(Investigative Ophthalmology and Visual Science. 2001;42:983-986.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
The Presence and Properties of Myocilin in the Aqueous Humor
Paul Russell1,
Ernst R. Tamm2,
Franz J. Grehn3,
Greda Picht3 and
Mark Johnson4
1 From the National Eye Institute, National Institutes of Health, Bethesda, MD; the
2 University of Erlangen-Nürnberg, Germany; the
3 University of Würzburg, Germany; and
4 Northwestern University, Evanston, Illinois.
 |
Abstract
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PURPOSE. To determine whether myocilin is present in the aqueous humor (AH) and
to examine certain properties of this protein.
METHODS. Human AH was obtained at the time of either glaucoma surgery or
cataract extraction. Monkey AH was obtained at the time of death, and
bovine aqueous was obtained from eyes delivered from an abattoir.
Column chromatography was performed on aqueous samples to determine the
approximate size of the myocilin present. Sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western
blot analysis were performed using antibody prepared against a peptide
sequence in myocilin. Analysis of the bovine proteins present in AH
that were retained by a microporous filter was also performed using
western blot analysis.
RESULTS. By western blot analysis, myocilin was present in human,
monkey, and bovine AH. The apparent molecular size of the myocilin
present in the AH were greater than 250,000 Da, when quantified with a
gel filtration column. Myocilin appeared to be hydrophobic and was one
of the proteins that was retained on microporous filters that were
obstructed by AH.
CONCLUSIONS. Myocilin is a constituent in the AH. It appears that myocilin is a
hydrophobic protein that may exist in an oligomeric state or in
association with other proteins. Myocilin is retained by microporous
filters and may be involved in the obstruction of these filters that
occurs when AH is perfused through them.
|
Introduction
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Aqueous humor (AH) flows through the chambers of the eye,
and there is a circadian modulation of its formation.1
There have been many studies concerning the ion, amino acid, and
protein composition of AH.2
3
4
Although the bulk of the
proteins probably enters the AH through the root of the iris, they can
also be released from other tissues in the eye.5
6
Recently, mutations in a gene on human chromosome 1 have been
genetically linked to primary open-angle glaucoma.7
The
protein encoded by this gene had been studied as the trabecular
meshwork inducible glucocorticoid response protein
(TIGR).8
9
Expression of this protein was also present in
other tissues in the eye, and the protein was named
myocilin.10
11
12
Because immunohistochemistry had indicated
localization of myocilin in cells of the nonpigmented ciliary
epithelium, corneal endothelium and iris stroma,12
it was
of interest to determine whether the myocilin was present in the AH and
the apparent molecular size of the protein.
Initial results indicated that this protein was very hydrophobic, and
additional studies were therefore undertaken. Previous studies had
suggested that there is a hydrophobic component in the AH that is
able to obstruct flow through microporous polycarbonate filters with
pore sizes similar to those that are found in the juxtacanalicular
connective tissue. This component appeared not to be present in serum,
because serum at concentrations equivalent to those found in AH would
not impede the flow through the filter to the extent that AH
would.13
14
To determine whether myocilin is associated
with obstruction of the microporous filters, filters were perfused with
AH, and the proteins tightly bound to those filters were examined.
|
Methods
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All procedures in this study conformed to the tenets of the
Declaration of Helsinki, the National Institutes of Health guidelines
on the care and use of animals in research and the ARVO Statement on
the Use of Animals in Ophthalmic and Vision Research. Human AH was
obtained at the time of either glaucoma surgery or cataract removal. An
institutionally approved human protocol governed the acquiring of the
human samples, and informed consent was obtained from all patients. AH
was obtained by paracentesis of the anterior chamber at the beginning
of the operation. Two samples from patients with primary open-angle
glaucoma and three samples from patients with cataract, but no history
of glaucoma were obtained. The AH was frozen at -70°C until use. To
determine whether the AH was contaminated with intracellular proteins
as a result of cells that were damaged during the paracentesis, a
commercially available assay for lactic acid dehydrogenase (LDH; Sigma,
St. Louis, MO) was performed on each of the AH samples. The assay
followed the manufacturer’s protocol, but the volumes were reduced to
allow the assay to be conducted in a 96-well plate. For gel
electrophoresis, after the human AH (0.75 µg) was thawed, it was
dried (SpeedVac system; Savant, Farmingdale, NY). The AH was then
reconstituted in sample buffer (62.5 mM Tris [pH 6.8]), 2% sodium
dodecyl sulfide [SDS], and 5% ß-mercaptoethanol) and placed in
boiling water. Samples were run on 12.5% gels (PhastGels; Pharmacia
Biotech, Piscataway, NJ) and blotted onto nitrocellulose (BioRad,
Hercules, CA) as directed by the manufacturer’s protocol. Protein
standards (BenchMark Protein Ladders) were obtained from Life
Technologies (Grand Island, NY). The western blot analyses were
developed with a kit (Chemiluminescence Reagent Plus; NEN Life
Sciences, Boston, MA) after incubation with primary antibody to
myocilin12
overnight at 4°C and incubation with
secondary antibody (Kirkegaard and Perry, Gaithersburg, MD) for 1 hour.
Chemiluminescence was detected using film (XAR; Kodak, Rochester, NY)
or with an image analysis system (Image Station 400; NEN Life
Sciences).
Bovine eyes were transported from the abattoir, and the AH was removed
within 5 hours of death. For column chromatography, the AH was
concentrated 10-fold (Centricon-10 concentrator; Amicon, Danvers, MA),
and sample was diluted 10-fold with water and reconcentrated. Samples
of up to 200 µl were applied to a gel filtration column (Zorbax
GF250; DuPont, Wilmington, DE) with a running buffer of 50 mM HEPES (pH
7.5), 0.2M NaCl, and 0.02% NaAzide. The flow rate through the column
was 330 µl/min. Fractions were collected each minute. Fractions were
concentrated on the concentrators, gel sample buffer was added, and the
samples were placed in boiling water. Samples were run on 12.5% gels
and western blot analysis developed according to the manufacturer’s
protocol (Pharmacia Biotech). The blotted gel was silver stained
according to the protocol.
Monkey AH was obtained at the time of death. The monkey AH was treated
in a manner similar to the bovine AH, with the exception that it was
not diluted with water after concentration.
To study the obstruction of microporous filters, the bovine AH was
first centrifuged at 100,000g for 45 minutes. Between 1 and
1.25 ml of the supernatant was passed through a polycarbonate 0.2-µm
filter that had not been treated with polyvinylpyrrolidone (PVP;
Nucleopore, Pleasanton, CA). These PVP-free filters are
hydrophobic.15
The filters were washed with 1 ml of
Dulbecco’s phosphate-buffered saline (PBS) after the aqueous had been
perfused through and then rinsed briefly in this saline. Ten filters
were placed in 1 ml of 0.1% Nonidet P40 (American Bioanalytical,
Natick, MA) overnight in the refrigerator. Samples of all the fractions
at each step of the process (uncentrifuged, centrifuged supernatant,
material perfused through the filter, and proteins eluted from the
filters) were concentrated (Centricon-10; Amicon) and then used in the
biochemical analysis.
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Results
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All the human samples had lactic acid dehydrogenase levels below
22 U/l. This value has previously been reported to be the upper level
of normal AH.16
This result suggests that the paracentesis
at the time of surgery did not cause rupture of cells in the eye and
was not contaminated with secondary aqueous. AH obtained from a donor
eye that arrived in the laboratory more than 24 hours after death had a
value of 55 U/l showing that the LDH assay of AH could detect
cellular damage. Using SDS-polyacrylamide gel electrophoresis (PAGE) of
the human samples and western blot analysis with an antibody developed
to a peptide sequence of human myocilin, two immunoreactive bands were
seen in the human AH samples (Fig. 1)
. The antibody has been used to detect myocilin in various tissues in
the eye.12
The topmost band in the AH from both the
patients with glaucoma and the ones undergoing cataract extraction was
located at approximately 65 kDa. The lower one was present at
approximately 55kDa. In a separate experiment, the proteins from human
serum were also run on SDS-PAGE, but western blot analysis did not
reveal any immunoreactive bands with this antibody, which suggests that
the level of myocilin was below the level of detectability (data not
shown).
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Figure 1. SDS-PAGE (left) and western blot (right)
of human AH samples from three patients who underwent cataract removal
(lanes 1, 2 and 3) from
two patients who underwent glaucoma surgery (lanes 4 and
5). The protein ladder (M) is the manufacturer’s
standard. The western blot analysis using antibody to myocilin showed
two immunoreactive bands at approximately 65 and 55 kDa in each of the
AH samples.
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Myocilin was also present in bovine and monkey AH. Both monkey and
bovine AH were used for the column chromatography experiments. Initial
attempts with the bovine AH to determine the apparent molecular size of
the myocilin were not successful. Gel exclusion columns that were based
on agarose beads appeared to interact with the myocilin in the aqueous.
Positive immunoreactivity on western blot analysis could be detected
from the void volume to the column volume fraction. This finding
suggests that the myocilin may interact hydrophobically with the beads.
Gel filtration was undertaken using a silica-based column (Zorbax GF
250; Dupont) to minimize the hydrophobic interactions. The pattern of
the bovine AH eluting from the column indicated that there was a small
peak of absorbance at 280 nm at the void volume (Fig. 2) . Blue dextran (2000 kDa) eluted from the column in this fraction, but
under the conditions of flow, proteins greater than approximately
250,000 Da could have been present in the first collected tube. A
considerably larger peak was observed at fraction 26 that corresponded
to the fraction in which the albumin standard eluted in another run.
There was a shoulder on the major peak and a couple of other peaks of
low molecular size that trailed the major peak.
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Figure 2. The elution pattern (top) of bovine AH from a gel
filtration column. Standards eluted from the column in the following
fractions: blue dextran, fraction 22; aldolase, fraction 24; albumin,
fraction 26; and carbonic anhydrase, fraction 31. SDS-PAGE
(bottom left) and western blot analysis
(bottom right) of the proteins eluting in
the different column fractions indicated that immunoreactive myocilin
was present in the void volume fraction. A small amount of
immunoreactive myocilin was present in fraction 24, although the
protein content of this fraction was small.
|
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SDS-PAGE and western blot analysis of the fractions were performed, and
immunoreactive myocilin was observed principally in the first fraction
that eluted from the column. Myocilin was not the only protein present
in this fraction. In addition to the bands that resolved on the gel,
stained material was also present at the stacking gel interface
suggesting that there might be some additional large proteins present
in the complex. A very small amount of immunoreactivity to myocilin was
observed in other fractions, particularly in fraction 24, that
corresponded to the fraction in which the standard aldolase eluted,
which suggests an apparent size of this myocilin of approximately
150,000 Da and may correspond to a dimeric form of this protein. The
silver-stained gel did not reveal many proteins in this fraction. In
fraction 26 at the expected elution of albumin, the most prominent
band on the silver-stained SDS-PAGE gel had an apparent molecular mass
of 67 kDa.
Monkey AH was collected at death and had LDH values less than 17 U/l.
Column chromatography was also performed with the monkey AH. With the
monkey AH, a distinct peak at the void volume was not seen, rather the
absorbance appeared to plateau until the peak at fraction 26. Because
the monkey sample was not washed to reduce ascorbate, a peak of very
low molecular weight material was present. Ascorbate, which has a very
high concentration in AH,2
readily absorbs light at 280 nm
(Fig. 3)
. The majority of the immunoreactive myocilin was present in the first
fraction eluting from the column. A very small amount of immunoreactive
material was present in other fractions from the column, as was true in
bovine AH.
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Figure 3. The elution pattern (top) of monkey AH from the gel
filtration column. SDS-PAGE (bottom left) and western
blot analysis (bottom right) of the proteins eluting in
the different fractions indicated that most of the immunoreactive
myocilin was present in the void volume fraction.
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Because the agarose column results suggested that myocilin might be a
very hydrophobic protein, the possibility was examined that myocilin
might be one of the proteins responsible for the reported capability of
AH to obstruct flow through hydrophobic filters (Nucleopore). Previous
work had shown that bovine AH obtained immediately after death or
obtained within a few hours after death caused obstruction of the
filters.14
The proteins present in the bovine AH, the
supernatant from the 100,000g centrifugation of the AH, the
AH proteins that passed through the filter, and the AH proteins that
were tightly bound to the filter were examined by SDS-PAGE (Fig. 4)
. Only approximately 2.2% of the proteins were retained on the filter.
Several of the proteins in the AH were present in the fraction that was
bound tightly to the filters; however, certain of the proteins were not
as prominent on the filter as in the material that went through the
filter, such as the protein migrating at approximately 180,000 Da.
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Figure 4. Silver-stained SDS-polyacrylamide gel of proteins present in the bovine
AH. Lane 1: The proteins present in the AH when it was
first obtained; lane 2: the proteins present in the
supernatant of the 100,000g centrifugation of bovine AH;
lane 3: the proteins that were perfused through the
filter; lane 4: proteins that were tightly bound to the
filter. The protein standard (M) is the manufacturer’s protein
ladder.
|
|
By western blot analysis, the myocilin could be detected in all these
fractions (Fig. 5)
. Other immunoreactive bands were also detected with lower molecular
masses. These bands, such as the one at approximately 25 kDa, may
represent some degraded fragments of the myocilin. Two of these bands
were prominent in the material that was closely associated with the
filter. One of the bands had a molecular mass of approximately 32 kDa
and the other of approximately 15 kDa.
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Figure 5. The blotted and silver-stained SDS- polyacrylamide gel of proteins
identical with those shown in Figure 3
(left) and the
western blot of this gel (right) showing the
immunoreactive myocilin present at 65 and 55 kDa in the fractions. In
the lane showing the fraction of the proteins tightly bound to the
filter, additional bands were seen at approximately 32 and 15 kDa.
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Discussion
|
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Myocilin was detected in AH. This protein was present in human,
monkey, and bovine AH at molecular masses of approximately 65 and 55
kDa according to results of SDS-PAGE. This result suggests that both
the glycosylated form and an unglycosylated form are present in the AH.
Both forms have been previously reported to be present in media from
human trabecular meshwork cells treated with
glucocorticoids.17
The ratios of the glycosylated and
unglycosylated forms appeared similar in all the human samples. There
is perhaps more glycosylated form present, but additional studies with
multiple human samples will have to be undertaken to accurately
quantitate any differences. The cells responsible for the release of
the myocilin into the AH are not known at this time. The absence of
immunoreactivity of the antibody with human serum suggests that the
amount of this protein is low or perhaps not normally present in the
blood. Because of the high turnover rate of the AH,1
the
possibility that myocilin is present in the AH as a result of the
diffusion of serum proteins at the iris root5
appears
remote. Myocilin has been reported to be in a number of tissues in the
eye,10
11
12
17 and therefore several tissues could release
myocilin into the AH, not just the trabecular meshwork. Candidates may
be cells of the corneal endothelium, nonpigmented ciliary epithelium,
or anterior iris stroma, in that positive immunolabeling with the
antibody against myocilin was shown in a previous
study.12
The apparent molecular mass of the myocilin in the bovine and monkey AH
appears to be greater than expected of a dimeric protein. The gel
filtration studies suggest that this protein could be oligomeric or
could be associated with other proteins in the AH. An aggregate of
proteins might also be suggested by the apparent hydrophobicity of the
protein itself. This possibility suggests future experiments to
determine the proteins that may be interacting with the myocilin in the
AH and whether the composition of these aggregated may be changed
during glaucoma. A small amount of myocilin appears to exist in the
dimeric state, because there was some immunoreactivity in fractions
that eluted after the void volume fraction.
The apparent hydrophobic interaction of the protein with the agarose
beads led us to re-examine the earlier findings of Johnson et
al.14
that AH contains proteins that can obstruct
hydrophobic, microporous membranes. They concluded that a protein with
molecular mass of approximately 28 kDa was involved with the filter
obstruction process. However, the possibility that the very prominent
spot seen at 67 kDa might contain not only albumin but also another
protein was not considered.
The tight association of the myocilin with the polycarbonate filters is
consistent with the apparent hydrophobic nature of this protein.
Although it is currently not possible to say whether the myocilin is
the component in the AH that is responsible for obstructing the
filters, it certainly is a candidate. Future experiments with purified
myocilin that is glycosylated or unglycosylated may answer this
question. It is conceivable that certain proteins selectively bind to
either one form or the other. The finding of small molecular weight
immunoreactive bands in the group of proteins tightly adhering to the
filter may indicate that some proteolysis of the myocilin is occurring
in the AH. This is particularly interesting in the case of the 32-kDa
band, which is close to the 28-kDa one reported by Pavao et
al.13
as being related to the filter-obstruction process.
It may be that these fragments of myocilin are more influential in
obstructing the filters than the full-length protein.
In summary, myocilin was present in AH. Both the glycosylated and
unglycosylated forms were found. The majority of the myocilin was found
in a fraction that included proteins with apparent molecular masses
greater than 250,000 Da. This result suggests that myocilin may exist
as an oligomer or may be in association with other proteins in the AH.
Myocilin appears to be a hydrophobic protein and is one of the proteins
that is tightly bound to polycarbonate filters that become obstructed
when AH is perfused through them.
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Footnotes
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Presented in part at the annual meeting of the Association for Research
in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2000.
Supported by Sonderforschungsbereich 539 of the Deutsche Forschungsgemeinschaft (ERT) and National Institutes of Health Grant R01EY09699 (MJ).
Submitted for publication June 12, 2000; revised November 22, 2000; accepted December 8, 2000.
Commercial relationships policy: N.
Corresponding author: Paul Russell, National Eye Institute, Building 6 Room 228, Center Drive MSC2735, Bethesda, MD 20892. russellp{at}intra.nei.nih.gov
|
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Top
Abstract
Introduction
