ADP-ribosylation of intermediate filament protein, desmin
Date
1996
Authors
Zhou, Hao
Major Professor
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Graves, Donald J.
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Abstract
Desmin is an intermediate filament protein which can be ADP-ribosylated by arginine specific mono(ADP-ribosyl) transferase. Stoichiometric modification of desmin by the transferase causes disassembly of intermediate filaments (Biochem. Biophys. Res. Comm. 197, 1993, 570-577). In this work, the site(s) of modification that can affect disassembly have been identified. ADP-ribosylated desmin (1.2 moles ADP-riboses/mole 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 the head domain were shown to be sites of modification, with arginine 48 the major ADP-ribosylation site.
Also in this work, ADP-ribosylated desmin (4 moles ADP-riboses/mole desmin) was treated with arginine specific mono(ADP-ribosyl) hydrolase. Removal of 2 or 3 groups results in partial restoration of formation of the filaments. It is necessary to remove all ADP-ribose groups to restore complete formation of intermediate filaments. The fact that the effect of ADP-ribosylation on filamentous properties of desmin is fully reversible suggests that ADP-ribosylation alone is responsible for the changes noted in desmin.
Desmin is a muscle specific intermediate filament protein which can be ADP-ribosylated by muscle arginine specific mono(ADP-ribosyl)transferase (70). Incubation of membrane fractions from 96-hr primary chick myotubes with [³²P] NAD resulted in labeling of two protein bands with molecular weight of 56 kDa and 36 kDa (94). Desmin was shown to be present in the 56 kDa band and labeling of both these two protein bands was due to ADP-ribosylation by muscle transferase (94). In this work, labeling membrane fractions of 5-day primary chick myotube with [³²P] NAD, resolving immunoprecipitated desmin on SDS-PAGE, and performing autoradiography showed the presence of radiolabeled desmin. This result strongly indicate that the labeling of 56 kDa protein band is indeed due to the ADP-ribosylation of desmin at the arginine residues by the muscle transferase from the membrane fractions of chick myotubes. Also in this work, by using R-28 polyclonal antibodies specific for ADP-ribosylated arginine, we have shown that soluble desmin immunoprecipitated from 4- or 5-day chick myotubes reacted specifically with this antibody and this reaction was weakened when soluble desmin was treated with neutral hydroxylamine. This result is consistent with ADP-ribosylation of arginine residue of desmin from the intact muscle cells. We also found that that desmin purified from turkey gizzard also strongly reacted with this R-28 antibody, which indicates that desmin from smooth muscle cell is ADP-ribosylated by the arginine specific mono(ADP-ribosyl)transferase.
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