Site-specific transamidation and deamidation of the small heat-shock protein Hsp20 by tissue transglutaminase

Crosslinking of small heat-shock proteins (sHsps) by tissue transglutaminase (tTG) is enhanced by stress and under pathological conditions. We here used hexapeptide probes to determine the amine donor (K) and acceptor (Q) sites for tTG in Hsp20. Mass spectrometric peptide mass fingerprinting and peptide fragmentation established that Q(31) and the C-terminal K-162 are involved in inter- and intramolecular crosslinking (transamidation). Q(31) is a conserved glutamine in sHsps where the neighboring residue determines its reactivity. Moreover, we detected highly efficient simultaneous deamidation of Q(66), which suggests that tTG-catalyzed transamidation and deamidation is specific for different glutamine residues.