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International Journal
of Ophthalmology
2017; 10(9): 1344-1348
·Basic Research·
Analysis of proteomic differences between
liquefied after-cataracts and normal lenses using two-dimensional gel
electrophoresis and mass spectrometry
Jia-Jia Ge, Yu-Sen Huang
Provincial
Eye Laboratory of Ophathalmology, Shandong Eye Institute, Qingdao 266071,
Shandong Province, China
Correspondence
to: Yu-Sen Huang. Provincial Eye Laboratory of Ophathalmology,
Shandong Eye Institute, Qingdao 266071, Shandong Province, China. huang-yusen@126.com
Received:
2017-04-29
Accepted: 2017-06-28
Abstract
AIM:
To analyze and identify the
proteomic differences between liquefied after-cataracts and normal lenses by
means of liquefied chromatography-tandem mass spectrometry (LC-MS/MS).
METHODS: Three
normal lenses and three liquefied after-cataracts were exposed to
depolymerizing reagents to extract the total proteins. Protein concentrations
were separated using two-dimensional gel electrophoresis (2-DE). The digitized
images obtained with a GS-800 scanner were then analyzed with PDQuest7.0
software to detect the differentially-expressed protein spots. These protein
spots were cut from the gel using a proteome work spot cutter and subjected to
in-gel digestion with trypsin. The digested peptide separation was conducted by
LC-MS/MS.
RESULTS: The
2-DE maps showed that lens proteins were in a pH range of 3-10 with a relative
molecular weight of 21-70 kD. The relative molecular weight of the more
abundant proteins was localized at 25-50 kD, and the isoelectric points were
found to lie between PI 4-9. The maps also showed that the protein level within
the liquefied after-cataracts was at 29 points and significantly lower than in
normal lenses. The 29 points were identified by LC-MS/MS, and ten of these
proteins were identified by mass spectrometry and database queries:
beta-crystallin B1, glyceraldehyde-3-phosphate dehydrogenase, carbonyl
reductase (NADPH) 1, cDNA FLJ55253, gamma-crystallin D, GAS2-like protein 3, sorbitol
dehydrogenase, DNA FLJ60282, phosphoglycerate kinase, and filensin.
CONCLUSION: The
level of the ten proteins may play an important role in the development of
liquefied after-cataracts.
KEYWORDS: capsular
block syndrome; liquefied after-cataract; liquid chromatography-tandem
mass spectrometry
DOI:10.18240/ijo.2017.09.02
Citation: Ge JJ, Huang YS. Analysis of proteomic differences between
liquefied after-cataracts and normal lenses using two-dimensional gel
electrophoresis and mass spectrometry. Int J Ophthalmol 2017;10(9):
1344-1348
INTRODUCTION
Capsular
block syndrome (CBS) is a rare complication of phacoemulsification with
continuous curvilinear capsulorhexis (CCC) and posterior chamber in-the-bag
intraocular lens (IOL) implantation[1-3].
It is categorized into three types depending on the time of onset:
intraoperative, early postoperative, and late postoperative[4].
Liquefied after-cataract (LAC) is a special type of late complication following
standard surgery, without a shallow anterior chamber and secondary glaucoma. On
average, late postoperative CBS occurs 3.8y after surgery and can be identified
by white deposits behind the IOL inside the capsular bag[5].
Eifrig[6] showed that the white liquid contained
high concentrations of alpha-crystallin and relatively low levels of albumin,
suggesting that the liquid may originate from the epithelial cells of the
cataract. However, the content of the white liquid is not fully understood. In
this study, we adopted two-dimensional gel electrophoresis (2-DE) to
investigate proteomic differences between LAC and normal lenses. We also
explored the pathogenesis of LAC from the perspective of quantitative
proteomics.
MATERIALS AND METHODS
Materials Ethical
clearance for the study was obtained from the institutional review board
according to the Declaration of Helsinki, and informed consent from all patients
was obtained. Three fresh transparent lenses obtained from the donor eyes were
provided by the Eye Bank of Shandong Eye Institute and used as controls (group
A). The study enrolled three cases which presented with painless, gradual
visual loss at 5-8y after uneventful cataract surgery and were diagnosed with
LAC at the Qingdao Eye Hospital, Shandong Eye Institute (group B). The three
patients in group B were aged 63, 69 and 72y respectively (Table 1). Standard
coaxial ultrasonic phacoemulsification was performed in all of them, with
capsulorhexis, hydrodissection, and enhanced cortical clean-up and in-the-bag
foldable IOL fixation with an anterior capsular overlap. Surgeries were
performed by a single experienced surgeon with a superior corneal incision. The
blurring of vision was gradual. On clinical examination, the intraocular
pressure (IOP) was normal (ranging from 15 to 19 mm Hg). Anterior segment
photographs showed fibrosis of CCC and anterior capsular opacity. The space
between the IOL and the posterior capsule was filled with a milky opalescent
fluid which in slim beam looked like a meniscus-shaped opaque space with
concave anterior and convex posterior borders. The optical section appeared as
though two lenses had been placed in the bag (Figure 1). Pentacam Scheimpflug
examination of the anterior segment of the eye demonstrated normal anterior
chamber depth. The milky white substance was located behind the IOL optic
(Figure 2).
Table 1 Clinical features of three cases
Figure 1 Slit-lamp photograph showing fibrosis of CCC, anterior capsular
opacity, a backward extension of the posterior capsule, and the presence of
milky-white fluid between IOL and the posterior capsule.
Figure 2 Scheimplug photography of the anterior segment of the eye
showing normal anterior chamber depth and relative density of the milky white substance
behind the IOL.
Surgical Technique Proper asepsis techniques were employed, and a blepharostat was placed
in position. A clear corneal incision was made. A 27-gauge needle was inserted
through the edge of the CCC into the capsular bag to extract aqueous humor from
the anterior chamber along with the milky white substance for biochemical study
(Figure 3). Then the liquid was centrifuged at 4℃, and the supernatant was drawn and preserved at -80℃. The fresh transparent lenses of the donor eyes were examined and shown to be without disease or signs of surgery or
other trauma. The lenses were ground in liquid nitrogen and then dissolved in 1
mL lysis buffer to extract proteins before the supernatant was drawn and
preserved at -80℃. The protein concentration
of each sample was measured using a Bio-Rad
protein assay method.
Figure 3 A 27-gauge needle was inserted through
the edge of the CCC into the capsular bag to extract aqueous humor from the
anterior chamber along with the milky white substance.
Reagents and Instruments The main reagents included isoelectric focusing (IEF) strip (18 cm, pH
3-10 linear range), dithiothreitol (DTT),
3-[(3-cholamidopropy)-dimethylammonio]-1-propane sulfonate (CHAPS),
sodiumdodecylsulfate (SDS), iodoacetamide, urea, Tris (Bio-Rad, USA), coomassie
blue, ammonium persulfate (Sigma, USA), and thiourea (Solarbio, China). The
instruments were Protean IEF cell Isoelectric focusing system, Protean II xi cell
Vertical electrophoresis tank, the Versa Doc 1000 gel imaging system,
PDQuest7.0 image analysis software (Bio-Rad), Labofuge 400R High-speed
refrigerated centrifuge (Heraeus, USA), and Biowave Ultraviolet
spectrophotometer (Biochrom, Cambridge UK).
Two-dimensional Gel Electrophoresis
According to the method of Gorg and
the instructions of Protean IEF Cell Isoelectric Focusing System, each sample
was joined with a loading buffer to 350 μL[7]. Hydration and isoelectric focusing were performed
automatically on a Protean IEF cell. The program was set for the following
intervals: 1) passive hydration for 12h; 2) at 250 V slow boost for 30min; 3)
at 1000 V fast boost for 2h; 4) at 1000 V fast boost for 2h; 5) at 10000 V
linear boost for 3h; and 6) at 10000 V holding to 6000 V for 1h. After IEF, the
strips were balanced in a solution. Electrophoresis was performed at 15℃ on a 25% SDS-PAGE
gel, before the electrophoresis gel was stained with coomassie blue for 50min,
decolored, and finally stored in 7% acetic acid solution.
Image Acquisition and Fibrin Glue Point Identification We used the Versa Doc 1000 gel imaging system to obtain images after the
gel was stained. PDQuest7.0 image analysis software was employed to analyze the
results including tailoring, filtering, and matching. We repeated the test
three times to ensure the reliability of the test results before filtering out
the common different protein points and using the mass spectrometry analysis to
identify the differential protein.
RESULTS
Two-dimensional Gel Electrophoresis Results and Analysis Using the method described above to carry out 2-DE of the two groups of
proteins, we repeated the process three times, and the distribution of the protein
was basically the same (Figure 4). Through PDQuest7.0 software analysis and
statistical analysis, a total of 29 different points were found in all matching
spots. Compared with group A (normal lens), the protein level in group B (LAC)
was down-regulated.
Figure 4 Two-dimensional electrophoresis of normal lenses (A) and
liquefied after-cataract (B) 1: Beta-crystallin B; 2: Glyceraldehyde-3-phosphate dehydrogenase; 3:
Carbonyl reductase (NADPH) 1; 4: cDNA FLJ55253; 5: Gamma-crystallin D; 6:
GAS2-like protein 3; 7: Sorbitol dehydrogenase; 8: cDNA FLJ60282; 9:
Phosphoglycerate kinase; 10: Filensin.
Differences
in Protein Mass Spectrum Identification Results A mass
spectrometer successfully appraised the 29 different protein spots belonging to
ten different proteins: filensin (2 spots), beta-crystallin B1 (2 spots),
gamma-crystallin D (14 spots), glyceraldehyde-3-phosphate dehydrogenase (4
spots), carbonyl reductase (NADPH) 1 (1 spot), cDNA FLJ55253, highly similar to
actin, cytoplasmic 1 (2 spots), GAS2-like protein 3 (fragment) (1 spot),
sorbitol dehydrogenase (1 spot), cDNA FLJ60282, highly similar to sorbitol
dehydrogenase (1 spot), and phosphoglycerate kinase (1 spot) (Table 2).
Table 2 Mass
spectra results
Mass spectrometer showing the 29 different protein spots belonging to 10
different proteins: 1, beta-crystallin B; 2, glyceraldehyde-3-phosphate
dehydrogenase; 3, carbonyl reductase (NADPH) 1; 4, cDNA FLJ55253; 5,
gamma-crystallin D; 6, GAS2-like protein 3; 7, sorbitol dehydrogenase; 8, cDNA
FLJ60282; 9, phosphoglycerate kinase; 10, filensin.
DISCUSSION
In recent
years, the use of mass spectrometry techniques combined with 2-DE protein
separation and identification has become an important means of protein research
and has been widely used in various fields of life sciences. The crystalline
lens contains high levels of proteins, which play an important part in maintaining
transparency, normal morphology, and function of the lens. Any change in the
structure or amount of specific crystallins can lead to cataract[8-12].
There have
been few studies about the components of the white milky material in LAC. Our
study aimed to apply protein research technology to investigate the
pathogenesis of LAC from the molecular level.
Miyake
postulated that the cortical cells underwent metaplastic changes and
proliferated in the bag during the late postoperative period[13].
This may lead to posterior capsular opacification and cause occlusion of the
capsular opening by sealing off the gap between the anterior capsule and the
lens implant. These metaplastic cells can also lead to the release of a turbid
fluid retained in the retro-lenticular space. Other related factors may be
surgery-induced disturbance of blood ocular barriers that lead to free access
of different molecules, growth factors, hormones, cells in the capsular bag or
deposition of various cell types inside the capsule during and after surgery,
and biocompatibility of IOL materials[14].
Accumulation of similar materials has been documented [15].
However, the components of the milky liquid are not clearly identified.
The results of our study showed that filensin, beta-crystallin B1,
gamma-crystallin D, glyceraldehyde-3-phosphate dehydrogenase, carbonyl
reductase (NADPH) 1, cDNA FLJ55253, GAS2-like protein 3 (fragment), sorbitol
dehydrogenase, cDNA FLJ60282, and phosphoglycerate kinase were all
down-regulated when compared with normal lenses. Perhaps the ten proteins play
a critical role in the formation process of the LAC.
The lenses
of the eyes are composed of two types of cells: epithelial cells, which form a
monolayer at the anterior surface of the lens, and lens fiber cells, which
originate from epithelial cells and are highly differentiated. Lens fiber cells
lack organelles, have lens-specific structures such as gap junctions and beaded
filaments, and synthesize lens-specific proteins. Beaded filaments are lens
fiber cell-specific intermediate filaments[16] composed
of proteins of filensin and phakinin. Beaded filaments are 15-20 nm in diameter
and consist of globular particles with a periodicity of 19-21 nm. Primary
amino-acid sequence analysis showed that filensin and phakinin were members of
the intermediate filament family of proteins[17].
Beaded filament proteins were found exclusively in the fiber cells of the
lenses in all vertebrate orders examined[18],
which suggests that beaded filaments play a critical role in lens function.
Phakinin and filensin are expressed upon initiation of fiber cell
differentiation, predominantly localizing to the fiber cell membrane in young
fiber cells in the shallow cortex, and are proteolytically processed and become
more cytoplasmic as the cells age and lose their organelles[19-20].
Previous
studies[21-22] have shown that
the deletion of filensin or phakinin expression in mice by gene targeting could
cause cataracts and that some forms of hereditary cataracts in humans are
caused by mutations of filensin or phakinin. The data suggest that beaded
filaments are related to lens transparency. Our data proved that in the LAC,
the filensin, beta-crystallin B1, and gamma-crystallin D were decreased, which
may play an important part in the formation of cataracts.
The
water-soluble protein of the lens is the main lens protein and is closely
related to the transparency of the lens and diopter. The lens proteins mainly
include alpha-crystallin, beta-crystallin, and gamma-crystallin. Our results
showed that beta-crystallin B1 and gamma-crystallin D were reduced
significantly in the LAC. Many human β- lens proteins occurred after they were
translationally modified, including deamidation, protein truncation, and
oxidation of methionine and tryptophan[23].
Accumulation of β-deamidation may damage lens protein interactions and reduce
their stability. This in turn leads to the accumulation of insoluble
beta-crystallin in the process of cataract development[24].
Gamma-crystallin is prone to deamidation[25].
Such modifications to lens proteins may lead to gamma-crystalline structural
changes, and then aggregation occurs. Our 2-DE results showed that the content
of gamma-crystallin D was reduced.
It should be
noted that this study examined only three cases, but the saving grace was in
three high consistency results. Despite its preliminary character, this study
may have a certain representativeness. We will further expand the sample size
to validate our results, and study the relationship between the specific
content of each protein change and LAC, seeking solutions for LAC.
ACKNOWLEDGEMENTS
Foundation: Supported by
National Natural Science Foundation of China (No.81370996).
Conflicts of
Interest: Ge JJ, None; Huang YS, None.
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