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 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.
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
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H
Zhou
T W
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Huiatt
R M
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Robson
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Sernett
D J
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Graves
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003893:Desmin / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D007382:Intermediate Filaments / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003893:Desmin; D010446:Peptide Fragments; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases