title: 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.
authors.affiliation: Department of Health Sciences and Technology, ETH Zürich, Vladimir-Prelog-Weg 4, 8093 Zürich (Switzerland) http://www.appliedmechanobio.ethz.ch.
authors.forename: Susanna M
authors.initials: SM
authors.lastname: Früh
authors.forename: Philipp R
authors.initials: PR
authors.lastname: Spycher
authors.forename: Maria
authors.initials: M
authors.lastname: Mitsi
authors.forename: Melanie A
authors.initials: MA
authors.lastname: Burkhardt
authors.forename: Viola
authors.initials: V
authors.lastname: Vogel
authors.forename: Ingmar
authors.initials: I
authors.lastname: Schoen
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D002478:Cells, Cultured; D057930:Click Chemistry; D004867:Equipment Design; D026122:Factor XIIIa / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D038761:Photobleaching
chemical_list: D001386:Azides; D005353:Fibronectins; D005456:Fluorescent Dyes; D015335:Molecular Probes; D026122:Factor XIIIa
keywords: bioconjugation; click chemistry; protein modifications; stepwise bleaching; transferases