Defined conjugation of glycans to the lysines of CRM197 guided by their reactivity mapping.
Systematic characterisation of the reactivity of the lysine moieties in CRM197 towards N-hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two-step conjugation of various glycans to CRM197 by click chemistry in a well-defined manner. By semiquantitative LC-MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine-mediated glycoconjugates with improved batch-to-batch reproducibility, thereby producing neo-glycoconjugates with more-consistent biological activity.
['Crotti|Stefano|S|', 'Zhai|Huili|H|', 'Zhou|Jing|J|', 'Allan|Martin|M|', 'Proietti|Daniela|D|', 'Pansegrau|Werner|W|', 'Hu|Qi-Ying|QY|', 'Berti|Francesco|F|', 'Adamo|Roberto|R|']
D000480:Alkynes D000595:Amino Acid Sequence D001386:Azides D001426:Bacterial Proteins D002851:Chromatography, High Pressure Liquid D057930:Click Chemistry D018360:Crystallography, X-Ray D019281:Dimerization D006001:Glycoconjugates D008239:Lysine D010455:Peptides D011134:Polysaccharides D017434:Protein Structure, Tertiary D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
glycoconjugates bioconjugation carbohydrates mass spectrometry vaccines