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J. Biol. Chem., Vol. 281, Issue 43, 32619-32629, October 27, 2006

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Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme^*

Vasanthy Vigneswara, Jonathan D. Lowenson, Claire D. Powell, Matthew Thakur, Kevin Bailey, Steven Clarke, David E. Ray, and Wayne G. Carter¹

From the Medical Research Council Applied Neuroscience Group, School of Biomedical Sciences, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom and the Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein L-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1^-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1^-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-³H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1^-/- animals compared with Pcmt1^+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, -synuclein, and -synuclein, are all substrates for the L-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1^-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Received for publication, June 6, 2006 , and in revised form, August 17, 2006.

^* This work was supported by a UK Medical Research Council program grant (to D. E. R.) and by National Institutes of Health Grant GM026020 (to S. C. and J. D. L.). Purchase of a Multichannel Plate Detector was funded by the UK Dept. of the Environment, Food, and Rural Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

¹ To whom correspondence should be addressed. Tel.: 44-(0)115-823-0176; Fax: 44-(0)115-823-0142; E-mail: wayne.carter{at}nottingham.ac.uk.

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