Functional modification of fibronectin by N-terminal FXIIIa-mediated transamidation

Abstract

A straightforward strategy is presented for the site-specific incorporation of fluorophores or reactive probes into the extracellular matrix (ECM) protein fibronectin (Fn) by using the enzyme-catalyzed transamidation by activated factor XIII. Characterization by SDS-PAGE, western blotting, absorption measurements, mass spectrometry, and stepwise photobleaching for labeling quantification at the single-molecule level showed that the labeling was efficient and restricted to the N-terminal tails. The introduction of labels did not interfere with Fn fibrillogenesis, as verified by the incorporation of fluorescently labeled Fn into ECM and manually pulled Fn fibers. Site-specific incorporation of an azide was used to create a template for bioorthogonal click chemistry reactions in a second bioconjugation step, thus offering versatile modification and application possibilities in the context of matrix biology and tissue engineering.

Keywords: bioconjugation; click chemistry; protein modifications; stepwise bleaching; transferases.