|Molecular Vision 2004;
Received 9 September 2004 | Accepted 6 November 2004 | Published 8 November 2004
Alkali burn causes aldehyde dehydrogenase 3A1 (ALDH3A1) decrease in mouse cornea
Yi Feng, Yumei Feng, Xudong Zhu,
Ying Dang, Qingjun Ma
Genetic Engineering Lab, Beijing Institute of Biotechnology, Beijing, Peoples Republic of China
Correspondence to: Yi Feng, Genetic Engineering Lab, Beijing Institute of Biotechnology, P. O. Box 130(8), Beijing, 100850, Peoples Republic of China; Phone: 086-010-66931809; email: firstname.lastname@example.org
Purpose: Aldehyde dehydrogenase 3A1 (ALDH3A1) is the most abundant soluble protein component in the mouse cornea, produced mainly by corneal epithelial cells. High levels of ALDH3A1 in cornea contribute to maintenance of a stable an d transparent corneal structure. Alkali burn is a common damage to the corneal surface, which produces an alkaline hydrolysis of matrix proteins and induces an inflammatory reaction. Our study was intended to detect changes in ALDH3A1 expression after corneal alkaline burn.
Methods: To address this issue we employed RTQ-PCR to monitor the transcriptional change of ALDH3A1 after alkali burn. We used zymography to test enzyme activity changes of ALDH3A1 in the alkali burn cornea; And SDS-PAGE and mass spectrometry technology were used to verify protein content changes and to identify ALDH3A1 protein.
Results: Using zymography, ALDH3A1 enzymic activity was observed to decrease immedialtely after corneal alkali burn and the levels recovered following healing. Proteins extracted from alkali burned corneas, when run on SDS-PAGE, showed the same sized band (about 54 kDa, which is the molecular weight of ALDH3A1) but in much smaller quantity, compared to normal corneas. This result was further verified by mass spectrometry fingerprinting of the in-gel lysis product. An immediate decrease of ALDH3A1 transcription after alkali burning of the cornea was also found using RTQ-PCR. This level of transcription was gradually restored during healing.
Conclusions: Alkali burn of the corneal surface caused a rapid decrease of ALDH3A1 in the corneal at both the RNA and protein levels, which leads to the loses of the protective component of the corneal surface and makes it vulnerable to further damage. The ALDH3A1 level in the cornea gradually recovered during the healing process. Use of an anti-oxidation reagent as a treatment ingredient for alkali burn of the corneal surface could compensate for the decrease of anti-oxidation protection potential caused by ALDH3A1 loss.
Aldehyde dehydrogenase 3A1 (ALDH3A1) is expressed at high concentration in the mammalian cornea, wherein it constitutes 5-40% of the total water soluble proteins in different species [1,2]. Its expression in mouse cornea is 500 fold higher than in other tissues . Histochemistry studies reveal that in mice high level expression occurs in the anterior corneal epithelial cells, with very little expression in stromal keratocytes and no expression in endothelial cells . The proposed functions of ALDH3A1 are thought to include the destruction of toxic products of lipid peroxidation , ultraviolet (UV) absorption , and to protect corneal epithelial cells from ultraviolet induced oxidative damage [7-9]. The mechanisms that control the constitutive expression of ALDH3A1 in the cornea are not fully defined, except that it has been shown that ALDH3A1 gene expression in corneal epithelium may be controlled by light inducible and light maintenance pathways [10,11]. Ultraviolet radiation decreases the expression of ALDH3A1 [12-14] and the expression of the ALDH3A1 gene is abrogated by hypoxia [11,15].
Severe alkali burns induce long-lasting corneal epithelial defects and ulcerations, which pose many problems with wound healing of the ocular surface and lead to a decrease in corneal transparency . Gene expression changes after cornea alkali burns have been studied and mainly focus on inflammatory cytokines, MMPs, and ECM components [17-19]. Investigations of ALDH3A1 gene expression changes during cornea alkali burns will give hints as to the function of ALDH3A1 in cornea oxidative stress.
Using real time quantitative PCR, peptide mass fingerprint and substrate SDS-PAGE methods, we report a dramatic down regulation of ALDH3A1 from alkali burns and its recovery during cornea healing.
Animal protocols and corneal alkali burn model
All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Animal Care and Use Committee of Beijing Biotechnology Institute.
C57Bl/6 mice, weighting 20 to 25 g, were provided by the Medical Experimental Animal Center. All procedures were performed under general anesthesia (subcutaneous injection of xylazine 10 mg/kg and ketamine 150 mg/kg). To monitor systemic side effects of the treatment, body weight and temperature were measured on every observation day. Animals were held in groups of 10 and fed regular laboratory chow and water. A 12 h day and night cycle was maintained.
Cornea alkali burn was performed as described , with some modification. Briefly, a filter disc (diameter 1 mm) soaked in 1 N NaOH for 1 min was placed on the center of the cornea for 1 min, after which the anterior surface and inner aspect of the eyelids were gently irrigated with 20 ml of sterile saline for 1 min. The mice were randomly divided into 2 groups.
Sample preparation of corneal soluble proteins
Six control corneas and six alkaline burned corneas were collected at day 3 post alkaline burn. Corneas were dissected and placed in 100 μl lysis buffer (20 mM imidazole HCl, 10 mM KCl and 1 mM EDTA [pH 6.8]) supplemented with a protease inhibitor cocktail. Following mechanical homogenization, the lysate was cleared of debris by centrifugation at 14,000 rpm for 15 min (4 °C) and the supernatant was collected. Total protein was determined by the BCA assay (Pierce, Rockford, IL). The sample was analysed by sodium dodecylsulfatePolyacrylamide gel electrophoresis (SDS-PAGE) followed by in-gel digestion or ALDH3A1 zymography analysis.
SDS-PAGE and in-gel trypsin digestion
Separating (T-10%) and stacking (T-4%) polyacrylamide gels containing 0.1% SDS were used for SDS-PAGE. The coomassie stained protein bands were cut out of the gel and destained with 50 μl of 50 mM ammonium bicarbonate/50% acetonitrile (ACN) three times for 30 min at ambient temperature. The destained gel pieces were dried in a Speedvac vacuum concentrator (Savant Instruments, Farmingdale, NY). Gels were treated with 2 μl of 25 mM ammonium bicarbonate containing 10 ng of trypsin at 4 °C for 1 h. After overnight incubation at 37 °C, the gels were dried in a high vacuum centrifuge to evaporate solvent. TFA (8 μl of 5%) was added and incubated at 40 °C for 1 h; The supernatant was transfered into a fresh tube. TFA/50% ACN (8 μl of 2.5%) was used for the second step of gel extraction at 30 °C for 1 h. This supernatant was combined with the initial one. The combined extracts were dried in a Speedvac vacuum concentrator, and redissolved in 3 μl of 0.1% TFA.
Peptide mass fingerprinting (PMF) by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)
MALDI-TOF MS measurements were performed on a Bruker Reflex®III MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) operating in reflectron mode. A saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% TFA was used for matrix. The matrix solution (1 μl) and sample solution were combined at a 1:1 ratio were mixed and applied onto the Score384 target well. MALDI-TOF MS analysis was performed at 20 kV accelerating voltage and 23 kV reflecting voltage.
The data obtained from PMF were used to search the protein database to determine the identity of the 54 kDa protein. The program Mascot, developed by Matrix Science Ltd., was used. The search parameters were set as follows: search type was Peptide Mass Fingerprint, the enzyme used was trypsin, the variable modification was oxidation (M), the mass values were monoisotopic, the protein mass was unrestricted, the peptide mass tolerance was ±0.1 Da, the peptide charge state was 1+, the maximum missed cleavages was 1, and the number of queries was 22.
Monoisotopic peptide masses were used to search the databases, allowing a peptide mass accuracy of 100 ppm and one partial cleavage. Oxidation of methionine as well as carbamidomethyl modification of cysteine was considered.
Enzyme activity detected by zymography with ALDH3A1 preferable substrates
Six control corneas and six alkaline burned mouse corneas were collected at day 3 post alkaline burn. Corneas were dissected and placed in 100 μl lysis buffer (20 mM imidazole HCl, 10 mM KCl and 1 mM EDTA [pH 6.8]) supplemented with a protease inhibitor cocktail. Following mechanical homogenization, the lysate was cleared of debris by centrifugation at 14,000 rpm for 15 min (4 °C), and the supernatant was collected. Total protein was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Zymogram analysis was performed as described . In brief, we used the ALDH3A1 specific substrates NADP+ and benzaldehyde, and protein samples were separated on 10% non-reduced SDS-PAGE gels. Gels were washed four times for 20 min each in 10 mM phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 2.5% Triton X-100 and then twice for 20 min in 10 mM phosphate buffer. ALDH3A1 bands were developed for 15 min at 37 °C with 10 ml of 10 mM phosphate buffer (pH 7.0) solution containing 20 mg NADP+, 8 mg of MTT, 0.4 mg of phenazine methosulfate and 20 μl of benzaldehyde, and the reaction was stopped by washing the gel with double de-ionized water.
RNA preparation and real time quantitative polymerase chain reaction (RTQ-PCR)
Total RNA was isolated from pooled corneal tissue (eight corneas per time point with pooling of samples to give repeatable responses between multiple experiments) from native and from cauterized corneas 48, 96, and 168 h after cautery, using the standard RNA extraction procedure outlined in the manufacture's protocol (TRIZOL, Sigma, Indianapolis, IN). Isolated RNA was treated with RNase-free DNase I to remove any contaminating genomic DNA. Total RNA was quantified with an Eppendorf Biophotometer® (Eppendorf, Hamburg, Germany). Complementary DNA synthesis was carried out using AMV Reverse Transcriptase and Oligo(dT)15 primer. This cDNA was used as template for real time quantitative PCR (RTQ-PCR) analysis. RNA in the absence of reverse transcriptase was used as a negative control.
The specific primers and Taqman® probe for mouse ALDH3A1 were designed using ABI Primer Express® Software version 2.0. the forward primer sequence was: 5'-ACC TGC GCA AGA ATG AAT GG-3' and the reverse primer sequence was: 5'-GCC CCT TAA TCG TGA AAT CG-3'. The TaqMan® MGB probes sequence was: Fam5'-CCT ACT ACG AGG AGG TGG CTC ACG TGC-3'TAMRA. The amplicon length was 80 bp. RTQ-PCR was performed with 5 μl samples in a total volume of 50 μl consisting of 1X TaqMan® Universal PCR Master Mix (containing AmpliTaq Gold DNA Polymerase, Amperase UNG, dNTPs with dUTP, passive reference, and optimized buffer components), 600 nM each of forward and reverse primer, and 400 nM Taqman® probe. Cycling conditions were 10 min at 95 °C, and then 40 cycles consisting of 15 s at 95 °C and 1 min for annealing and extension at 60 °C. RTQ-PCR for GAPDH was performed simultaneously in our experiments as an internal control. The ALDH3A1 mRNA level was expressed as the transcription proportion, which is determined by the ALDH3A1 copy number divided by the GAPDH copy number obtained from the same sample.
Statistical analysis was performed using the two tailed student t-test. Results are presented as mean±standard error of the mean of three or more experiments and a p<0.05 was considered statistically significant.
Identification of water soluble proteins decreased in alkali burned mouse cornea
Water soluble proteins were extracted from normal or alkali burned mouse corneas and the samples were examined by SDS-PAGE (Figure 1B). Compared to samples from normal corneal tissue, a prominent protein with apparent molecular mass of approximately 50 kDa consistent with the molecular weight of mouse ALDH3A1 was markedly reduced in the water soluble protein from alkali burned mouse corneas (Figure 1B, arrow). We then performed in-gel trypsin digestion and analyzed the peptide mixture with MALDI-TOF MS. The obtained PMF (Figure 2) was used to search the NCBI database using the Mascot program. The search result identified this protein as aldehyde dehydrogenase family 3, subfamily A1 [Mus musculus], NM_007436, the search score was 106, (score is -10*log[P], where P is the probability that the observed match is a random event). Protein scores greater than 62 are significant (p<0.05).
Zymography using ALDH3A1 substrate showed a decreased ALDH3A1 activity in alkali burned cornea
Zymography with ALDH3A1 preferred substrates was used to directly show enzyme activity in the gel. Following non-reducing SDS-PAGE, the gels were washed and developed with the ALDH3A1 preferred substrates benzaldehyde and NADP+, and the reduction of NADP+ was visualized colorimetrically. In the sample pool from normal mouse cornea, zymography detected a sharp band that migrates at about 50 kDa (Figure 1A), identical to mouse ALDH3A1. This band almost disappeared in the sample from alkali burn cornea (Figure 1A, AB), and after corneal wound healing the ALDH3A1 activity was mainly restored at about day 15 post alkali burn (Figure 3, Day 15). When the ALDH3A1 specific substrate NADP+ was not included, no colorimetric signal was observed in any lane (not shown).
Real time quantitative PCR to monitor ALDH3A1 mRNA changes in alkali burned corneas
The transcription level of ALDH3A1 in mouse cornea was determined by RTQ-PCR. Typically, the measurement used in RTQ-PCR is the number of cycles required to pass a given fluorescent threshold. The cycle threshold and the actual DNA content is determined from a standard curve prepared with purified DNA. However this measurement might introduce additional errors during the preparation of template. Therefore, in our experiments the relative transcription proportion was used as the measurement of ALDH3A1 transcription level, GAPDH transcription levels served as the internal control. The ALDH3A1 absolute transcription value was divided by the GAPDH absolute transcription value obtained from the same template, and the resulting transcription proportion was calculated. In normal mouse cornea the ALDH3A1 transcription level was high, as reported previously , and the relative transcription level was about 11.481±0.436 (Figure 4). One day after alkali burn of the cornea, the transcription level dropped to 0.421±0.019 (p<0.001), then restored during cornea healing, returning to 0.747±0.078 (p <0.001) at day 4 post alkali burn and increasing to 1.692±0.105 (p<0.01) at about day 9.
ALDH3A1 is preferentially expressed in mammalian corneal epithelium and it is thought that this enzyme contributes to corneal transparency through its protective effects against UV radiation, and through the prevention of oxidative damage in corneal epithelial cells [5-9]. Consistent with this proposal is the observation that decreased ALDH3A1 activity has been reported in association with human corneal disease . The importance of ALDH in maintaining the stability of corneal structure is well known. Previous reports indicate that both UVB and hypoxia down regulate ALDH3A1 expression in cornea epithelia [11,14,15]. The possible hazard of UVB to the cornea through damage to ALDH3A1 has been extensively discussed [9,12-14], but other factors affecting corneal ALDH3A1 expression have seldom been discussed. The results reported here provide direct evidence that the expression of ALDH3A1 in mouse cornea is down regulated by alkali burn of the cornea surface, and the expression of ALDH3A1 is restored gradually during corneal wound healing.
In our study, ALDH3A1 enzyme activity decrease following alkali burn of mouse cornea, as determined by spectrophotometry (unpublished), was at first thought to be caused by the direct loss and damage of corneal tissue. However the alkali burn only damages about 10% of corneal surface tissue and ALDH3A1 transcription decreased by about 85.3% at Day 1 after alkali burn (Figure 4). This result suggests that there is down regulation of ALDH3A1 expression after alkali burn of mouse cornea. In addition, when total protein extracts of mouse cornea were run on SDS-PAGE gels, the band at about 50 kDa (Figure 1B), which was identified by PMF (Figure 2) as ALDH3A1, almost disappears after alkali burn. This was further confirmed by zymography (Figure 1A).
At this stage our experiments cannot elucidate the mechanism by which alkali burn down regulates ALDH3A1 expression in mouse cornea. There are studies suggesting that hypoxia down regulates ALDH3A1 expression [11,15] and that alkali burn of cornea leads to hydrolysis of stroma matrix protein and induce an inflammation response . It is therefore possible that the acute damage by alkali in the cornea might lead to local hypoxia, and this could be the reason for corneal ALDH3A1 down regulation. A more detailed study should be carried out to analyse factors that influence ALDH3A1 expression in the cornea.
From the proposed function of ALDH3A1 in mouse cornea, the ALDH3A1 decrease caused by alkali burn of the corneal surface will lead to loss of the anti-oxidation self protection potential of cornea. Therefore, it will be interesting to observe the effect of using anti-oxidation reagents on corneal alkali burns. The loss and restoration of ALDH3A1 expression in the corneal alkali burn and wound healing processes described in this study suggest the significance of ALDH3A1 in normal mouse cornea. We propose that ALDH3A1 could be a useful indicator for intact corneal epithelial function that can be used in ocular disease experimental models and correlative ocular treatment studies.
We thank Dr. Liqun Cao and Dr. Jing Liu for their professional help with mouse cornea surgery and immunohistochemistry sample preparation. We also grateful to the National Center of Biomedical Analysis for their assistance with the peptide mass fingerprint analysis.
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