4. Results
4.8. Protein nitrotyrosine in chondrocytes
4.8.3. Detection of protein-nitrotyrosine in one- and two-dimensional gel
As already shown nitrotyrosine was present in both cartilage samples and isolated chondrocytes, therefore further analysis of protein nitration was performed employing electrophoresis and Western blotting.
One dimensional electrophoresis and Western blotting revealed presence of nitrotyrosine in studied samples (Figure 75). Especially samples from chondrocytes stimulated with IL-1β were nitrotyrosine positive. There was one protein band with ca.
40kDa, which was repetitively nitrotyrosine positive in IL-1 stimulated cells. However the staining of other proteins was not intensive and clear. Because of low abundancy of nitrated proteins signal detection was possible only with SuperSignal West Femto chemiluminescent substrate and long exposure times, therefore the standard band with nitrated BSA is very intensive.
6 – control
7 – IL-1β 1nM, 24h 8– control
9 - IL-1β 1nM, 24h 1 – protein standard : nitro-BSA
2 - control
3 – IL-1β 1nM, 24h 4 – control
5 - IL-1β 1nM, 24h 250
98
50
16 148
22 36 64
kDa
1 2 3 4 5 6 7 8 9
Figure 75. Western blot of chondrocyte protein lysates immunostained with anti-nitrotyrosine antibody (Cayman 10µg/ml).
To further investigate protein nitration in human chondrocytes two-dimensional gels were used. Chondrocytes used for experiments were isolated from cartilage, cultured in monolayer for three days and then for further 24h under experimental conditions.
Western blotting and incubation with anti-nitrotyrosine antibody was used for detection of nitrated proteins. Two distinct anti-nitrotyrosine antibodies were used: polyclonal antibody from Chemicon and monoclonal antibody clone 1A6 from Upstate. In experiments where samples needed to be compared, the gels were blotted on the same membrane and exposed simultaneously.
Proteins detected with antibodies in Western blot as nitrated were matched with Coomassie blue stained protein spots in parental acrylamide gels. Corresponding spots were then excised from the gels, digested and identified using MALDI-TOF mass spectrometry.
At the beginning of 2-D gel analysis we used anti-nitrotyrosine polyclonal antibody from Chemicon, but the signal obtained with this antibody was week. However we were able to identify cellular filament proteins: actin and vimentin as nitrated (Figure 76). This staining was also confirmed by the anti-nitrotyrosine monoclonal antibody from Cayman (data not shown).
kDa 97,4
45,0
31,0 21,5 vimentin actin
Figure 76. Anti-nitrotyrosine positive proteins in human chondrocytes stimulated with IL-1β 1nM, 24h detected with polyclonal antibody from Chemicon.
Arrows indicate immunopositive spots, which were identified by mass spectrometry as vimentin and actin.
144µg protein
pH 3 - 6
Further analysis was performed using monoclonal 1A6 antibody, which meanwhile had been well characterised (Aulak et al., 2001; Koeck et al., 2004; Miyagi et al., 2002) and revealed clear nitrotyrosine staining in our experimental settings (Figure 77). The excision and analysis of immunopositive protein spots was performed. Via this analysis pyruvate kinase M1/M2 isoenzymes, fructose-bisphosphate aldolase A, glyceraldehydes - 3-phosphate dehydrogenase, MnSOD and proteasome subunit β1 were identified as nitrated.
a) b)
1 - pyruvate kinase M1/M2 isoenzymes 2 - fructose-bisphosphate aldolase A 3 – glyceraldehydes - 3-phosphate dehydrogenase
4- MnSOD and proteasome subunit β1 SIN-1
Figure 77. a) anti-nitrotyrosine positive proteins in human chondrocytes detected with
monoclonal antibody 1A6 (Upstate), b) the 2D acrylamide gel stained with Coomassie blue of IL-1β stimulated chondrocytes.
Spots corresponding with
immunopositive proteins (indicated by arrows) were excised from the parent gel, digested and analysed by mass spectroscopy,
pH 3 - 10
Because most of nitrotyrosine-positive spots were detected in the pH range 6 - 7,5 (Figure 77) gels with pH range 6,2 – 7,5 were used for further analysis, to better separate proteins located in this area.
Use of gels with pH range 6,2 – 7,5 allowed identification of further nitrated proteins:
annexin A2, alpha enolase, and confirmed nitration of MnSOD (Figure 78).
a) b)
control
7
6 5
IL-1β
SIN-1
MS identification:
Figure 78. a) anti-nitrotyrosine positive proteins in human chondrocytes detected with
monoclonal antibody 1A6 (Upstate), b) the 2D acrylamide gel of IL-1β
stimulated chondrocytes stained with Coomassie blue.
Spots corresponding with
immunopositive proteins (indicated by arrows) were excised from the parent gel, digested and analysed by mass spectroscopy,
5 - alpha enolase 6 – MnSOD 7 – annexin A2
pH 6,2 – 7,5
4.8.3.1.2D-gel analyses revealed no COX-2 nitration
To investigate possible COX-2 protein nitration in human chondrocytes two-dimensional gels were used. Chondrocytes used for experiments were isolated from cartilage, cultured in monolayer for three days and then for further 24h with IL-1β 0,5nM or SIN-1 250µM.
For comparison (control, IL-1, SIN-1) the gels were blotted on the same membrane and exposed simultaneously. Western blots were first incubated with monoclonal anti-nitrotyrosine antibody (1A6, Upstate) for detection of nitrated proteins; afterwards the membrane was stripped and stained with polyclonal COX-2 antibody. Several nitrotyrosine positive spots were detected in all examined samples. A COX-2 expression was detected only in IL-1β stimulated cells. There was no correlation between anti-nitrotyrosine and COX-2 staining.
These data indicate that at least in our experimental setting COX-2 is not nitrated.
Figure 79. Anti-nitrotyrosine staining detected with monoclonal antibody 1A6 (Upstate) on the left and COX-2
staining on the right for comparison.
One representative experiment of three replicates is presented.
Anti-nitrotyrosine positive spots corresponding with COX-2 spot were not detected.
To sum up, two-dimensional gels showed that nitrated proteins are present in human chondrocytes. Nitrotyrosine was detected in unstimulated chondrocytes and protein nitration was slightly increased after stimulation with IL-1β or incubation with peroxynitrite generating compound: SIN-1. With further mass-spectrometric analysis several proteins were identified as nitrated: MnSOD, a principal antioxidant enzyme of mitochondria, which converts superoxide radical to hydrogen peroxide; annexin A2, a member of a large family of annexins characterized by their ability to bind to phospholipids in a calcium dependent manner (Liemann and Lewit-Bentley, 1995; Moss et al., 1991). Interestingly alpha enolase, pyruvate kinase M1/M2 isoenzymes, fructose-bisphosphate aldolase A, glyceraldehydes- 3-phosphate dehydrogenase found to be nitrated are all enzymes of glycolytic pathway. To our knowledge this is the first report identifying nitrated proteins in human chondrocytes.
4.9. Superoxide
We were able to show the presence of nitrotyrosine and to identify some nitrated proteins in chondrocytes originating from OA tissue. 3-nitrotyrosine is the stable product of tyrosine residue nitration by peroxynitrite and therefore used as marker for reactions involving peroxynitrite, which has a very short half-life.
Peroxynitrite is a product of a very rapid reaction of NO with superoxide, so nitrotyrosine is indirectly an indicator of oxidative stress in the tissue.
As production of high levels of nitric oxide by chondrocytes had been shown we were interested in the rate of superoxide generation.
Superoxide anion was determined by two methods: spectrophotometric measurement of cytochrome c reduction and by electron spin resonance (ESR). Several spin traps were tested for ESR measurements on chondrocytes: CPH, DMPO, DEPMO and CMH.
Finally we decided to use CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine hydrochloride) as a high-cell permeable and non-toxic spin probe, adequate for the quantification of slow released intracellular superoxide anion production. Oxidation of CMH by reactive oxygen species (ROS) leads to the formation of 3-methoxy-carbonyl-proxyl (CM•), which is a stable radical and can be detected by ESR spectroscopy. Each ESR measurement contained one probe in which cells were treated with polyethylene glycol-superoxide dismutase (PEGSOD) to quantify SOD-inhibitable formation of CM•. This radical formation inhibited by SOD was considered as superoxide generation. Dieethylenetriamine-pentaacetic acid was added to all samples to inhibit iron-catalysed reactions.
4.9.1. Measurement of ESR signal in chondrocytes supernatant
In our first attempt to quantify the rate of superoxide generation by human chondrocytes cells grown in monolayer were stimulated with IL-1β 6h prior to incubation with spin trap CMH. ESR spectra were recorded in ESR buffer every 30min of incubation with the cells. The three-line ESR spectrum of CM• was observed after incubation of CMH with chondrocytes indicating superoxide generation. The ESR signal increased with the time