AJP - Heart pressure measurements
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H2220-H2227, 2006. First published January 6, 2006; doi:10.1152/ajpheart.01293.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2220    most recent
01293.2005v1
Right arrow Alert me when this article is cited
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 Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (34)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, S.
Right arrow Articles by Cohen, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, S.
Right arrow Articles by Cohen, R. A.

Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging

Shanqin Xu,1 Jia Ying,1 Bingbing Jiang,1 Wei Guo,1 Takeshi Adachi,1 Viktor Sharov,4 Harold Lazar,2 James Menzoian,2 Tatyana V. Knyushko,3 Diana Bigelow,3 Christian Schöneich,4 and Richard A. Cohen1

1Vascular Biology Unit, Whitaker Cardiovascular Institute, Evans Department of Medicine and 2Department of Surgery, Boston University Medical Center, Boston, Massachusetts; 3Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington; and 4Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas

Submitted 8 December 2005 ; accepted in final form 2 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

nitrotyrosine; manganese superoxide dismutase; sarcoplasmic endoplasmic reticulum calcium adenosinetriphosphatase; antibody; immunohistochemistry

OXIDATIVE STRESS affects the function of cells in tissues affected by a variety of diseases. 3-Nitrotyrosine is a posttranslational oxidant modification of proteins, which has increased abundance in diseased tissues, offering a marker of oxidant stress (3). Tyrosine nitration does not appear to occur indiscriminately because screens for proteins nitrated in animal models of sepsis or diabetes detect on the order of 100 affected proteins (6, 10). Nitration can influence the function of proteins, and some proteins such as manganese superoxide dismutase (MnSOD; SOD2) (8) and prostacyclin synthase (9) that are nitrated in their active catalytic sites are inactivated by nitration of single tyrosine. Other proteins such as synuclein (4) and the sarcoplasmic reticulum calcium ATPase (SERCA) (1, 11) appear to be nitrated in diseased tissues on multiple tyrosines. Nitrotyrosine usually occurs on a fraction of the total tyrosine residues on any one protein, making it difficult to detect protein-specific nitration. Western blotting is often negative or shows nonspecific staining, but immunoprecipitation together with immunoblotting has permitted detection of nitration on specific proteins (1, 5). Studies of rats infused with ANG II for 7 days showed that MnSOD accounted for 20% of the total tyrosine-nitrated proteins in renal tissue and was associated with a 50% reduction of enzymatic activity. In addition, atherosclerotic rabbit and human aorta showed tyrosine nitration of SERCA that was associated with impaired vasodilator function and oxidative stress.

The purpose of this study was to develop and test polyclonal antibodies to specific peptides containing nitrotyrosine at MnSOD-Tyr34, shown to be the residue most susceptible to nitration by peroxynitrite (8), and a di-nY in SERCA Tyr-294,Tyr-295 identified in aging skeletal (11) and cardiac muscle (7). Tyrosine nitration of both of these proteins appears to be associated with protein dysfunction.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primary antibodies. Affinity-purified rabbit anti-MnSOD nY-34, and anti-SERCA2 di-nY-294,295 were provided by Bethyl Laboratories (Montgomery, TX). Antibodies were raised against the tyrosine-nitrated peptides: MnSOD, 25LHHSKHHAA(nY)VNNLNV40; SERCA, 281DPVHGGSWIRGAI(nY)(nY)FKIAV300. The peptides were chemically synthesized with the constituent amino acids including nitrotyrosine. Each antigen was injected into rabbits, and serum was obtained approximately every 2 wk over a 5-mo period. To remove antibodies to the nonnitrated sequences, antisera were processed over immunosorbents consisting of nonnitrated peptides immobilized on agarose. Subsequently, specific antibodies against nitrated peptides were column purified from the processed antisera by using immunosorbents consisting of the nitrated peptides immobilized on agarose. Yields of antibody ranged from 5 to 20 µg/ml. Polyclonal anti-nitrotyrosine antibody was obtained from Upstate Biotechnology (Lake Placid, NY). A polyclonal anti-SERCA antibody conjugated to Sepharose was obtained from Bethyl Laboratories. A polyclonal anti-MnSOD antibody was obtained from Upstate Biotechnology, and a monoclonal anti-SERCA2 antibody (clone IID8) was obtained from Affinity Bioreagents (Golden, CO).

Tissue preparation. Kidney from rats infused with ANG II (0.72 mg·kg–1·day–1) for 7 days and thoracic aorta from New Zealand rabbits fed a diet with 0.5% cholesterol and 4% peanut oil for 13 wk were obtained as described previously (1, 5). Samples of aging rat skeletal muscle and cardiac sarcoplasmic reticulum membrane fraction were obtained from 34-mo-old male Fischer 344 x Brown Norway F1 hybrid rats purchased from the National Institute on Aging, from colonies maintained at Harlan Sprague-Dawley (Indianapolis, IN). These have previously been shown by mass spectrometry studies to contain increased amounts of SERCA di-nY-294,295 (7, 11). The rats were allowed to adapt for 2 wk after arrival in a 12:12-h light-dark cycle and were provided with water and food ad libitum. The animals were anesthetized by CO2 and killed by decapitation, the heart was obtained, and hindlimb skeletal muscle was rapidly sliced into 5-mm-thick samples and fixed in 10 vol of 10% buffered formalin acetate (Fisher). Human aortic punch biopsies, discarded femoral artery segments, and cardiac atrial biopsies were obtained at the time of surgery from patients with atherosclerosis and diabetes mellitus. The tissues were fixed in 10% buffered formalin acetate and embedded in paraffin. Study of these specimens was approved by the Boston University Medical Center Institutional Review Board.

Immunohistochemistry. After removal of paraffin and rehydration, tissue sections (5 µm thick) were treated with 10 mmol/l citric acid (pH 6.0) and were microwave heated (2 min, 3 times at 700 W) to recover antigenicity. Nonspecific binding was blocked with 10% normal goat or horse serum in PBS (pH 7.4) for 30 min before incubation with individual primary antibodies, i.e., polyclonal anti-nitrotyrosine antibody (1 µg/ml) termed "pan-nY" here, polyclonal anti-MnSOD antibody (3 µg/ml), monoclonal anti-SERCA2 antibody (2.8 µg/ml), polyclonal anti-MnSOD nY-34 antibody (4 µg/ml), or polyclonal anti-SERCA2 di-nY-294,295 antibody (2 µg/ml), each in PBS with 1% BSA overnight at 4°C. Tissue sections were then incubated for 30 min at room temperature with a biotinylated anti-rabbit IgG secondary antibody (1:800 for nitrotyrosine or 1:200 for others) by using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Vector Red alkaline phosphatase substrate (Vector) was used to visualize positive immunoreactivity. Specificity of anti-pan-nY, anti-MnSOD nY-34, or anti-SERCA2 di-nY-294,295 antibodies was confirmed by treating sections for 40 min with sodium dithionite (100 mmol/l in 100 mmol/l sodium borate, pH 9.0) to reduce nitrotyrosine to aminotyrosine or by preincubating the antibodies with free MnSOD nY-34 peptide or SERCA2 di-nY-294,295 peptide (antibody:peptide, 1:5 by weight, respectively). Antibodies against nonnitrated MnSOD (polyclonal) or SERCA2 (monoclonal, clone IID8) were used for testing specificity of staining. Each tissue section was stained in parallel with an adjacent section of the same tissue stained by using a nonimmune IgG (Vector Laboratories, Burlingame, CA) as the primary antibody as a negative IgG isotype control. The development of staining with the immune IgG was stopped before any nonspecific staining occurred with the nonimmune IgG to ensure specificity.

Immunoblotting. A homogenate was prepared from an atherosclerotic human femoral artery obtained at the time of leg amputation necessitated by severe peripheral arterial atherosclerotic disease. Homogenization and immunoprecipitation of SERCA was performed with a polyclonal anti-SERCA antibody (Bethyl) conjugated to Sepharose beads as previously described (2) and after SDS-PAGE was detected with the anti-SERCA2 di-nY-294,295 antibody. To eliminate staining of both the immunoprecipitation antibody and human IgG by the secondary antibody, the SERCA2 di-nY antibody was conjugated to horseradish peroxidase, used at a concentration of 0.2 µg/ml and directly visualized with ECL solution (Amersham). Protein concentrations were estimated by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) by using bovine serum albumin as the standard. To convert nitrotyrosine to aminotyrosine, blots were incubated for 5 min with sodium dithionite (100 mmol/l in 100 mmol/l sodium borate, pH 9.0) and rinsed 2 x 10 min with 0.1 mmol/l PBS with 0.1% Tween-20 before immunoblotting.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Immunohistochemical staining for 3-nitrotyrosine.

Positive staining with polyclonal anti-nitrotyrosine (pan-nY) antibody was demonstrated in tubules within the renal inner medulla of an ANG II-infused rat (Fig. 1A), as well as in atherosclerotic rabbit aorta (Fig. 1C) as shown previously (1, 5). The staining was abolished if the antibody was preincubated with 3-nitrotyrosine (10 mmol/l, data not shown) (1, 5). In addition, staining throughout the kidney (Fig. 1B) and in the aorta (Fig. 1D) was largely prevented by pretreatment of the tissue sections with sodium dithionite (100 mmol/l, 40 min) to reduce nitrotyrosine to aminotyrosine.


Figure 1
View larger version (130K):
[in this window]
[in a new window]
  		Fig. 1. Positive immunohistochemical staining with polyclonal anti-nitrotyrosine (pan-nY) antibody in tubules within the renal inner medulla of an ANG II-infused rat (A) and in atherosclerotic rabbit aorta (C). The staining was diminished by pretreatment of the tissue sections with sodium dithionite in the kidney (B) and in the aorta (D).

 
MnSOD nY-34.

ANG II infusion for 7 days dramatically increased staining of the renal medullary tubular cells with the MnSOD nY-34 antibody (Fig. 2, A and B). Lesser increases were noted in the cortical distal tubules and glomeruli (Fig. 2, C and D). Treatment of the renal tissue sections with dithionite nearly eliminated staining by the MnSOD nY-34 antibody (Fig. 3, A and B). Dithionite treatment had no apparent effect on staining the sections with a polyclonal antibody against MnSOD (Fig. 3, C and D), indicating the specificity of dithionite treatment to eliminate nitrotyrosine. Staining of ANG II-infused rat kidneys with anti-MnSOD nY-34 antibody was also blocked by prior treatment of the antibody with the MnSOD nY-34 peptide but not with the SERCA2 di-nY-294,295 peptide, further demonstrating specificity of the antibody (Fig. 4A).


Figure 2
View larger version (143K):
[in this window]
[in a new window]
  		Fig. 2. ANG II infusion increases MnSOD nY-34 immunoreactivity in rat kidney. Intensive staining with anti-MnSOD nY-34 antibody was observed in distal tubule (DT), collecting duct (CD), and glomerular epithelial cells, podocytes, and mesangial cells from ANG II-infused rat. Medulla (A and B) and glomerulus (C and D) are shown from control (A and C) and ANG II-infused rat (B and D).

 

Figure 3
View larger version (148K):
[in this window]
[in a new window]
  		Fig. 3. Positive staining in renal medulla from ANG II-infused rat with anti-MnSOD nY-34 antibody, but not with antibody against nonnitrated MnSOD, was eliminated by sodium dithionite, indicating the specificity of anti-MnSOD nY-34 antibody for tyrosine-nitrated MnSOD.

 

Figure 4
View larger version (69K):
[in this window]
[in a new window]
  		Fig. 4. A: staining in renal medulla from ANG II-infused rat with anti-MnSOD nY-34 antibody was blocked by MnSOD nY-34 peptide but not by the nonnitrated MnSOD Y-34 peptide or sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2) di-nY peptide, indicating specific staining for MnSOD nY-34. A negative control with nonspecific IgG is also included. B: immunoblot with anti-MnSOD nY-34 antibody of (left to right) normal rat kidney homogenate (50 µg protein), the same sample spiked with 1 µg nitrated recombinant MnSOD, same spiked sample after the blotted membrane was treated with sodium dithionite (DT), ANG II-infused rat kidney homogenate (50 µg), and recombinant nitrated MnSOD (1 µg). Primary antibody was used at 0.5 µg/ml and secondary antibody at 0.005 µg/ml.

 
Immunoblotting of homogenates of normal or ANG II-infused rat kidney failed to detect any MnSOD nY-34 (Fig. 4B). The MnSOD nY-34 antibody did detect a 26-kDa protein, as well as several higher molecular weight species in recombinant MnSOD treated with peroxynitrite (50 µmol/l) (Fig. 4B, rightmost lane). The nitrated recombinant protein could also be detected when spiked into normal kidney homogenate (Fig. 4B, second lane from left), and the staining was prevented by dithionite.

SERCA2 di-nY-294,295.

Staining of the media and intimal lesion of atherosclerotic rabbit aorta with the SERCA2 di-nY-294,295 antibody (Fig. 5A) was similar to those in Fig. 1C and previously observed with the polyclonal anti-pan-nY antibody (1). Dithionite strongly decreased staining by the anti-SERCA2 di-nY-294,295 antibody throughout the aortic cross section (Fig. 5B). Dithionite did not affect staining of the aortic media by the monoclonal antibody for SERCA2 (Fig. 5, C and D). Staining of atherosclerotic rabbit aorta with anti-SERCA2 di-nY antibody was blocked by pretreating the SERCA2 di-nY antibody with the di-nY SERCA2 peptide but not with the MnSOD nY-34 peptide (Fig. 6A).


Figure 5
View larger version (132K):
[in this window]
[in a new window]
  		Fig. 5. Positive staining with anti-SERCA2 di-nY antibody of atherosclerotic rabbit aorta (A) was diminished by sodium dithionite (B). Staining with monoclonal (mono) antibody against nonnitrated SERCA showed a pattern similar to that with SERCA2 di-nY antibody, except the staining was less in the neointima (C). Staining with the monoclonal SERCA2 antibody was not diminished by pretreatment with dithionite (D), indicating the specificity of anti-SERCA2 di-nY antibody for tyrosine-nitrated SERCA. A, inset, shows lack of staining with a nonspecific IgG used at the same concentration.

 

Figure 6
View larger version (77K):
[in this window]
[in a new window]
  		Fig. 6. A: staining of atherosclerotic rabbit aorta with anti-SERCA2 di-nY antibody was blocked by the SERCA2 di-nY peptide but not by the MnSOD nY-34 peptide, indicating specific staining of SERCA2 di-nY. Staining with the SERCA2 di-nY antibody is greater in hypercholesterolemic than in normal rabbit aortic sections. B: Western blot of aging rat cardiac sarcoplasmic reticulum membrane protein (0.5 or 1.0 µg per lane) with SERCA2 di-nY antibody (0.1 µg/ml, secondary antibody 0.05 µg/ml) is attenuated by preincubation of the antibody with the di-nY peptide (nY-294,295 peptide; 0.5 µg/ml, ~30-fold molar excess, 1–2 h at room temperature) but not the nonnitrated SERCA2 di-Y peptide.

 
An immunoblot performed on sarcoplasmic reticulum obtained from aging rat cardiac muscle with the SERCA2 di-nY antibody showed a prominent 110-kDa band. The staining was decreased by pretreating the antibody with the SERCA2 di-nY-294,295 antigenic peptide but not the nonnitrated SERCA2 peptide (Fig. 6B).

Strong immunohistochemical staining for SERCA2 di-nY was observed in aging rat skeletal muscle, and the staining was decreased by prior incubation of the antibody with the di-nY-294,295, but not the nonnitrated peptide, as well as by treating the tissue section with dithionite (Fig. 7).


Figure 7
View larger version (108K):
[in this window]
[in a new window]
  		Fig. 7. Staining of aging rat (34 mo old) skeletal muscle with SERCA2 di-nY antibody is blocked by the di-nY peptide but not the nonnitrated di-Y peptide. It is also attenuated by dithionite. Staining of adjacent sections with a nonspecific IgG or the SERCA clone IID8 monoclonal antibody is shown for comparison.

 
Human diabetic atherosclerotic aorta biopsies stained positively with the anti-SERCA2 di-nY antibody in a pattern similar to that seen in the atherosclerotic rabbit aorta (Fig. 8A). Both plaque and smooth muscle beneath it were stained. Dithionite treatment eliminated staining (Fig. 8B). In addition, SERCA that was immunoprecipitated with Sepharose-conjugated anti-SERCA antibody from a homogenate of a segment of human atherosclerotic superficial femoral artery stained positively with anti-SERCA2 di-nY antibody (Fig. 8C), and detection by the antibody was prevented by treatment of the blot with dithionite.


Figure 8
View larger version (62K):
[in this window]
[in a new window]
  		Fig. 8. Positive staining for SERCA2 di-nY in biopsy of aortic wall from a diabetic patient with atherosclerotic coronary artery disease (A). Staining can be seen in both the plaque in the top portions and in the smooth muscle lamellae in the bottom portions of the image. Staining was prevented by sodium dithionite (B). C: immunoblot (IB) for SERCA2 di-nY of immunoprecipitate (IP) obtained with Sepharose-conjugated polyclonal anti-SERCA antibody from lysate of atherosclerotic diabetic human femoral artery, showing a 110-kDa band consistent with SERCA2 di-nY staining. The primary antibody, horseradish-peroxidase-conjugated anti-SERCA2 di-nY antibody, was used at a concentration of 0.2 µg/ml. The immunoblotting was attenuated by treating the membrane with sodium dithionite.

 
Biopsies of human diabetic atherosclerotic cardiac atrium stained positively both for MnSOD nY-34 and SERCA2 di-nY (Fig. 9). The staining was located in myocardial cells and was prevented by pretreatment of the tissue sections with dithionite.


Figure 9
View larger version (109K):
[in this window]
[in a new window]
  		Fig. 9. Positive staining for SERCA2 di-nY (A and B) and MnSOD nY-34 (C and D) in a biopsy of cardiac atrium of a diabetic patient with atherosclerotic coronary artery disease. Staining for both antigens is restricted to myocardium (A and C) and is largely prevented by treatment of adjacent sections with dithionite (B and D).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrates the feasibility of developing antibodies toward oxidant modifications of specific proteins, identifying not only the type of modification, in this case nitrotyrosine, but also the protein affected. Staining for such oxidant-modified proteins is widespread in tissues of pathological and aging animal models and humans with cardiovascular disease. The two antibodies introduced here were shown to specifically detect nitrotyrosine in MnSOD and SERCA in tissues in which the modification had previously been demonstrated by other methods. Tyrosine-nitrated MnSOD was demonstrated to increase in ANG II-infused rat kidney where it represented ~20% of the total nitrated proteins in the kidney and was implicated in decreased MnSOD activity (5). However, as in the present study, Western blotting of whole tissue homogenate failed to detect the nitrated protein unless the protein was first immunoprecipitated and then immunoblotted. Immunoprecipitation of SERCA from homogenates of atherosclerotic rabbit aorta was also required for detection of nitrotyrosine (1). The requirement for immunoprecipitation to reach the required sensitivity for detection of protein-specific nitrotyrosine may be explained by the fact that despite the high fraction of tyrosine nitration of some specific proteins like MnSOD or SERCA, the total amount of nitrotyrosine on any one protein is very low. Very often when nitrotyrosine is reported on the basis of whole tissue immunoblots, the staining has not been verified with the multiple controls used here. Another problem with SERCA in particular is that because it is prone to aggregation on heating, immunoblots are usually performed on unboiled protein, and immunoglobulin heavy chain dimers under these conditions migrate at the same apparent molecular weight. Special attention has to be paid to this possibility. Immunohistochemistry is far more sensitive than Western blotting for the detection of nitrotyrosine. The sites of tyrosine nitration to which the antibodies utilized in this study were directed were previously shown to be nitrated in the models studied. MnSOD nY-34 was shown to be the most sensitive to nitration by peroxynitrite, and the protein modification was demonstrated by mass spectrometry (8). Likely due to the fact that the tyrosine residue is within the active site of the enzyme and involved in its catalytic activity, nitration of this tyrosine residue is associated with inactivation of the enzyme. In the ANG II-infused rat kidney, MnSOD activity was decreased by 50% without a change in protein expression (5), consistent with Tyr-34 nitration demonstrated in this study. In addition, by using the sequence-specific MnSOD-nY antibody described here, an increase in staining was demonstrated in the kidneys of diabetic apolipoprotein E-deficient mice (12). The increase in staining correlated with a 30% decrease in MnSOD activity, further linking tyrosine nitration of MnSOD to its inactivation in vivo.

SERCA2 di-nY-294,295 was demonstrated by chemical methods and mass spectrometry to be present in aging skeletal and cardiac muscle sarcoplasmic reticulum (7, 11), and in unpublished studies we have identified by mass spectrometry the di-nY-294,295 in SERCA from atherosclerotic rabbit aorta (T. Adachi and J. Ying, unpublished data). Earlier studies showed that tyrosine nitration of SERCA is associated with decreased activity of the enzyme (1), although the specific functional effect of di-nY-294,295 is not yet known. This pair of tyrosine residues is located on the inner aspect of the sarcoplasmic reticulum membrane on one of the transmembrane loops that constitute the calcium pore of the enzyme, suggesting the potential functional importance of this nitrated pair of tyrosines. When tyrosine nitration was decreased in atherosclerotic rabbit aorta after treatment with an antioxidant, SERCA activity was restored, suggesting a relationship between tyrosine nitration and activity (1).

The specificity of the two antibodies used in this study was demonstrated by showing that staining was decreased by dithionite, which reduces nitrotyrosine to aminotyrosine. In addition, we considered whether an antibody raised to the tyrosine-nitrated peptides used here might be immunoreactive toward nitrotyrosine residues in general. However, this possibility was discounted by showing that immunogenicity of the antibody was blocked by the specific nitrated peptide but not the nitrated peptide of the other protein. Giasson et al. (4) took another approach in raising monoclonal antibodies directed toward synuclein that was tyrosine nitrated in vitro. Unlike the anti-peptide antibodies developed here, those antibodies showed reactivity toward several sites of tyrosine nitration on synuclein.

Use of antibodies directed toward site-specific protein oxidation uniquely compliments methods already developed. These include immunochemical, biochemical, and mass spectral techniques. For instance, gel electrophoresis with immunoblotting has the potential to identify proteins with nitrotyrosine modifications but not the specific sites of nitration. Mass spectral and chemical techniques are able to definitively identify sites of tyrosine nitration, although the sensitivities of the methods are, so far, less than immunochemical detection. In addition, there is an added advantage to immunohistochemical detection because the tissue required is minimal, as demonstrated in this study by the positive staining detected in diseased patient tissue biopsies.

The proteins to which the antibodies were developed here were chosen for their suspected relevance for function in tissues affected by oxidant stress. The ability to detect tyrosine nitration of specific proteins like MnSOD and SERCA in tissues will allow investigators to detect if these or other proteins are affected by oxidants in vivo. The advantage afforded by antibodies toward specific oxidant-modified sites provides the ability to infer a change in protein function that has been identified to be associated with the modification. This should add significantly to our knowledge of the effects of oxidant stress on tissues and cells in vivo.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The studies were supported by National Institutes of Health (NIH) Grants R01-HL-31607–21, R01-HL-55620–08, and R01-AG-27080–01, the NIH Boston University Cardiovascular Proteomics Center (N01-HV-28178), American Heart Association Grant-in-Aid 0455799T, and NIH Grant 2P01-AG-12993 (V. Sharov, C. Schöneich, and D. Bigelow).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Cohen, Vascular Biology Unit X708, Boston Univ. Medical Center, 650 Albany St., Boston, MA 02118 (e-mail: racohen{at}bumc.bu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adachi T, Matsui R, Xu S, Kirber M, Lazar HL, Sharov VS, Schoneich C, and Cohen RA. Antioxidant improves smooth muscle sarco/endoplasmic reticulum Ca2+-ATPase function and lowers tyrosine nitration in hypercholesterolemia and improves nitric oxide-induced relaxation. Circ Res 90: 1114–1121, 2002.[Abstract/Free Full Text]
  2. Adachi T, Weisbrod RM, Pimentel D, Ying J, Sharov VS, Schoneich C, and Cohen RA. S-glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide. A mechanism targeted by oxidants in vascular disease. Nat Med 10: 1200–1207, 2004.[CrossRef][Web of Science][Medline]
  3. Beckman JS and Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol Cell Physiol 271: C1424–C1437, 1996.[Abstract/Free Full Text]
  4. Giasson BI, Duda JE, Murray IVJ, Chen Q, Souza JM, Hurtig HI, Ischiropoulos H, Trojanowski JQ, and Lee Y. Oxidative damage linked to neurodegeneration by selective {alpha}-synuclein nitration in synucleinopathy lesions. Science 290: 985–989, 2000.[Abstract/Free Full Text]
  5. Guo W, Adachi T, Matsui R, Xu S, Jiang B, Zou MH, Kirber M, Lieberthal W, and Cohen RA. Quantitative assessment of tyrosine nitration of manganese superoxide dismutase in angiotensin II-infused rat kidney. Am J Physiol Heart Circ Physiol 285: H1396–H1403, 2003.[Abstract/Free Full Text]
  6. Kanski J, Behring A, Pelling J, and Schoneich C. Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effect of biological aging. Am J Physiol Heart Circ Physiol 288: H371–H381, 2005.[Abstract/Free Full Text]
  7. Knyushko TV, Sharov VS, Williams TD, Schoneich C, and Bigelow DJ. 3-Nitrotyrosine modification of SERCA2a in the aging heart: a distinct signature of the cellular redox environment. Biochemistry 44: 13071–13081, 2005.[CrossRef][Medline]
  8. MacMillan-Crow LA, Crow JP, and Thompson JA. Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues. Biochemistry 37: 1613–1622, 1998.[CrossRef][Medline]
  9. Schmidt P, Youhnovski N, Daiber A, Balan A, Arsic M, Bachschmid M, Przybylski M, and Ullrich V. Specific nitration at tyrosine-430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase. J Biol Chem 278: 12813–12819, 2003.[Abstract/Free Full Text]
  10. Turko IV, Li L, Aulak KS, Stuehr DJ, Chang JY, and Murad F. Protein tyrosine nitration in the mitochondria from diabetic mouse heart. Implications to dysfunctional mitochondria in diabetes. J Biol Chem 278: 33972–33977, 2003.[Abstract/Free Full Text]
  11. Viner RI, Ferrington DA, Williams TD, Bigelow DJ, and Schoneich C. Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle. Biochem J 340: 657–669, 1999.
  12. Xu S, Jiang B, Maitland-Toolan KA, Bayat H, Gu J, Nadler JL, Corda S, Lavielle G, Verbeuren TJ, Zuccollo A, and Cohen RA. The thromboxane receptor antagonist, S18886, attenuates renal oxidant stress and proteinuria in diabetic apolipoprotein E-deficient mice. Diabetes 55: 110–119, 2006.



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
P. Wenzel, S. Schuhmacher, J. Kienhofer, J. Muller, M. Hortmann, M. Oelze, E. Schulz, N. Treiber, T. Kawamoto, K. Scharffetter-Kochanek, et al.
Manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction
Cardiovasc Res, November 1, 2008; 80(2): 280 - 289.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. S. Reifenberger, K. L. Arnett, C. Gatto, and M. A. Milanick
The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity
Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1191 - F1198.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
V. Randriamboavonjy, F. Pistrosch, B. Bolck, R. H.G. Schwinger, M. Dixit, K. Badenhoop, R. A. Cohen, R. Busse, and I. Fleming
Platelet Sarcoplasmic Endoplasmic Reticulum Ca2+-ATPase and {micro}-Calpain Activity Are Altered in Type 2 Diabetes Mellitus and Restored by Rosiglitazone
Circulation, January 1, 2008; 117(1): 52 - 60.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
J. An, Z. J. Bosnjak, and M. T. Jiang
Myocardial Protection by Isoflurane Preconditioning Preserves Ca2+ Cycling Proteins Independent of Sarcolemmal and Mitochondrial KATP Channels
Anesth. Analg., November 1, 2007; 105(5): 1207 - 1213.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
G. Peluffo and R. Radi
Biochemistry of protein tyrosine nitration in cardiovascular pathology
Cardiovasc Res, July 15, 2007; 75(2): 291 - 302.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
P. Pacher, J. S. Beckman, and L. Liaudet
Nitric Oxide and Peroxynitrite in Health and Disease
Physiol Rev, January 1, 2007; 87(1): 315 - 424.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Viappiani and R. Schulz
Detection of specific nitrotyrosine-modified proteins as a marker of oxidative stress in cardiovascular disease
Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2167 - H2168.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2220    most recent
01293.2005v1
Right arrow Alert me when this article is cited
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 Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (34)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, S.
Right arrow Articles by Cohen, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, S.
Right arrow Articles by Cohen, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.