Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M606958200 on September 7, 2006

J. Biol. Chem., Vol. 281, Issue 44, 33802-33813, November 3, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/44/33802    most recent
M606958200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, J. X.
Right arrow Articles by Aswad, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, J. X.
Right arrow Articles by Aswad, D. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Protein Repair in the Brain, Proteomic Analysis of Endogenous Substrates for Protein L-Isoaspartyl Methyltransferase in Mouse Brain*

Jeff X. Zhu{ddagger}1, Hester A. Doyle§, Mark J. Mamula§, and Dana W. Aswad{ddagger}2

From the {ddagger}Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697 and the §Section of Rheumatology, Yale University School of Medicine, New Haven, Connecticut 06510

Received for publication, July 21, 2006 , and in revised form, August 30, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-3H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 3H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Formation of isoaspartate (isoAsp)3 is a major source of spontaneous protein damage under physiological conditions, arising in conjunction with deamidation of asparaginyl residues and isomerization of aspartyl residues (1-6). This non-enzymatic process occurs via the formation of a succinimide intermediate (cyclic imide) following nucleophilic attack of the Asx side-chain carbonyl group by the amide nitrogen at the C-flanking amino acid (see Fig. 1). Hydrolysis of the succinimide leads to formation of isoaspartyl (isoAsp) and aspartyl products in a typical ratio of 3 to 1. The beta-linkage characteristic of the predominant isoaspartyl form introduces a kink into the polypeptide backbone that can disrupt normal protein folding and activity. Isoaspartyl formation is strongly influenced by the amino acid that immediately follows (is C-flanking to) an Asn or Asp residue, by the degree of local polypeptide flexibility, and by environmental stressors such as high pH, heat shock, radiation, and oxidation.

The enzyme activity we now call protein L-isoaspartyl methyltransferase (PIMT, EC 2.1.1.77 [EC] ) was first encountered in 1965 by Axelrod and Daly (7) as an S-adenosyl-L-methionine (AdoMet)-dependent methanol-forming enzyme activity in pituitary extracts. By 1973 it became apparent that this enzyme was a protein methyltransferase whose immediate products were protein methyl esters that spontaneously hydrolyze over a period of minutes to hours at physiological pH (8-10). For the following 10 years, it was generally assumed that methylation occurred on the side-chain carboxyls of normal aspartyl or glutamyl residues and that methylation was probably serving to regulate protein activity in a manner analogous to protein phosphorylation. It was not until 1984 that this widely distributed methyltransferase was found to have a high degree of specificity for atypical L-isoaspartyl residues (11, 12). Subsequently, it was shown that isoaspartate formation is a common form of protein damage at physiological pH and temperature, fostering the idea that PIMT functions as an important intracellular repair enzyme. As predicted by the repair hypothesis, one finds that isoaspartate levels rise dramatically above their normally low steady-state levels when PIMT activity is reduced in vivo by pharmacological inhibition of cultured cells (13, 14) or when the PIMT gene is disrupted in mice (15, 16).


Figure 1
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 1.
Mechanism of isoaspartate formation and its repair by PIMT. Deamidation of asparaginyl residues and isomerization of aspartyl residues result in the formation of a metastable succinimide intermediate that hydrolyzes to generate a mixture of L-isoaspartyl and L-aspartyl linkages. PIMT catalyzes selective methylation of the L-isoaspartyl residues, converting them back to the succinimide. Each cycle of methylation/demethylation/succinimide hydrolysis reduces the isoaspartate level by 15-30% relative to the previous cycle, resulting in a net repair of ≥85% after 10 or more cycles. Accordingly, substantial reduction of PIMT activity in vivo leads to the accumulation of isoaspartyl proteins as the rate of de novo isoaspartate formation exceeds the rate of repair.

 
As illustrated in Fig. 1, the repair mechanism is a three-stage process: 1) enzymatic methylation of the {alpha}-carboxyl group of the L-isoaspartyl site, 2) spontaneous formation of a succinimide intermediate, and 3) spontaneous hydrolysis of the succinimide to generate a mixture of 15-30% L-aspartyl and 70-85% L-isoaspartyl forms. Although each cycle through this repair pathway is only 15-30% efficient, recycling of the isoAsp sites through the PIMT reaction results in an overall repair efficiency of 85% or greater. This has been shown in vitro with synthetic isoaspartyl peptides (17, 18) and with isoAsp-containing proteins such as calmodulin (19) and synapsin I (20). Moreover, recent studies demonstrating an unusual D/L enantiomer ratio of Asp-25 in histone H2B (an endogenous substrate for PIMT) strongly suggest that repair of isoAsp formation at this site occurs in vivo with an overall efficiency of ~87% (21). The inability of PIMT to restore the normal side-chain amide of deamidated asparagine sites may be of little significance given the much greater importance of having a normal peptide backbone for proper protein folding.

Despite considerable progress made over the past 41 years in elucidating the function of "methanol-forming enzyme," there has been relatively little information regarding the identity of its major endogenous substrates. This situation is attributable to the fact that isoAsp levels are normally held to very low levels by PIMT action, and the fact that the protein methyl ester products of the PIMT reaction are so unstable that they do not survive most commonly used methods of high resolution protein separation. Until now, only four proteins have been found to serve as targets of PIMT action in vivo: calmodulin (20, 22), tubulin (23, 24), histone H2B (14), and synapsin I (20, 25). The discovery of all four of these proteins came not from systematic proteomics but rather from a combination of serendipity and guess work.

The recent creation of mice deficient in PIMT activity provides a unique opportunity to carry out an unbiased search for endogenous substrates for this enzyme. The PIMT-KO mouse is characterized by a phenotype that is so far predominantly neurological: progressively increasing brain size (16, 26), abnormal synaptic and neuronal organization (16, 27, 28), alterations in the insulin receptor signaling pathway (26), atypical patterns of motor activity (29) and neurophysiology (27, 30), and death from epileptic seizure at 4-10 weeks after birth (15, 16). Abnormalities in the immune system of PIMT-KO have also been found; namely increased T-lymphocyte proliferation and increased antibody production (31).

To understand more about the special need for PIMT activity in brain, we embarked on a proteomics approach to obtain a more global picture of the major substrates for PIMT in this organ. Our approach to this problem is founded on the observation that endogenous substrates for PIMT accumulate high levels of isoAsp in the PIMT-KO mouse (15, 16). When extracts of the KO mouse brain are 3H-methylated by PIMT in vitro and separated by SDS-PAGE under acidic conditions, it is apparent that a diverse group of proteins are acting as endogenous substrates. Unfortunately, until now, the low chemical stability of the [3H]methyl esters has made it difficult to employ the high resolution separation techniques needed to identify the individual radiolabeled proteins. To circumvent this problem, we separated the unmethylated proteins by two-dimensional-gel electrophoresis, transferred them to a polyvinylidene difluoride (PVDF) membrane, and then 3H-methylated them on the membrane with an overlay of recombinant PIMT and [methyl-3H]AdoMet. Autofluorography of the membrane provided information on which Coomassie-stained spots on a parallel two-dimensional gel corresponded to major methyl acceptors. Peptide mass fingerprinting of these spots allowed us to identify 22 proteins that are major endogenous substrates for PIMT.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Materials—Bio-Lyte 3/10 and Bio-Lyte 5/8 ampholytes were purchased from Bio-Rad. Immobilon-P PVDF membranes (0.45 µm) were purchased from Millipore. S-Adenosyl-[methyl-3H]L-methionine (AdoMet) and EN3HANCETM spray surface autoradiography enhancer were purchased from PerkinElmer Life Sciences. Rabbit anti-synapsin II polyclonal antibody (catalog no. 56382-100) was purchased from QED Bioscience Inc. (San Diego, CA). Secondary ECL detection antibody (horseradish peroxidase-linked donkey anti-rabbit IgG (catalog no. NA934V) was from Amersham Biosciences. Sequencing grade modified trypsin was purchased from Promega.

Mice—Heterozygous PIMT+/- founder pairs (from which PIMT wild-type, heterozygous, and knock-out mice were bred) were kindly provided by Dr. Edward Kim of the Gladstone Institute of Cardiovascular Disease, San Francisco, CA, in conjunction with the laboratories of Dr. Stephen Young at the same institution, and Dr. Steven Clarke at the University of California, Los Angeles (15). The presence of the neo gene and presence/absence of the PIMT gene were detected by PCR of tail DNA using the following primers: PIMT forward, 5'-GCAGCGACGGCAGTAACAGC-3'; PIMT reverse, 5'-ACCCTCTTCCCATCCACATC-3'; neo forward, 5'-GCACGAGGAAGCGGTCAGCCCATTC-3'; and neo reverse, 5'-CGCATCGAGCGAGCACGTACTCGG-3'. Mice were sacrificed at ~4-6 weeks of age by cervical dislocation, and brains were rapidly removed and frozen at -80 °C.

Preparation of Mouse Brain Extract—Approximately 2 g of mouse brain (PIMT-/- or PIMT+/+) tissue was used in each preparation. The tissue sample was cut into small pieces and mixed with 9 volumes of cold homogenization buffer (5 mM K-Hepes, pH 7.6, 0.5 mM EDTA, 0.1 mM dithiothreitol, 10% (w/v) sucrose, and 1% mammalian protease inhibitor mixture (Sigma catalog no. P3840)). The suspension was homogenized for 10 strokes (up and down) using a Potter-Elvejhem homogenizer with a rotating Teflon pestle. The homogenate was filtered through two layers of cheesecloth and centrifuged at 800 x g for 30 min. The supernatant was removed and stored at -80 °C.

Cloning, Expression, and Purification of Recombinant Rat PIMT—Rat PIMT cDNA was PCR-amplified from a rat liver cDNA pool (Clontech) and subcloned into pET28a (+) between BamH1 and XhoI. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) cells. Expression of rat PIMT was induced in LB media with the addition of 1 mM isopropyl-beta-D-thiogalactoside at 37 °C for 3 h. Cells were harvested and stored at -80 °C. The 6xHis PIMT was purified by nickel-nitrilotriacetic acid affinity chromatography (Qiagen) as recommended by the manufacturer. Eluted proteins were dialyzed at 4 °C overnight against 25 mM Tris-Cl, pH 7.0, 150 mM NaCl, 10% glycerol, and 5 mM 2-mercaptoethanol. The specific activity of the purified PIMT was ~14,000 pmol/min/mg protein using bovine {gamma}-globulin as the methyl acceptor (32).

Methylation of Mouse Brain Extracts by Recombinant PIMT—To label isoaspartyl proteins in mouse brain extracts, methylation reactions were performed in the presence of recombinant rat PIMT with [methyl-3H]AdoMet as the methyl donor. A typical reaction (20 µl) consisted of 100 mM Na-MES (pH 6.2), 40 µg of mouse brain extract, 2.5 µM recombinant PIMT, and 100 µM [methyl-3H]AdoMet. The reaction was initiated by adding AdoMet, incubating at 30 °C for 10 min, and stopped by adding 6 µl of the 4x-concentrated SDS-PAGE sample buffer (pH 6.8) described by Laemmli and Favre (33). After heating at 50 °C for 10 min, samples were subjected to electrophoresis in 4-12% NuPAGE gels (Invitrogen) using the MES running buffer at 50 V, 4 °C for 4 h. The gel was stained in the Coomassie blue, destained, impregnated with sodium salicylate (34), and dried under vacuum. The dried gel was exposed to preflashed (35) BioMAX XAR film (Kodak) at -80 °C.

Two-dimensional PAGE and Electroblotting—Non-equilibrium pH gradient electrophoresis (NEPHGE) was based on the method of O'Farrell (36). First dimension gels were polymerized in 13.5 x 0.45-cm glass tubes containing 4% acrylamide/bisacrylamide (29:1), 9.2 M urea, 2% (w/v) Nonidet P-40, 0.4% Bio-Lyte 3/10, 1.6% Bio-Lyte 5/8. ~160 µg of mouse brain extract was solubilized in NEPHGE sample buffer (9 M urea, 0.8% Bio-Lyte 5/8, and 0.2% Bio-Lyte 3/10). The solubilized samples were centrifuged for 5 min at 14,000 rpm, and the clear supernatant (~50 µl) was applied to the tube gel, overlaid with 20 µl of NEPHGE sample buffer, and filled with anode solution (10 mM H3PO4). The upper chamber of the electrophoresis apparatus was filled with 10 mM H3PO4 solution, and the lower chamber was filled with degassed cathode solution (20 mM NaOH). Tube gels were run at 500 V for 4 h at room temperature. After electrophoresis, the tube gel was extruded and equilibrated in 12 ml of SDS sample buffer (62.5 mM Tris-Cl, pH 6.8, 10% glycerol, 2% SDS, and 5% 2-mercaptoethanol) for 1 h at room temperature, and stored at -80 °C.

For the second dimension, SDS-PAGE slab gels utilized the Tris-glycine buffer system described by Laemmli and Favre (33). The separating gel was 8.5%, and stacking gel was 4%. Electrophoresis was carried out at 40 mA per gel in a Bio-Rad PROTEAN II Xi cell at room temperature. After electrophoresis, the gel was equilibrated with transfer buffer (25 mM Tris, 193 mM glycine, pH 8.3, 20% ethanol) for 30 min. Proteins were then transferred onto an Immobilon-P PVDF membrane at 50 V, 4 °C for 4 h. After blotting, the membrane was either stained with Coomassie Blue or used directly for on-membrane methylation.

On-blot Methylation and Fluorography—Prior to on-blot methylation, the membrane was incubated with 0.2 mg/ml bovine serum albumin in 10 mM Na-MES (pH 6.2) for 30 min and then placed on a glass plate in the hood. Methylation solution (6 ml) consisting of 100 mM Na-MES, pH 6.2, 4 µM recombinant rat PIMT, 4 µM [3H]AdoMet (10,000 dpm/pmol), and 0.1 mg/ml bovine serum albumin was warmed to 30 °C and then pipetted onto the membrane surface. After a reaction time of 20 min at room temperature, the membrane was rinsed briefly with water, then washed twice with stop buffer (10 mM Na-MES, pH 6.2, 0.3 M NaCl, 50 µM S-adenosyl-L-homocysteine, 0.05% (v/v) Tween 20) for 15 min. Finally the membrane was soaked in water for 5 min and in methanol for 20 s, and then air-dried. The dry membrane was taped to a filter paper backing, sprayed with EN3HANCETM, and exposed to preflashed KODAK BioMAX XAR film at -80 °C.

In-gel Digestion—Two-dimensional gels were lightly stained (20 min) with Coomassie Blue. Spots of interest from the two-dimensional gel were excised using a OneTouch manual spot picker (The Gel Company, San Francisco, CA), destained in 50% acetonitrile, reduced with 20 mM dithiothreitol at 45 °C for 30 min, and alkylated with 100 mM iodoacetamide in 50 mM ammonium bicarbonate for 30 min at 37 °C in the dark. The processed gel spots were digested with trypsin at 37 °C overnight. The tryptic peptides were extracted twice with 50 µl of extraction solution (50% (v/v) acetonitrile and 0.1% trifluoroacetic acid in water). The combined extracts were concentrated to ~20 µl and cleaned up with ZipTip (C18, Millipore Inc., Bedford, MA) according to the product instructions. The peptides were eluted with 4 µl of extraction solution from the Zip-Tip for analysis by mass spectrometry.

MALDI-TOF Mass Spectrometry and Database Search—Tryptic digests were analyzed by MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) mass spectrometry. Analytical samples consisted of 1 part tryptic digest, 1 part peptide calibration mix, and 2 parts matrix (10 mg/ml {alpha}-cyano-4-hydroxycinnamic acid in water). Mass data were acquired in the positive reflector mode on a Voyager-DE STR Biospectrometry workstation (Applied Biosystems, Foster City, CA). To identify proteins, the calibrated de-isotoped peak list for each digest was queried against the UniProt data base using the ProteinPropector MS-FIT program (University of California, San Francisco) at propector.ucsf.com.

Western Blots—The PVDF membrane from either a one-dimensional gel or two-dimensional gel was first blocked with 5% nonfat milk in Tris-buffered saline containing Tween 20 (0.05%, v/v) for 30 min. The membrane was then incubated first with primary antibody (1:10,000) in 5% nonfat milk for 2 h followed, then with secondary ECL detection antibody (1:10,000) for 1 h at room temperature. The membrane was washed 3x in Tris-buffered saline containing Tween 20 (0.05%, v/v) following antibody incubation. The ECL-Plus Western Detection System (Amersham Biosciences) was used to develop and detect the signals.

Generation of Anti-mouse CRMP2 Polyclonal Antibody—A synthetic peptide, acetyl-DITEWHKGIQEEGC-NH2 containing mouse CRMP2 140-151 sequence, was designed for antibody generation. Two rabbits were immunized with peptide conjugated to keyhole limpet hemocyanin. The polyclonal anti-body was then purified from antisera using a peptide-coupled affinity column. The purified antibodies were dialyzed in phosphate-buffered saline buffer at 4 °C overnight. Peptide synthesis and antibody production were contracted to AnaSpec Inc. (San Jose, CA).

Protein Concentration—Protein concentrations were determined by the method of Lowry et al. (37) using bovine serum albumin as standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Development of Methodology for Global Separation and Detection of Isoaspartyl Proteins in Brain Extracts of the PIMT-KO Mouse—To achieve a global analysis of proteins that serve as major substrates for PIMT in mammalian brain we considered two strategies, both of which are based on the fact that isoaspartyl proteins accumulate to high levels in the PIMT-KO mouse brain and that such proteins can be selectively labeled by PIMT-catalyzed methylation using [methyl-3H]AdoMet as the methyl donor. The first approach we tested was to pass brain extracts from PIMT-KO mice slowly over a PIMT-agarose column at 4 °C in the presence of unlabeled AdoMet. Because PIMT has a low catalytic turnover number (~0.4 min-1 at 30 °C) we reasoned that isoaspartyl proteins might form a sufficiently stable ternary complex (isoaspartyl protein·PIMT·AdoMet) to bind a number of isoaspartyl proteins to the column. Elution of the isoaspartyl proteins could then be carried out by addition of the strong competitive inhibitor S-adenosyl-L-homocysteine to displace the AdoMet and weaken the protein-PIMT interaction as predicted by the relevant enzyme kinetics (38). Attempts to employ this strategy were unsuccessful; no binding or significant retardation of isoaspartyl proteins on the column was observed.

Our second strategy was to separate proteins from the PIMT-KO mouse brain by two-dimensional gel electrophoresis, transfer the proteins to PVDF membranes, and then detect the positions of the PIMT substrates by on-blot 3H-methylation followed by fluorographic imaging on x-ray film. Our motivation to try this approach was bolstered by the success of Lee and Bedford (39) who used on-blot 3H-methylation to detect substrates for protein arginine methyltransferases on a commercial membrane-bound array of expressed proteins. We favored post-separation, on-blot 3H-methylation, as opposed to 3H-methylation prior to separation, because we feared significant loss of the labile [3H]methyl esters during the separations. The first dimension of isoelectric focusing or NEPHGE, especially, would expose the more basic proteins to high pH resulting in loss of the 3H label as proposed by O'Connor and Clarke (40).

Our ability to detect PIMT substrates by on-blot methylation was first tested using one-dimensional SDS-PAGE for protein separation. Fig. 2A shows the results of a control experiment in which proteins from PIMT+/+ or PIMT-/- mice were analyzed for isoAsp content by the traditional pre-gel-labeling approach in which 3H-methylated brain extracts are subjected to SDS-PAGE under conditions designed to minimize protein methyl ester hydrolysis. After electrophoresis, the wet gel is then impregnated with scintillant, dried, and exposed to x-ray film to image the 3H-labeled isoaspartyl proteins. This control experiment confirmed that PIMT-deficient mice accumulate high levels of isoaspartyl proteins compared with normal mice.

Fig. 2B shows the results obtained using an on-blot 3H-methylation approach. The same brain extracts used in Fig. 2A were separated by the same SDS-PAGE system, but the Fig. 2B extracts were not methylated prior to separation. Instead, the proteins were transferred onto a PVDF membrane and 3H-methylated on-blot using purified recombinant rat PIMT and [methyl-3H]AdoMet as described under "Experimental Procedures." After methylation, the membrane was extensively washed to remove residual AdoMet, dried, sprayed with scintillant, and exposed to x-ray film. We were gratified to see robust methylation of numerous proteins from the PIMT-deficient mice (-/-lane) and only minimal methylation of proteins from the wild-type mice (+/+ lane). This indicated that on-blot methylation of PIMT substrates works and that the methyl esters survive relatively intact. Importantly, a comparison of the conditions used to obtain the two sets of images shown in Fig. 2 indicates that on-blot methylation is significantly more sensitive than pre-gel methylation. As indicated in the legend to Fig. 2, the film exposure time for the image in panel B was only half that required for that in panel A, even though 1) the amount of protein loaded per lane in panel B was about half that used in panel A, and 2) both the specific activity and concentration of AdoMet was lower in panel B than in panel A. The only significant disadvantage of the panel B method is that it consumes considerably more [methyl-3H]AdoMet and recombinant PIMT per analysis than does the method in panel A.


Figure 2
View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 2.
Comparison of in-gel and on-blot fluorograms from one-dimensional gels of 3H-methylated brain extracts from PIMT+/+ and PIMT-/- mice. A, in-gel fluorogram. Extracts were methylated with recombinant PIMT and [methyl-3H]AdoMet (100 µM, 20,000 dpm/pmol) prior to SDS-PAGE (22.5 µg of protein per lane). The wet gel was impregnated with sodium salicylate, dried, and subjected to fluorography (41-h exposure) as described under "Experimental Procedures." B, on-blot fluorogram. Unlabeled brain extracts were separated by SDS-PAGE (10 µg per lane) and electroblotted onto a PVDF membrane. The membrane was incubated with recombinant PIMT and [methyl-3H]AdoMet (4 µM, 10,000 dpm/pmol). After washing, the membrane was sprayed with EN3HANCETM scintillant for fluorography (18-h exposure).

 
In an attempt to achieve a higher resolution separation of PIMT substrates, we subjected brain extracts to two-dimensional gel electrophoresis essentially as described by O'Farrell (36). We used NEPHGE (as opposed to isoelectric focusing) for the first dimension because of its superior ability to separate both basic and acidic proteins. We also felt that the lower voltages and shorter run times needed for NEPHGE would help to minimize artifactual generation of isoaspartyl sites. After the second dimension, proteins were transferred to PVDF and subjected to on-blot methylation and fluorographic imaging as described above. Analyses were carried out with extracts of PIMT+/+ and PIMT-/- mouse brain extracts, and the results are shown in Fig. 3. A large number of well separated PIMT substrates are evident in the -/- extract. Significantly less methylation of these proteins is seen in the +/+ extracts. These results demonstrate that two-dimensional gel electrophoresis, combined with on-blot methylation, provides a powerful approach to proteomic analysis of endogenous PIMT substrates in brain.

To judge the relative extent of isoaspartate formation among proteins in the KO brain extracts, we carried out a [3H]methyl blot similar to that shown in Fig. 3 and stained the membrane with Coomassie Blue (Fig. 4A). The membrane was then destained and subjected to fluorography to visualize the methyl acceptors (Fig. 4B). Protein spots that were ultimately identified by peptide-mass fingerprinting are bounded by a rectangle, diamond, or circle. These shapes were chosen to distinguish proteins for which the stoichiometry of methylation appears to be high, moderate, or low, respectively. Synapsins Ia, Ib, IIa, and IIb, for example, all exhibit a series of intense spots on the methyl blot, but are almost undetectable by Coomassie staining, suggesting a high stoichiometry of isoaspartyl sites. Indeed, we recently reported that synapsin I purified from the brains of 4- to 6-week-old PIMT-KO mice contains 0.9 ± 0.3 mol of isoaspartate per mole of synapsin (20). The metabolic enzymes aconitase, pyruvate kinase, and enolase exemplify the opposite extreme: low level methylation but intense staining. It is clear from these results that individual brain proteins vary widely in both their absolute and relative contributions to isoaspartate formation.

Identification of PIMT Substrates by Peptide Mass Fingerprinting—In an attempt to identify specific PIMT substrates, brain extracts from the PIMT-KO mouse were separated by two-dimensional gel electrophoresis as described above. The wet slab gel from the second dimension was stained lightly with Coomassie Blue, and spots of interest (corresponding to methyl acceptors) were excised for tryptic digestion followed by mass spectrometry via MALDI-TOF. Peptide data for each spot were analyzed against the UniProt protein data base using the MS-FIT portion of ProteinProspector 4.1.3 at the University of California, San Francisco (prospector.ucsf.edu).

Of the 30-35 spots (or closely spaced series of spots) that were analyzed, good matches were found for 22 (Fig. 4 and Table 1). All of the listed matches produced a good MOWSE (molecular weight search) score, high sequence coverage (21% or greater), and had molecular weights and isoelectric points consistent with the location of the protein on the two-dimensional gel. Several methyl acceptors were not identified, because, either we could not reliably find a Coomassie spot corresponding to the fluorographic spot, or the mass data did not yield a match with a high MOWSE score or good sequence coverage. Unidentified methyl acceptors include a group of five prominent spots located between aldolase A and VDAC-1 (voltage-dependent anion channel 1), two spots in the mid-NEPHGE dimension with a molecular mass of ~24 kDa, a spot at 10 o'clock for aspartate aminotransferase, and a spot at 7 o'clock for actin.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Identification of isoaspartyl proteins from PIMT-KO mouse brain extract

Coomassie-stained spots corresponding to major methyl acceptors (Fig. 4) were subjected to peptide mass fingerprinting by MALDI-TOF. The UniProt database was searched using the Protein Prospector MS-FIT program at prospector.ucsf.edu.

 
Previous to this study, only four mammalian proteins have been recognized as endogenous substrates for PIMT: synapsins Ia and I (20, 25), tubulin (23, 24), histone H2B (14), and calmodulin (20, 22). The re-identification of synapsins Ia and Ib, and of tubulin, in the present study serve as useful positive controls to validate the on-blot labeling approach. Histone H2B has previously been identified in the nuclear fraction of PC12 cells and mouse brain but was not expected here because the present study was carried out on a post-nuclear cytosolic fraction of mouse brain. In fact, we observed no significant methyl acceptors below 20 kDa. Calmodulin, a 17-kDa acidic protein, was also not expected, because it has been shown to accumulate only trace levels of isoaspartate in the KO mouse brain (20).


Figure 3
View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 3.
Comparison of methyl-accepting capacity of brain extracts from PIMT+/+ and PIMT-/- mice after two-dimensional separation and on-blot methylation. Equal amounts (160 µg/tube) of +/+ and -/-mouse brain extract were subjected to NEPHGE separation followed by SDS-PAGE. After transfer to PVDF membranes, on-blot 3H-methylation and fluorography were carried out as in Fig. 2B. The relatively low level of 3H-methylation seen in the +/+ extract demonstrates that a minimal amount of artifactual isoaspartate is generated by the two-dimensional separations.

 


Figure 4
View larger version (93K):
[in this window]
[in a new window]
  		FIGURE 4.
A comparison of protein staining and 3H-methylation patterns indicates a wide range of isoaspartate content in proteins from the PIMT-/- mouse. Brain extracts from the PIMT-/- mouse were analyzed as in Fig. 3. A, Coomassie Blue stain of a PVDF membrane after 3H-methylation and fluorography. B, 3H fluorogram of the same membrane prior to staining. Protein spots that were subsequently identified by peptide-mass fingerprinting are marked. Rectangles mark proteins that appear to have a relatively high level of isoaspartate per unit protein; diamonds and circles mark proteins with intermediate and low levels of isoaspartate, respectively. C, enlargement from A in the region containing synapsins IIa and IIb, and CRMP2 (as labeled in B). D, enlargement from B showing the same synapsin II/CRMP2 region. E, Western blot of the same synapsin II/CRMP2 region obtained with a mixture of rabbit anti-mouse synapsin II and rabbit anti-mouse CRMP2 (both at 1:10,000). All five panels are from the same PVDF membrane.

 
Four newly discovered methyl acceptors identified in Fig. 4 and Table 1 (dynamin I, synapsin IIa, synapsin IIb, and CRMP2) were of particular interest to us because of their high isoAsp content and their unique roles in neuronal function. The latter three proteins appear in groups of three to four closely spaced spots in the NEPHGE dimension. Fingerprinting of individual spots from a given series confirmed that they were isoelectric variants of the same protein. All three proteins are known to be phosphorylated at multiple sites in vivo, which probably contribute to the isoelectric heterogeneity of these proteins. Deamidation of asparagine residues is also likely to contribute, because deamidation adds a negative charge and is often associated with the formation of isoaspartyl sites. To further confirm the identity of synapsin IIa, synapsin IIb, and CRMP2, the PVDF membrane shown in Fig. 4B was washed free of the scintillant spray used for fluorography and then subjected to Western blot analysis using a primary antibody mixture consisting of 1:10,000 anti-synapsin IIa/IIb and 1:10,000 anti-CRMP2. The Western/ECL image seen in Fig. 4E shows immunoreactivity that superimposes perfectly over the fluorographic images of these methyl acceptors in panel D. Individual Western blots were also carried out using only antisynapsin II or only anti-CRMP2 and these gave the expected results (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Although PIMT is present in all mammalian tissues examined to date, the phenotype of the PIMT-KO mouse is primarily neurological: slightly enlarged brain, atypical synaptic physiology and motor behavior, and dramatically shorted life span resulting from fatal epileptic seizures at 4-10 weeks of age, with an average lifetime of 42 days. Lowenson et al. (41) produced a transgenic mouse in the PIMT-KO background in which PIMT activity was expressed exclusively in neurons under the control of a neuron-specific enolase promoter. The average lifetime of these mice increased 5-fold to 225 days, even though transgenic expression of PIMT activity in their brains was restored to only 7-13% of that found in wild-type mice. Similar results were reported by Shimizu et al. (25). The life extension afforded by the neuronal transgene expression confirms the vital importance of PIMT in the brain. The unique sensitivity of brain function to this common form of protein damage may prove relevant to one or more neurological diseases ranging from childhood neurological disorders to psychiatric illness and age-related dementias. To learn more about the susceptibility of brain function to isoAsp formation, we felt it was worthwhile to undertake a global analysis of the proteins in mammalian brain that are most susceptible to this type of spontaneous damage.

Proteomic analysis of isoaspartyl proteins has been a challenging task. Because there are no antibodies that generally recognize isoaspartyl proteins, PIMT-catalyzed radiolabeling is the current method of choice for their detection. Radiolabeling the proteins prior to separation is of limited use, however, because of the instability of the methyl esters that carry the label. Isoaspartyl methyl esters have a typical half-life at pH 7.4 and 37 °C of only 7 min (42, 43). O'Connor and Clarke (40) partially overcame this instability problem by subjecting 3H-methylated isoaspartyl proteins from human erythrocytes (labeled by incubating the intact cells with [methyl-3H]L-methionine) to two-dimensional gel electrophoresis using isoelectric focusing in the first dimension and an acidic (pH 2.4) SDS-PAGE system for the second dimension. Because of the need to maintain acidic conditions, protein separations were limited in the isoelectric focusing dimension to the pH range of 4-8. Because the cells used were wild type with regard to PIMT activity, the degree of radiolabeling was expectedly quite low, requiring fluorographic exposure times of 50-150 days. The main conclusion from this study was that "most, if not all, erythrocyte membrane and cytosolic proteins can act as sub-stoichiometric methyl acceptors."

The availability of PIMT-KO mice provides a unique opportunity to investigate the differential susceptibility of isoaspartate formation among all the proteins in a given tissue or cell type. In vitro labeling of protein extracts from KO and WT tissue allows one to clearly distinguish which proteins are acting as substrates for PIMT in vivo. This is not a trivial issue, because some isoaspartyl proteins in the normal brain contain significant levels of isoAsp due to inaccessibility to PIMT in vivo. A case in point is the protein phosphacan, a high molecular weight chondroitin-sulfate proteoglycan that resides in the extracellular matrix of brain and harbors about seven methylaccepting isoAsp sites per polypeptide in the adult rat (44). The high level of isoAsp in phosphacan of normal animals is explained by the fact that the PIMT repair system appears to be exclusively intracellular, whereas phosphacan is mainly, if not exclusively, an extracellular protein. By comparing in vitro 3H-methylation patterns of proteins in extracts of KO and WT mouse brain, Kim et al. (15) have shown using acidic one-dimensional SDS-PAGE gels that isoAsp levels are 5- to 10-fold higher in KO brains. Similar, but less dramatic enhancement is seen in other tissues such as heart, skeletal muscle, liver, testis, and erythrocytes. Unfortunately, the limited spatial resolution of the labeled bands seen after acidic one-dimensional SDS-PAGE of KO versus WT mouse brain extracts has facilitated the discovery of only one new endogenous substrate for PIMT, namely histone H2B (14).

To employ a high resolution two-dimensional separation of isoaspartyl proteins without risking the loss of preformed [3H]methyl esters, we separated unlabeled brain extract proteins by the classic two-dimensional method of O'Farrell (36) prior to blotting the proteins onto PVDF membranes. The isoaspartyl proteins were then 3H-methylated on-blot using recombinant PIMT. To maximize preservation of the methyl esters, we employed a pH 6.2 methylation buffer and a pH 6.2 post-methylation membrane wash procedure. The membrane was then sprayed with the organic solvent-based EN3HANCETM scintillant and dried prior to fluorography. The on-membrane methylation approach was inspired by Lee and Bedford (39) who demonstrated successful on-membrane methylation of a commercial protein expression array to discover new substrates for protein arginine methyltransferases I and IV. Arginine methylation is a stable modification, so it was initially unclear if adoption of this approach would also work for detection of isoAsp methyl esters.

The pattern of labeled proteins we observed in KO mouse brain extracts allowed us to locate the corresponding Coomassie-stained proteins on a wet gel. The spots were excised and subjected to peptide-mass fingerprinting using MALDI-TOF. We were able to identify 22 of the major methyl acceptors as indicated in Fig. 4B and Table 1. Relevant properties of all these proteins are summarized in Table 2. (Closely related proteins, e.g. {alpha} and beta tubulin, appear as single entries, so the 22 entries in Table 1 appear as 19 proteins in Table 2.) Of these 19 proteins, 9 are expressed exclusively or predominantly in the brain. The 19 proteins represent a range of specific cellular functions, including components of the cytoskeleton, proteins associated with synaptic vesicles, and enzymes involved in energy metabolism, nitrogen metabolism, pH balance, and protein folding. Three of the 19 proteins (glutamine synthetase, CA II, and Hsc71) can be considered as having a protective role in cells by virtue of their respective abilities to prevent accumulation of ammonia, to correct pH imbalance, and to assist in the proper folding or refolding of proteins.


View this table:
[in this window]
[in a new window]
  		TABLE 2
Distribution, functions, and other relevant properties of endogenous PIMT substrates

 
As indicated in Fig. 4, the identified PIMT substrates vary widely in their apparent stoichiometry of isoAsp content. This information is also included in Table 2 in the column labeled "M/P," a subjective estimate of the ratio of [3H]methyl incorporation (Fig. 4B) to protein stain intensity (Fig. 4A). We assume that proteins with the greatest structural damage (isoAsp content) are also the most likely to have some functional deficit that would contribute to the neurological abnormalities in the KO mouse. 5 of the 19 proteins in Table 2 rank in the highest category (+++) for isoAsp content: CRMP2, dynamin I, synapsins Ia and Ib, synapsins IIa and IIb, and fructose bisphosphate aldolase A. It is interesting, and perhaps highly relevant, that the first four of these proteins are found mainly or exclusively in neurons. Tubulin, a ubiquitous protein highly abundant in neurons, is also of special interest because it appears to be the single largest contributor to isoaspartate in the PIMT-KO mouse brain.

CRMP2—CRMP2 is one of five proteins in the collapsin-response mediator family, all of which are highly expressed in neurons during brain development (45). CRMP2 is also well expressed in adult neurons (more so than the other family members), especially in areas that retain a high degree of plasticity, such as the hippocampus, olfactory bulb, and cerebellum. CRMP2 binds to {alpha},beta-tubulin heterodimers to promote their polymerization into microtubules (46). CRMP2 appears to mediate the collapse of neuronal growth cones when they encounter semaphorin-3, an extracellular protein that inhibits neurite extension (47, 48) and has also been implicated in regulation of axonal transport (49), cell surface receptor internalization (50), and apoptosis (51). Because CRMP2 plays a key role in neuronal development, it is easy to imagine that CRMP2 damage could be a major factor in the neuronphysiological and behavioral abnormalities characteristic of PIMT-KO mice. A defect in the putative role of CRMP2 in mediating neuronal apoptosis might also help to explain the enlargement of the PIMT-KO mouse brain.

In Fig. 4 (C-E), we see three charge variants of CRMP2. Interestingly, the acidic forms appear to be more highly methylated than the most basic form (Fig. 4D). One possible explanation is that the acidic forms arise from deamidation of labile asparagine sites as diagrammed in Fig. 1. As such, it is expected that these forms would be highly enriched in isoAsp sites. Methylation of the predominant basic form (based on protein staining, Fig. 4C) would reflect generation of isoAsp by isomerization of one or more aspartyl sites, a process that would not entail a significant increase in negative charge. An alternative explanation is that the more acidic forms represent the mono- and di-phosphorylated forms of CRMP2. The CRMP2 protein is regulated in vivo by sequential phosphorylation (52). The Cdk5 kinase adds the first phosphate in what is considered to be a priming reaction, which then permits a second phosphorylation catalyzed by GSK3beta. This latter explanation raises the interesting possibility that phosphorylation might render CRMP2 more susceptible to isoaspartate formation, or vice versa. Hyperphosphorylation of related signaling proteins in PIMT-KO mice have recently been reported by two laboratories. Doyle et al. (31) observed hyperphosphorylation of several proteins, including GSK3{alpha}, in the T-cell receptor signaling pathway of activated T lymphocytes. In brain extracts, Farrar et al. (26) observed hyperphosphorylation of several proteins, including GSK3beta, in the phosphatidylinositol 3-kinase/Akt signal transduction pathway.

Dynamin-1—Dynamin-1 is a large GTPase, concentrated in presynaptic terminals of neurons, that is believed to function in the "pinching-off" phase of synaptic vesicle production and in the recovery of spent vesicles after membrane fusion (53). The Drosophila phenotype known as shibire has a temperature-sensitive mutation in dynamin that causes paralysis at the non-permissive temperature. Because of its essential role in synaptic transmission, disruption of dynamin function stemming from isoaspartate accumulation might well contribute to the abnormal behavior and neurophysiology of the PIMT-KO mouse. Like CRMP2, dynamin-1 is regulated in vivo by phosphorylation at two distinct sites (54); however, we do not see any clear evidence for multiple charged forms of dynamin in Fig. 4.

Synapsins I and II—The synapsins are a family of neuronspecific phosphoproteins that bind to the cytoplasmic surface of small synaptic vesicles (55). The synapsins anchor vesicles to the cytoskeleton and regulate their availability and "ready" state. There are three synapsin genes (I, II, and III), each of which express two major splice variants termed a and b forms. Synapsins I and II are abundant in adult synapses and are differentially expressed in subsets of neurons. Synapsin III is expressed most highly in the developing nervous system where it is concentrated in growth cones (56). Synapsin I and II single or double KO mice have a normal lifespan indicating that neither of them are essential for a life-sustaining level of brain function in a laboratory environment (57). Of particular interest to us is the finding that the synapsin I/II double KO mice exhibit an increased susceptibility to epileptic seizures and have significant abnormalities in synaptic physiology. The susceptibility to seizures is strongly dependent on the total number of mutant alleles in the synapsin I and II genes.

We recently reported that synapsin I purified from PIMT-KO mice contains near stoichiometric levels of isoaspartate (20). The present study confirms this finding and shows further that synapsin II is also a major endogenous substrate for PIMT. The methylation patterns (Fig. 4D) for synapsins IIa and IIb indicate that isoaspartate is generated on multiple charge forms with a bias toward higher isoAsp levels in the more acidic forms. At least five different protein kinases have been implicated in the regulation of synapsin function. As with CRMP2, the heterogeneity of charge and methylation levels seen in synapsins I and II most likely reflect a combination of phosphorylation and deamidation states. Because of their important roles in regulating synaptic transmission, isoaspartate-related damage to the synapsins may contribute significantly to the abnormal behavior and neurophysiology of the PIMT-KO mouse.

Tubulin—The most intense fluorographic spot in Fig. 4B is tubulin. Tubulin was first identified as an endogenous substrate for PIMT in normal rat brain by Ohta et al. (23) who labeled rat brain slices with [methyl-3H]L-methionine. Because there was no attempt to reduce PIMT activity in these slices, the methylation they observed presumably reflects a low, steady-state level of methyl esters that characterize the balance between isoAsp formation and repair (Fig. 1). Najbauer et al. (24) treated rat PC12 cells with a methylation inhibitor and then analyzed the taxol-dependent polymerized tubulin fraction for isoAsp content. A significant increase in isoaspartyl tubulin was found in extracts of inhibitor-treated cells compared with control cells, indicating that tubulin does indeed serve as an in vivo substrate for PIMT-dependent repair. Lanthier et al. (59) found that PIMT activity was decreased by 50% in surgical samples of the hippocampus from patients with mesial temporal lobe epilepsy compared with surgical and post-mortem samples from hippocampi of patients without epilepsy. Interestingly, isoaspartate levels in beta-tubulin from the low PIMT epilepsy samples was approximately twice that found in the control samples. Yamamoto et al. (16) observed disorganized microtubules on histological examination of the cerebral cortex of the PIMT-KO mouse.

Although tubulin generates the most intense labeling in our two-dimensional fluorograms, tubulin is also one of the three most abundant proteins seen in Fig. 4A, leading us to rank it as "intermediate" with regard to its apparent isoAsp level. Tubulin is well known as one of the major components of the cytoskeleton in cells, and plays a key role in dynamic processes such as chromosome movement in cell division and in axonal transport. It is easy to imagine that isoaspartate-related damage to a subpopulation of tubulin within the neuron could interfere with neuronal development or synaptic transmission, helping to explain the phenotype of the PIMT-KO mouse. The appearance of tubulin in Fig. 4 (A and B) contrasts with that of actin, another abundant cytoskeletal protein whose overall level of isoAsp accumulation appears to be much lower.

Isoaspartate and Oxidation—Protein oxidation and isoaspartate formation are leading sources of spontaneous protein damage both in vitro and in vivo, and both processes have been linked to age-related neurodegeneration and Alzheimer's disease. Ingrosso et al. (60) observed increased isoaspartate formation in human erythrocyte proteins in response to oxidative stress. We wondered if there is any overlap between the set of brain proteins that are prone to isoaspartate formation and those that are prone to oxidation in vivo. A search of the recent literature revealed three proteomic studies of oxidized proteins in Alzheimer brains and age-matched controls (61-63). A total of 16 oxidized proteins were identified by these studies, and 7 of those 16 were found in our present study as indicated in the OX column of Table 2. One of the four high-isoAsp proteins we found, CRMP2, was identified by both laboratories involved in the oxidation studies as being significantly oxidized in aged human brain. As noted above, CRMP2 is believed to play an important role in neurite regrowth in the adult brain. It would be interesting to know if the isoaspartate content of CRMP2 increases in response to oxidative stress of cultured neurons, or if CRMP2 is more highly oxidized in the PIMT-KO mouse than in the WT.

Isoaspartate and Autoimmunity—In autoimmune diseases, an organism's immune system mistakenly attacks it own biomolecules as if they were foreign antigens. Autoimmunity to proteins can be induced by a variety of post-translational modifications, including the substitution of an isoaspartyl site in place of a normal aspartyl site (64). Robust T-lymphocyte responses and antibody production have been observed when mice are injected with an isoaspartyl peptide related to mouse cytochrome c, but not with the normal aspartyl form of this same peptide. A similar response is seen with a peptide derived from the D protein component of the U1/Sm small ribonuclear protein complex, an autoantigen in human systemic lupus erythematosus, and in murine models of this disease (65). Autoantibodies to neuronal proteins with associated neurological or psychiatric disorders are found in a wide range of disease states, including primary autoimmune diseases, cancers, and microbial infections. MRL autoimmune mice, which serve as a model for human lupus erythematosus, have significantly higher levels of isoaspartyl proteins in their erythrocytes and brain tissue than do non-immune mice (66).

We wondered if there is any overlap between the set of brain proteins we found that are prone to isoaspartate formation in vivo and those that have been encountered as autoantigens in humans. A literature search revealed that 10 of the 19 entries in Table 2 have been encountered as autoantigens in various human pathological conditions. Among the high isoAsp proteins in Table 2, only synapsin I and aldolase A have been reported as autoantigens. Synapsin I antibody was found in a subset of patients with discoid lupus erythematosus, in patients with double-stranded DNA-positive system lupus erythematosus, and in the patients whose sera were positive for rheumatoid factor (67). It was not determined if these patients also had antibody to synapsin II. We found no reports of autoantibodies to dynamin 1 or CRMP2; however, Honnorat and coworkers (68) have reported antibodies to CRMP3 in patients with paraneoplastic neurological syndrome, a debilitating neurodegenerative autoimmune condition associated with certain types of malignancies, especially small cell lung cancer. These workers specifically tested the same sera for antibody to CRMP2 but found none. A search for antibodies to CRMP2, dynamin 1, and synapsin II might prove beneficial in patients with neurological and psychiatric syndromes that are suspected to have an autoimmune component.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In PIMT-KO mice, all tissues exhibit large increases in damaged, isoaspartyl proteins, but only the brain shows an overt manifestation of the lost repair activity. One possible explanation is that there are a limited number of neuronal proteins whose damage cannot be fully abrogated by protein turnover. The location of presynaptic proteins, for example, might be a factor given their distance from the soma where most protein synthesis occurs. It also seems possible that certain neuronal proteins are subject to an unusual degree of molecular "wear and tear" in locations such as rapid firing synaptic terminals that harbor a high degree of molecular motion and energy utilization associated with neurotransmitter release, vesicle recycling, and generation of synaptic potentials. The proteomic study reported here has identified 19 proteins whose isoaspartate levels are significantly increased in the brains of PIMT-KO mice compared with PIMT-WT mice. Those that seem most likely to contribute to the phenotype of the PIMT-KO mouse include CRMP2, dynamin-1, synapsin I, synapsin II, and tubulin, proteins that are rich in neurons, that play an important role in neuronal development and/or synaptic transmission, and that have high relative or absolute levels of isoaspartyl sites. We observed an additional nine PIMT substrates that have not yet been identified. Several of these appear to be in the high isoAsp category and therefore merit further attempts at identification. Additional study of PIMT and isoaspartate-prone proteins in the brain may lead to progress in understanding the molecular basis of one or more developmental and/or age-related neurological diseases.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant (NIH) NS17269 (to D. W. A.), NIH Grants AI36529 and AI48120 (to M. J. M.), and Arthritis Foundation (the Ethel F. Donaghue Foundation grant) and NIH postdoctoral fellowship F32-AR47759 (to H. A. D.). Pfizer La Jolla provided financial support and work release time (to J. X. Z.). 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. Back

1 Present address: Biochemical Pharmacology, Pfizer La Jolla, 10628 Science Center Dr., San Diego, CA 92121. Back

2 To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, 3205 McGaugh Hall, University of California, Irvine, CA 92697-3900. Tel.: 949-824-6866; Fax: 949-824-8551; E-mail: dwaswad{at}uci.edu.

3 The abbreviations used are: isoAsp, isoaspartate/isoaspartyl; AdoMet, S-adenosyl-L-methionine; CRMP2, collapsin response mediator protein 2; KO, knock-out; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MES, 2-(N-morpholino)ethanesulfonic acid; NEPHGE, non-equilibrium pH gradient gel electrophoresis; PIMT, protein L-isoaspartate O-methyltransferase; PVDF, polyvinylidene difluoride; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Edward Kim and Stephen Young (Gladstone Institute of Cardiovascular Disease, San Francisco, CA) and Dr. Steven Clarke (University of California, Los Angeles) for providing PIMT+/- founder mice essential for the research reported here.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Volkin, D. B., Mach, H., and Middaugh, C. R. (1997) Mol. Biotechnol. 8, 105-122[Medline] [Order article via Infotrieve]
  2. Lindner, H., and Helliger, W. (2001) Exp. Gerontol. 36, 1551-1563[CrossRef][Medline] [Order article via Infotrieve]
  3. Clarke, S. (2003) Ageing Res. Rev. 2, 263-285[CrossRef][Medline] [Order article via Infotrieve]
  4. Reissner, K. J., and Aswad, D. W. (2003) Cell. Mol. Life Sci. 60, 1281-1295[CrossRef][Medline] [Order article via Infotrieve]
  5. Robinson, N. E., Robinson, Z. W., Robinson, B. R., Robinson, A. L., Robinson, J. A., Robinson, M. L., and Robinson, A. B. (2004) J. Pept. Res. 63, 426-436[CrossRef][Medline] [Order article via Infotrieve]
  6. Shimizu, T., Matsuoka, Y., and Shirasawa, T. (2005) Biol. Pharm. Bull. 28, 1590-1596[CrossRef][Medline] [Order article via Infotrieve]
  7. Axelrod, J., and Daly, J. (1965) Science 150, 892-893[Abstract/Free Full Text]
  8. Morin, A. M., and Liss, M. (1973) Biochem. Biophys. Res. Commun. 52, 373-378[CrossRef][Medline] [Order article via Infotrieve]
  9. Kim, S. (1973) Arch. Biochem. Biophys. 157, 476-484[CrossRef][Medline] [Order article via Infotrieve]
  10. Diliberto, E. J., Jr., and Axelrod, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1701-1704[Abstract/Free Full Text]
  11. Aswad, D. W. (1984) J. Biol. Chem. 259, 10714-10721[Abstract/Free Full Text]
  12. Murray, E. D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732[Abstract/Free Full Text]
  13. Johnson, B. A., Najbauer, J., and Aswad, D. W. (1993) J. Biol. Chem. 268, 6174-6181[Abstract/Free Full Text]
  14. Young, A. L., Carter, W. G., Doyle, H. A., Mamula, M. J., and Aswad, D. W. (2001) J. Biol. Chem. 276, 37161-37165[Abstract/Free Full Text]
  15. Kim, E., Lowenson, J. D., MacLaren, D. C., Clarke, S., and Young, S. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6132-6137[Abstract/Free Full Text]
  16. Yamamoto, A., Takagi, H., Kitamura, D., Tatsuoka, H., Nakano, H., Kawano, H., Kuroyanagi, H., Yahagi, Y., Kobayashi, S., Koizumi, K., Sakai, T., Saito, K., Chiba, T., Kawamura, K., Suzuki, K., Watanabe, T., Mori, H., and Shirasawa, T. (1998) J. Neurosci. 18, 2063-2074[Abstract/Free Full Text]
  17. Johnson, B. A., Murray, E. D., Jr., Clarke, S., Glass, D. B., and Aswad, D. W. (1987) J. Biol. Chem. 262, 5622-5629[Abstract/Free Full Text]
  18. McFadden, P. N., and Clarke, S. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2595-2599[Abstract/Free Full Text]
  19. Johnson, B. A., Langmack, E. L., and Aswad, D. W. (1987) J. Biol. Chem. 262, 12283-12287[Abstract/Free Full Text]
  20. Reissner, K. J., Paranandi, M. V., Luc, T. M., Doyle, H. A., Mamula, M. J., Lowenson, J. D., and Aswad, D. W. (2006) J. Biol. Chem. 281, 8389-8398[Abstract/Free Full Text]
  21. Young, G. W., Hoofring, S. A., Mamula, M. J., Doyle, H. A., Bunick, G. J., Hu, Y., and Aswad, D. W. (2005) J. Biol. Chem. 280, 26094-26098[Abstract/Free Full Text]
  22. Ota, I. M., and Clarke, S. (1990) Arch. Biochem. Biophys. 279, 320-327[CrossRef][Medline] [Order article via Infotrieve]
  23. Ohta, K., Seo, N., Yoshida, T., Hiraga, K., and Tuboi, S. (1987) Biochimie (Paris) 69, 1227-1234
  24. Najbauer, J., Orpiszewski, J., and Aswad, D. W. (1996) Biochemistry 35, 5183-5190[CrossRef][Medline] [Order article via Infotrieve]
  25. Shimizu, T., Ikegami, T., Ogawara, M., Suzuki, Y.-i., Takahashi, M., Morio, H., and Shirasawa, T. (2002) J. Neurosci. Res. 69, 341-352[CrossRef][Medline] [Order article via Infotrieve]
  26. Farrar, C., Houser, C. R., and Clarke, S. (2005) Aging Cell 4, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  27. Ikegaya, Y., Yamada, M., Fukuda, T., Kuroyanagi, H., Shirasawa, T., and Nishiyama, N. (2001) Hippocampus 11, 287-298[CrossRef][Medline] [Order article via Infotrieve]
  28. Farrar, C. E., Huang, C. S., Clarke, S. G., and Houser, C. R. (2005) J. Comp. Neurol. 493, 524-537[CrossRef][Medline] [Order article via Infotrieve]
  29. Vitali, R., and Clarke, S. (2004) Behav. Brain Res. 153, 129-141[CrossRef][Medline] [Order article via Infotrieve]
  30. Kim, E., Lowenson, J. D., Clarke, S., and Young, S. G. (1999) J. Biol. Chem. 274, 20671-20678[Abstract/Free Full Text]
  31. Doyle, H. A., Gee, R. J., and Mamula, M. J. (2003) J. Immunol. 171, 2840-2847[Abstract/Free Full Text]
  32. Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726[Medline] [Order article via Infotrieve]
  33. Laemmli, U. K., and Favre, M. (1973) J. Mol. Biol. 80, 575-599[CrossRef][Medline] [Order article via Infotrieve]
  34. Chamberlain, J. P. (1979) Anal. Biochem. 98, 132-135[CrossRef][Medline] [Order article via Infotrieve]
  35. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56, 335-341[Medline] [Order article via Infotrieve]
  36. O'Farrell, P. Z., Goodman, H. M., and O'Farrell, P. H. (1977) Cell 12, 1133-1142[CrossRef][Medline] [Order article via Infotrieve]
  37. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  38. Johnson, B. A., and Aswad, D. W. (1993) Neurochem. Res. 18, 87-94[CrossRef][Medline] [Order article via Infotrieve]
  39. Lee, J., and Bedford, M. T. (2002) EMBO Rep. 3, 268-273[CrossRef][Medline] [Order article via Infotrieve]
  40. O'Connor, C. M., and Clarke, S. (1985) Anal. Biochem. 148, 79-86[CrossRef][Medline] [Order article via Infotrieve]
  41. Lowenson, J. D., Kim, E., Young, S. G., and Clarke, S. (2001) J. Biol. Chem. 276, 20695-20702[Abstract/Free Full Text]
  42. Johnson, B. A., and Aswad, D. W. (1985) Biochemistry 24, 2581-2586[CrossRef][Medline] [Order article via Infotrieve]
  43. Barber, J. R., and Clarke, S. (1985) Biochemistry 24, 4867-4871[CrossRef][Medline] [Order article via Infotrieve]
  44. David, C. L., Orpiszewski, J., Zhu, X. C., Reissner, K. J., and Aswad, D. W. (1998) J. Biol. Chem. 273, 32063-32070[Abstract/Free Full Text]
  45. Charrier, E., Reibel, S., Rogemond, V., Aguera, M., Thomasset, N., and Honnorat, J. (2003) Mol. Neurobiol. 28, 51-64[CrossRef][Medline] [Order article via Infotrieve]
  46. Fukata, Y., Itoh, T. J., Kimura, T., Menager, C., Nishimura, T., Shiromizu, T., Watanabe, H., Inagaki, N., Iwamatsu, A., Hotani, H., and Kaibuchi, K. (2002) Nat. Cell Biol. 4, 583-591[Medline] [Order article via Infotrieve]
  47. Goshima, Y., Nakamura, F., Strittmatter, P., and Strittmatter, S. M. (1995) Nature 376, 509-514[CrossRef][Medline] [Order article via Infotrieve]
  48. Brown, M., Jacobs, T., Eickholt, B., Ferrari, G., Teo, M., Monfries, C., Qi, R. Z., Leung, T., Lim, L., and Hall, C. (2004) J. Neurosci. 24, 8994-9004[Abstract/Free Full Text]
  49. Kimura, T., Watanabe, H., Iwamatsu, A., and Kaibuchi, K. (2005) J. Neurochem. 93, 1371-1382[CrossRef][Medline] [Order article via Infotrieve]
  50. Nishimura, T., Fukata, Y., Kato, K., Yamaguchi, T., Matsuura, Y., Kamiguchi, H., and Kaibuchi, K. (2003) Nat. Cell Biol. 5, 819-826[CrossRef][Medline] [Order article via Infotrieve]
  51. Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Barzilai, A. (1999) J. Neurochem. 73, 961-971[CrossRef][Medline] [Order article via Infotrieve]
  52. Cole, A. R., Causeret, F., Yadirgi, G., Hastie, C. J., McLauchlan, H., McManus, E. J., Hernandez, F., Eickholt, B. J., Nikolic, M., and Sutherland, C. (2006) J. Biol. Chem. 281, 16591-16598[Abstract/Free Full Text]
  53. Praefcke, G. J., and McMahon, H. T. (2004) Nat. Rev. Mol. Cell. Biol. 5, 133-147[CrossRef][Medline] [Order article via Infotrieve]
  54. Anggono, V., Smillie, K. J., Graham, M. E., Valova, V. A., Cousin, M. A., and Robinson, P. J. (2006) Nat. Neurosci. 9, 752-760[CrossRef][Medline] [Order article via Infotrieve]
  55. Hilfiker, S., Pieribone, V. A., Czernik, A. J., Kao, H. T., Augustine, G. J., and Greengard, P. (1999) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 354, 269-279[Abstract/Free Full Text]
  56. Ferreira, A., Kao, H. T., Feng, J., Rapoport, M., and Greengard, P. (2000) J. Neurosci. 20, 3736-3744[Abstract/Free Full Text]
  57. Rosahl, T. W., Spillane, D., Missler, M., Herz, J., Selig, D. K., Wolff, J. R., Hammer, R. E., Malenka, R. C., and Südhof, T. C. (1995) Nature 375, 488-493[CrossRef][Medline] [Order article via Infotrieve]
  58. Dale, R. C., Candler, P. M., Church, A. J., Wait, R., Pocock, J. M., and Giovannoni, G. (2006) J. Neuroimmunol. 172, 187-197[CrossRef][Medline] [Order article via Infotrieve]
  59. Lanthier, J., Bouthillier, A., Lapointe, M., Demeule, M., Beliveau, R., and Desrosiers, R. R. (2002) J. Neurochem. 83, 581-591[CrossRef][Medline] [Order article via Infotrieve]
  60. Ingrosso, D., D'Angelo, S., di Carlo, E., Perna, A. F., Zappia, V., and Galletti, P. (2000) Eur. J. Biochem. 267, 4397-4405[Medline] [Order article via Infotrieve]
  61. Castegna, A., Aksenov, M., Aksenova, M., Thongboonkerd, V., Klein, J. B., Pierce, W. M., Booze, R., Markesbery, W. R., and Butterfield, D. A. (2002) Free Radic. Biol. Med. 33, 562-571[CrossRef][Medline] [Order article via Infotrieve]
  62. Castegna, A., Aksenov, M., Thongboonkerd, V., Klein, J. B., Pierce, W. M., Booze, R., Markesbery, W. R., and Butterfield, D. A. (2002) J. Neurochem. 82, 1524-1532[CrossRef][Medline] [Order article via Infotrieve]
  63. Pamplona, R., Dalfo, E., Ayala, V., Bellmunt, M. J., Prat, J., Ferrer, I., and Portero-Otin, M. (2005) J. Biol. Chem. 280, 21522-21530[Abstract/Free Full Text]
  64. Doyle, H. A., and Mamula, M. J. (2005) Ann. N. Y. Acad. Sci. 1050, 1-9[CrossRef][Medline] [Order article via Infotrieve]
  65. Mamula, M. J., Gee, R. J., Elliott, J. I., Sette, A., Southwood, S., Jones, P. J., and Blier, P. R. (1999) J. Biol. Chem. 274, 22321-22327[Abstract/Free Full Text]
  66. Yang, M.-L., Doyle, H. A., Gee, R. J., Lowenson, J. D., Clarke, S., Lawson, B. R., Aswad, D. W., and Mamula, M. J. (2006) J. Immunol. 177, 4541-4549[Abstract/Free Full Text]
  67. Gitlits, V. M., Sentry, J. W., Matthew, M. L. S. M., Smith, A. I., and Toh, B. H. (2001) J. Investig. Med. 49, 276-283[Medline] [Order article via Infotrieve]
  68. Honnorat, J., Byk, T., Kusters, I., Aguera, M., Ricard, D., Rogemond, V., Quach, T., Aunis, D., Sobel, A., Mattei, M. G., Kolattukudy, P., Belin, M. F., and Antoine, J. C. (1999) Eur. J. Neurosci. 11, 4226-4232[CrossRef][Medline] [Order article via Infotrieve]
  69. Tanaka, Y., Nakamura, M., Matsui, T., Iizuka, N., Kondo, H., Tohma, S., Masuko, K., Yudoh, K., Nakamura, H., Nishioka, K., Koizuka, I., and Kato, T. (2006) Microbiol. Immunol. 50, 117-126[Medline] [Order article via Infotrieve]
  70. Chretien-Leprince, P., Ballot, E., Andre, C., Olsson, N. O., Fabien, N., Escande, A., Oksman, F., Dubuquoi, S., Jego, S., Goetz, J., Chevailler, A., Sanmarco, M., Humbel, R. L., and Johanet, C. (2005) Ann. N. Y. Acad. Sci. 1050, 266-273[CrossRef][Medline] [Order article via Infotrieve]
  71. Hattori, T., Takeuchi, M., Ohno, K., Ogawara, M., Asatani, T., Usui, Y., Muramatsu, R., Inagaki, M., Usui, M., and Taguchi, O. (2006) Invest. Ophthalmol. Vis. Sci. 47, 249-255[Abstract/Free Full Text]
  72. Rajasalu, T., Teesalu, K., Janmey, P. A., and Uibo, R. (2004) Immunol. Lett. 94, 153-160[CrossRef][Medline] [Order article via Infotrieve]
  73. Tontsch, D., Pankuweit, S., and Maisch, B. (2000) Clin. Exp. Immunol. 121, 270-274[CrossRef][Medline] [Order article via Infotrieve]
  74. Ochi, H., Horiuchi, I., Araki, N., Toda, T., Araki, T., Sato, K., Murai, H., Osoegawa, M., Yamada, T., Okamura, K., Ogino, T., Mizumoto, K., Yamashita, H., Saya, H., and Kira, J. (2002) FEBS Lett. 528, 197-202[CrossRef][Medline] [Order article via Infotrieve]
  75. Mor, F., Izak, M., and Cohen, I. R. (2005) J. Immunol. 175, 3439-3445[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Plant CellHome page
L. Oge, G. Bourdais, J. Bove, B. Collet, B. Godin, F. Granier, J.-P. Boutin, D. Job, M. Jullien, and P. Grappin
Protein Repair L-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor in Arabidopsis
PLANT CELL, November 1, 2008; 20(11): 3022 - 3037.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/44/33802    most recent
M606958200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, J. X.
Right arrow Articles by Aswad, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, J. X.
Right arrow Articles by Aswad, D. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement