CHARACTERIZATION OF ADP-RIBOSYLATION SITES ON DESMIN AND RESTORATION OF DESMIN INTERMEDIATE FILAMENT ASSEMBLY BY DE-ADP-RIBOSYLATION

Desmin is an intermediate filament protein that can be ADP-ribosylated by arginine-specific mono(ADP-ribosyl) transferase. Stoichiometric mo dification of desmin by the transferase causes inhibition of assembly of desmin into 10-nm intermediate filaments (Huang ct al., 1993, Bioch em. Biophys. Res. Commun. 197, 570-577). In this work, the sites of mo dification that can affect disassembly have been identified. ADP-ribos ylated desmin (1.2 mol ADP-ribose/mol desmin) was digested with lysyl endopeptidase followed by trypsin. Two ADP-ribosylated peptides were o btained, sequenced by Edman degradation, and analyzed by the use of ma trix-assisted laser desorption/ionization mass spectrometry. Arginines 48 and 68 of desmin's head domain were shown to be sites of modificat ion, with arginine 48 the major ADP-ribosylation site. ADP-ribosylated desmin (4 molADP-ribose/mol desmin) was treated with ADP-ribosylargin ine hydrolase. Removal of more than three ADP-ribose groups results in partial restoration of desmin's ability to form intermediate filament s. It is necessary to remove all ADP-ribose groups from desmin to rest ore 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 res ponsible for the changes noted in desmin. (C) 1996 Academic Press, Inc .