Site-Selective Functionalization of Flagellin by Steric Self-Protection: A Strategy To Facilitate Flagellin as a Self-Adjuvanting Carrier in Conjugate Vaccine
Journal
ChemBioChem
Journal Volume
19
Journal Issue
8
Pages
805-814
Date Issued
2018
Author(s)
Abstract
Flagellin (FliC) can act as a carrier protein in the preparation of conjugate vaccines to elicit a T-cell-dependent immune response and as an intrinsic adjuvant to activate the toll-like receptor 5 (TLR5) to enhance vaccine potency. To enable the use of FliC as a self-adjuvanting carrier, an effective method for site-selective modification (SSM) of pertinent amino-acid residues in the D2 and D3 domains of FliC is explored without excessive modification of the D0 and D1 domains, which are responsible for activating and binding with TLR5. In highly concentrated Na2SO4 solution, FliC monomers form flagellar filaments, in which the D0 and D1 domains are situated inside the tubular structure. Thus, the lysine residues (K219, K224, K324, and K331) in the D2 and D3 domains of flagellin are selectively modified by a diazo-transfer reaction with imidazole-1-sulfonyl azide. The sites with azido modification are confirmed by MALDI-TOF-MS, ESI-TOF-MS, and LC–MS/MS analyses along with label-free quantitation. The azido-modified filament dissolves to give FliC monomers, which can conjugate with alkyne-hinged saccharides by the click reaction. Transmission electron microscopy imaging, dynamic light scattering measurements, and the secreted embryonic alkaline phosphatase reporter assay indicate that the modified FliC monomers retain the ability either to bind with TLR5 or to reassemble into filaments. Overall, this study establishes a feasible method for the SSM of FliC by steric self-protection of the D0 and D1 domains. ? 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Subjects
antigens; conjugate vaccines; flagellins; self-assembly; steric hindrance
SDGs
Other Subjects
alkaline phosphatase; drug carrier; flagellin; monomer; sodium sulfate; toll like receptor 5; flagellin; immunological adjuvant; TLR5 protein, human; toll like receptor 5; vaccine; Article; chemical modification; controlled study; electrospray mass spectrometry; enzyme activation; enzyme binding; human; liquid chromatography-mass spectrometry; matrix assisted laser desorption ionization time of flight mass spectrometry; photon correlation spectroscopy; priority journal; protein function; stereospecificity; transmission electron microscopy; vaccination; liquid chromatography; mass spectrometry; metabolism; procedures; ultrastructure; Adjuvants, Immunologic; Chromatography, Liquid; Drug Carriers; Flagellin; Mass Spectrometry; Microscopy, Electron, Transmission; Toll-Like Receptor 5; Vaccines, Conjugate
Type
journal article