Characterization of ADP-Ribosylation Sites on Desmin and Restoration of Desmin Intermediate Filament Assembly by De-ADP-Ribosylation

Abstract

Desmin is an intermediate filament protein that can be ADP-ribosylated by arginine-specific mono(ADP-ribosyl) transferase. Stoichiometric modification of desmin by the transferase causes inhibition of assembly of desmin into 10-nm intermediate filaments (Huang et al., 1993, Biochem. Biophys. Res. Commun. 197, 570-577). In this work, the sites of modification that can affect disassembly have been identified. ADP-ribosylated desmin (1.2 mol ADP-ribose/mol desmin) was digested with lysyl endopeptidase followed by trypsin. Two ADP-ribosylated peptides were obtained, sequenced by Edman degradation, and analyzed by the use of matrix-assisted laser desorption/ionization mass spectrometry. Arginines 48 and 68 of desmin's head domain were shown to be sites of modification, with arginine 48 the major ADP-ribosylation site. ADP-ribosylated desmin (4 mol ADP-ribose/mol desmin) was treated with ADP-ribosylarginine hydrolase. Removal of more than three ADP-ribose groups results in partial restoration of desmin's ability to form intermediate filaments. It is necessary to remove all ADP-ribose groups from desmin to restore its complete ability to form intermediate filaments. The fact that the effect of ADP-ribosylation on the filament-forming properties of desmin is fully reversible suggests that ADP-ribosylation alone is responsible for the changes noted in desmin.