicaRESUMEN
The synthesis of glycosylated Fmoc amino acids by reaction of mono- and disaccharide peracetates with Fmoc amino acids having free carboxyl groups was rapidly promoted by Lewis acids (SnCl(4), BF(3)·Et(2)O) under microwave irradiation. The products are useful building blocks for the synthesis of glycopeptides.
