From the Membrane Biophysics Laboratory, Department of Medicine, MCP Hahnemann University School of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania 15212-4772 and the § Department of Biochemistry, Kirksville College of Osteopathic Medicine, Kirksville, Missouri 63501
Received for publication, November 6, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Previous studies in our laboratory have provided
direct evidence for the existence of distinct cholesterol domains
within the plasma membranes of human ocular lens fiber cells. The fiber cell plasma membrane is unique in that it contains unusually high concentrations of cholesterol, with cholesterol to phospholipid (C/P)
mole ratios ranging from 1 to 4. Since membrane cholesterol content is
disturbed in the development of cataracts, it was hypothesized that
perturbation of cholesterol domain structure occurs in cataracts. In
this study, fiber cell plasma membranes were isolated from both normal
(control) and cataractous lenses and assayed for cholesterol and
phospholipid. Control and cataractous whole lens membranes had C/P mole
ratios of 3.1 and 1.7, respectively. Small angle x-ray diffraction
approaches were used to directly examine the structural organization of
the cataractous lens plasma membrane versus control. Both
normal and cataractous oriented membranes yielded meridional
diffraction peaks corresponding to a unit cell periodicity of 34.0 Å,
consistent with the presence of immiscible cholesterol domains.
However, comparison of diffraction patterns indicated that cataractous
lens membranes contained more pronounced and better defined cholesterol
domains than controls, over a broad range of temperature (5-40 °C)
and relative humidity (52-92%) levels. In addition, diffraction
analyses of the sterol-poor regions of cataractous membranes indicated
increased membrane rigidity as compared with control membranes.
Modification of the membrane lipid environment, such as by oxidative
insult, is believed to be one potential mechanism for the formation of
highly resolved cholesterol domains despite significantly reduced
cholesterol content. The results of this x-ray diffraction study
provide evidence for fundamental changes in the lens fiber cell plasma
membrane structure in cataracts, including the presence of more
prominent and highly ordered, immiscible cholesterol domains.
The human ocular lens is an optical tissue that contributes to
normal visual physiology by providing a means of light refraction and
accommodation (variable refraction). The utility of these basic
functions is critically dependent on lens transparency, which is
subserved by several unique features of the lens. First, the mammalian
lens is comprised almost entirely of a large number of elongated,
prismatic cells, known as fibers or fiber cells, that are deposited
throughout life in highly ordered concentric lamellae. These fiber
cells have a very regular cell shape and cell volume, and their very
tight structural arrangement in the lens body minimizes extracellular
space (a potential light scattering region). Second, fiber cell
precursors lose all nuclei and other cytosolic organelles during their
elongation and differentiation into mature fiber cells (1, 2). Thus,
potential intracellular light scattering elements are removed as fiber
cells migrate into more optically significant positions in the lens.
Third, a direct consequence of fiber cell differentiation is that
plasma membrane becomes the principal organelle of the lens and
contains essentially all lens lipid (3). Since plasma membrane is the
sine qua non of lens organization, it is conceivable that
the molecular and structural organization of the lens membrane itself
is essential to lens transparency. Indeed, numerous studies suggest
that cataractogenesis is associated with perturbation of lens membrane
composition (4-7), structure (8-12), and function (13-17).
Plasma membranes of the human ocular lens are distinguished from other
eukaryotic cell membranes by their unique lipid composition. Lens
plasma membranes contain very high relative concentrations of
long-chain saturated or monounsaturated fatty acids (18, 19);
polyunsaturated fatty acids are present only in trace amounts (20, 21).
In addition, sphingomyelin and sphingomyelin derivatives make up
greater than 50% of the total lens membrane phospholipid composition
(22, 23). A deficit of polyunsaturated fatty acids and an abundance of
sphingomyelin are believed to contribute to lens membrane rigidity and
structural order.
Another remarkable feature of the lens plasma membrane is its unusually
high relative concentration of cholesterol. The cholesterol to
phospholipid (C/P)1 mole
ratio of the human fiber cell plasma membrane ranges from 1 to 4, with
the greatest concentration of cholesterol found in the central or
nuclear region of the lens (24-26). Membrane cholesterol levels of
this magnitude have raised the speculation that the lipid organization
of the lens membrane is highly specialized, consisting of
cholesterol-poor and cholesterol-rich planar regions. Using small angle
x-ray diffraction approaches, we recently provided direct evidence that
cholesterol is organized into discrete clusters or domains within
normal, non-cataractous lens plasma membranes (27). These cholesterol
domains were identified by prominent scattering signals corresponding
to a reproducible unit cell periodicity of 34.0 Å, consistent with a
bilayer structure comprised of cholesterol monomers arranged in a
tail-to-tail orientation (28, 29). Cholesterol domains were observed in
both native and reconstituted lens membranes and remained stable over a
broad range of temperature and relative humidity levels (27). These
findings were consistent with the idea that pure cholesterol phases
form within cell membranes at C/P mole ratios in excess of 1.0 (30), as
confirmed by a number of experiments employing theoretical, model, and
native membrane systems (31-37).
Little is known about the structure and molecular organization of the
lens fiber cell plasma membrane in cataracts. The presence of
cholesterol domains in normal lens membranes would suggest that they
are important in maintaining lens transparency. If cholesterol domain
formation in normal lens membranes is a biophysical necessity given
such extremely high levels of cholesterol, it would be reasonable to
expect that perturbation of the cholesterol content of the lens plasma
membrane would result in disturbance of cholesterol domain and lens
membrane structure. Thus, changes in membrane cholesterol composition
and structural organization could contribute to cataractogenesis.
In this study, small angle x-ray diffraction approaches were used to
examine the relationship between cataracts and the structural organization of cholesterol in the lens fiber cell plasma membrane. Whole lens plasma membranes were isolated from normal and cataractous human lenses for these experiments. Cholesterol and phospholipid quantitation revealed that the C/P mole ratio of cataractous lens membranes was ~54% lower than that of normal lens membranes.
Immiscible cholesterol monohydrate domains were present in both normal
and cataractous whole lens plasma membranes and remained stable over a
broad range of temperature and relative humidity conditions. However,
the diffraction peaks corresponding to cholesterol domains in the
cataractous lens membranes were more intense as compared with controls,
suggesting that membrane-restricted cholesterol domains are a more
prominent feature of this disease. These results were quite surprising
since it was initially hypothesized that the lower C/P mole ratio in
the cataractous membranes would yield relatively weaker or smaller
cholesterol domains. Data collected in this study suggest that lower
levels of cholesterol paradoxically produce more stable domains in
cataractous lens membranes. The presence of prominent and more stable
cholesterol domains in the cataract lens membrane may reflect changes
in phospholipid content and distribution as a function of aging and
cataractogenesis. Modification of the lens membrane by age-related
insults, such as oxidative stress, may also promote cholesterol domain formation.
Isolation of Human Lens Plasma Membranes--
Three normal,
control lenses were obtained from the Kentucky Lions Eye Foundation and
Eye Bank (Louisville, KY). Donors ranged in age from 73 to 80 years.
Two lenses containing mixed cortical and nuclear mature cataracts were
kindly provided by Dr. Vittorio Rasi (Udine, Italy). Donors were 78 and
80 years of age.
Control and cataractous lenses were decapsulated and separately
homogenized in 3 ml of 5 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM Biochemical Characterization of Lens Plasma Membranes--
Small
aliquots of the lens membrane preparations were taken for protein
quantitation using a modified Lowry protein assay (39). Protein
concentrations for both control and cataractous lens membranes samples
are listed in Table I. Main intrinsic protein of 26 kDa and its
degradation product of 22 kDa were the principle proteins present in
both control and cataractous membrane samples, as characterized using
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown).
Total lipids from approximately one-third of each lens membrane
suspension were Folch extracted as previously described (40) and
dissolved in chloroform. Aliquots of these lipid extracts were taken
for gas-chromatograph quantitation of cholesterol (41) and colorimetric
assay of phospholipid (42). Compositions of the control and cataract
membrane suspensions are listed in Table I.
Preparation of Oriented Lens Plasma Membrane Samples for X-ray
Diffraction--
Control and cataractous lens plasma membrane samples
were oriented for x-ray diffraction analysis as described previously (27, 43). Briefly, plasma membrane samples (in buffer B) were loaded
into Lucite sedimentation cells using sample volumes to achieve 230 µg of phospholipid per aliquot. Each sedimentation cell contained an
aluminum foil substrate upon which the membrane pellets were collected.
The membrane samples were centrifuged in a Sorvall AH-629 swinging
bucket ultracentrifuge rotor (DuPont Corp., Wilmington, DE) at
35,000 × g for 50 min at 5 °C. Samples were washed
three times with diffraction buffer (0.5 mM HEPES + 150 mM NaCl, pH 7.3). After the final washing cycle, the
supernatants were aspirated and the aluminum foil substrates,
supporting the membrane pellets, were removed from the sedimentation
cells and mounted on curved glass slides. The samples were then placed
in hermetically sealed brass canisters in which temperature and
relative humidity were controlled during x-ray diffraction experiments.
Small Angle X-ray Diffraction Analysis--
The oriented lens
plasma membrane samples were aligned at grazing incidence with respect
to a collimated, monochromatic x-ray beam produced by a Rigaku Rotaflex
RU-200, high-brilliance microfocus generator (Rigaku USA, Danvers, MA).
Analytical x-rays are generated by electron bombardment of a rotating
copper anode and filtered through a thin nickel foil to provide
monochromatic CuK
The unit cell periodicity, or d-space, of the membrane lipid
bilayer is the measured distance from the center of one lipid bilayer
to the next, including surface hydration. The d-spaces for
the membrane multibilayer samples were calculated using Bragg's Law,
Saturated salt solutions were used to define the relative
humidity (RH) levels employed in these x-ray diffraction analyses. The
following salt solutions (with associated RH in parentheses) were used
in these experiments:
Mg(NO3)26H2O (52%),
K2C4H4O61/2H2O (74%), NaKC4H4O64H2O
(87%), and
Na2C4H4O62H2O
(92%).
Analysis of Normal and Cataractous Human Lens Plasma Membrane
Structure--
Small angle x-ray diffraction approaches were used to
characterize the structural organization of fiber cell plasma membranes isolated from both normal and cataractous human lenses. Representative x-ray diffraction profiles obtained from oriented normal and
cataractous lenses at 30 °C, 87% RH are shown in Fig.
1. These meridional diffraction patterns
are consistent with a biphasic structural organization in which
cholesterol monohydrate domains, corresponding to diffraction orders 1'
and 2', exist within a cholesterol-poor liquid crystalline membrane
bilayer phase, corresponding to diffraction orders 1 and 2 (where
present). Comparison of normal versus cataractous lens
membrane diffraction patterns reveals that the overall membrane resolution is greater in the cataractous preparations, with diffraction peaks that are more intense and better resolved. The relative intensities of the diffraction peaks corresponding to cholesterol domains are also significantly greater in cataractous lens membranes, suggesting a more ordered lipid membrane structure. These data were
confirmed with two-dimensional diffraction analysis as shown in Fig.
2.
Effects of Relative Humidity on Normal and Cataractous Lens
Membrane Structure--
Control and cataractous lens membranes were
examined over a broad range of relative humidity levels (Fig.
3). Diffraction peaks corresponding to
cholesterol domains (periodicity of 34.0 Å) were observed in control
membranes at each relative humidity level, with the greatest
cholesterol peak intensity occurring at 52% RH. The periodicity of the
cholesterol-poor phospholipid membrane bilayer increased by 43% (57.3 to 82.1 Å) as a function of increasing relative humidity. Cholesterol
domains were also identified in cataractous lens plasma membranes and
the scattering peaks associated with these domains were noticeably more
intense as compared with control profiles. As for the normal lens
membranes, the calculated periodicity of the cholesterol domains in
cataractous lenses was unaffected by relative humidity, with a
reproducible d-space of 34.0 Å. The periodicity of the
phospholipid bilayer phase in the cataractous membrane sample increased
by 25% (51.2 to 64.0 Å) over the same relative humidity range used
for control lens membranes.
Effects of Temperature on Normal and Cataractous Lens Membrane
Structure--
Cholesterol domains were also observed in normal and
cataractous lens plasma membranes over a broad range of temperature
levels (Fig. 4). With relative humidity
held constant at 87%, diffraction peaks corresponding to cholesterol
domains were more intense in cataractous membrane profiles as compared
with controls. A general decrease in phospholipid bilayer periodicity
was observed for control membranes as a function of increasing
temperature, although lower temperatures resulted in some attenuation
of phospholipid bilayer scattering intensity. The phospholipid bilayer
d-space of the cataractous lens membranes decreased from
81.4 to 71.3 Å as the temperature was increased from 15 to 40 °C.
Control and cataractous lens membrane x-ray scattering data were also
collected at 52% RH over the same temperature range. As observed at
87% RH, cholesterol peak intensities were greater in cataractous
membranes as compared with controls. The d-spaces associated
with the phospholipid bilayer regions decreased by 8.9 Å (15%) and
1.2 Å (2%), control and cataractous lens membranes respectively, as
temperature was increased from 5 to 40 °C.
Among its several unique properties, the lens fiber cell plasma
membrane is particularly noted for its unusually high relative concentrations of cholesterol. Lens plasma membranes have C/P mole
ratios that range from 1.0 to 4.0, with higher cholesterol content
found in the lens nucleus (24, 25). Several reports have provided
evidence that the C/P mole ratio increases with age (25), but changes
in the C/P mole ratio in cataractous lens membranes are not well
understood. The amount of cholesterol present in whole lenses has been
measured in cataracts, but with disparate results. Some reports provide
evidence that the relative amount of cholesterol in cataractous lenses
is not significantly different than the amount of cholesterol in normal
lenses (6, 24, 40, 44). However, some investigators have found
increased cholesterol levels (5, 45, 46), while others have reported
decreased cholesterol content in cataracts (4).
There is some evidence to suggest that cholesterol depletion or
restriction of cholesterol availability may be associated with the
development of cataracts. Pharmacologic inhibition of lens
cholesterolgenesis has been shown to induce cataracts in dogs (47),
rats (48-50), and humans (51-53). Treatment of rats with U18666A, an
inhibitor of 2,3-oxidosqualene cyclase (an enzyme upstream of
cholesterol in its biosynthetic pathway), has been shown to decrease
cholesterol synthesis in the lens (54), reduce the cortical C/P mole
ratio (55), disrupt the ultrastructure of lens fiber cell membranes
(11), and promote the development of irreversible nuclear cataracts
(49). Furthermore, congenital defects in cholesterol metabolism
(e.g. Smith-Lemli-Opitz syndrome and cerebrotendinous
xanthamatosis) have also been associated with increased risk of
cataracts (reviewed by Cenedella (56)). Collectively, these data
suggest that reduction or other changes in lens cholesterol content can
have deleterious effects on lens function (i.e.
transparency). However, the relationship between cataracts, altered
cholesterol content, and lens plasma membrane structural organization
has not been adequately characterized.
In this study, small angle x-ray diffraction approaches were used to
directly examine the structure of plasma membranes isolated from
cataractous lens fiber cells. The C/P mole ratio measured for these
membranes was 54% lower than that of controls (Table I), which is consistent with cholesterol
depletion in cataracts or to selective loss of other membrane fractions
due to lens membrane disintegration and vesiculation (6). It is
important to note that the C/P mole ratio measured in these cataractous
whole lens plasma membranes or membrane fractions is still sufficient
to induce cholesterol domain formation, since C/P mole ratios in excess
of 1.0 have been shown to promote the formation of separate cholesterol
domains in cell membranes (30). However, it was hypothesized that the
lower C/P mole ratio of the cataractous lens membrane would result in
less stable cholesterol domains and diminished x-ray scattering
intensity as compared with control lens membranes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol (buffer A)
using a glass Dounce homogenizer. Plasma membranes were isolated using
an approach based on the method of Russell et al. (38).
Briefly, the homogenates were initially centrifuged at 10,000 × g for 20 min at 8 °C. Sample pellets were washed three
times with buffer A using the same centrifugation settings. The pellets
were then extracted twice with 7 M urea in 50 mM Tris-HCl (pH 7.4), extracted twice with NaOH buffer (0.1 N NaOH + 1 mM
-mercaptoethanol), and washed
twice with 5 mM Tris-HCl (pH 8.0), 1 mM EDTA,
150 mM NaCl, 0.02% NaN3 (buffer B). Final control and cataract sample pellets were each resuspended in 1-2 ml of
buffer B.
radiation (K
1 and K
2
unresolved;
= 1.54 Å). Collimation of the x-ray beam is
achieved using a double-focusing Franks mirror (diffraction camera).
Diffraction data were collected on a one-dimensional,
position-sensitive electronic detector (Innovative Technologies,
Newburyport, MA) using a sample to detector distance of 150 mm.
Representative two-dimensional diffraction patterns for each sample
were also collected on Kodak storage phosphorscreen film (europium
matrix, Eastman Kodak Co., Rochester, NY) and analyzed using a
computerized PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
Two-dimensional data were collected at a sample to detector distance of
80 mm. Crystalline cholesterol monohydrate was used to verify the
calibration of the detectors.
in which h is the diffraction order number,
(Eq. 1)
is
the wavelength of the x-ray radiation (1.54 Å), d is the
membrane lipid bilayer unit cell periodicity, and
is the Bragg
angle equal to one-half the angle between the incident beam and
scattered beam.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 1.
Representative x-ray diffraction patterns
obtained from oriented normal and cataractous human lens fiber cell
plasma membrane samples. Data were collected on a one-dimensional,
position-sensitive electronic detector at 30 °C and 87% RH.
Diffraction profiles were generated from (A) control, normal
lens membrane samples and (B) cataractous lens membrane
samples. In each panel, diffraction peaks labeled as 1' and 2'
correspond to immiscible cholesterol domains (periodicity of 34.0 Å);
peaks labeled as 1 and 2 represent coherent
scattering from the surrounding membrane lipid bilayer phase.
View larger version (64K):
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Fig. 2.
Two-dimensional diffraction patterns obtained
from oriented clear and cataractous human lens fiber cell plasma
membrane samples. Data were collected on x-ray film at 20 °C,
87% RH. Meridional diffraction patterns were generated from whole lens
membranes isolated from normal control lenses (A) and
cataractous lenses (B). Diffraction bands are labeled as for
Fig. 1.
View larger version (31K):
[in a new window]
Fig. 3.
Comparative effects of relative humidity on
the structure of normal (A) and cataractous
(B) lens fiber cell plasma membranes. X-ray
diffraction patterns were collected on a one-dimensional,
position-sensitive electronic detector at 20 °C and over a broad
range of relative humidity levels (52-92%). Diffraction peaks
yielding a unit cell periodicity of 34.0 Å were present at every
relative humidity level (marked by arrows in each panel),
indicating the presence of membrane-restricted cholesterol domains.
Bragg peaks associated with cholesterol domains are more pronounced in
the cataractous tissue.
View larger version (70K):
[in a new window]
Fig. 4.
Comparative effects of temperature on the
structure of normal and cataractous lens fiber cell plasma
membranes. X-ray diffraction patterns were collected on a
one-dimensional, position-sensitive electronic detector at low water
levels and over a broad range of temperatures (5-40 °C).
Diffraction profiles were generated from normal (A) and
cataractous (B) lens membrane samples at 87% RH.
Diffraction peaks associated with cholesterol domains were present at
every temperature level (marked by arrows in each panel) and
were more intense in the cataractous membrane diffraction profiles.
Similar results were obtained with normal (C) and
cataractous (D) membrane samples at 52% RH.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Composition of whole lens membrane samples
The principal finding of this study is that cataractous lens plasma membranes, contrary to our initial hypothesis, contained prominent cholesterol domains. Bragg peaks corresponding to cholesterol domains were more intense and better resolved in the cataract diffraction profiles than cognate peaks in control profiles, suggesting that cholesterol domains are better defined and more ordered in the cataractous lens membrane. These comparative effects were consistent across all experimental conditions (temperature and relative humidity). Cholesterol domains in the cataractous lens membrane were stable over a broad range of temperature and relative humidity levels, as were cholesterol domains in normal lens membranes.
In both normal and cataractous fiber cell plasma membranes, cholesterol domains were observed to coexist with a phospholipid, liquid crystalline bilayer phase that was significantly influenced by changes in temperature and humidity. Such broad changes in the sterol-poor membrane bilayer environment may be due to complex acyl chain, lipid head group, and protein composition of the lens membrane. Several additional and interesting features of the sterol-poor membrane bilayer were observed in this study. First, the relative intensities of the cholesterol peaks were inversely related to relative humidity levels; maximal cholesterol peak intensities were obtained at 52% RH. This relationship was observed in both normal and cataractous lens membranes (see Figs. 3 and 4) and is also consistent with earlier findings from x-ray diffraction analyses of non-cataractous, nuclear and cortical lens membranes (27). One possible explanation of this phenomenon is that optimum structural organization in the lens is achieved at a lower water content. The average water content of the human lens is ~65% (57), and maintenance of proper hydration levels is crucial for optimal refractive power and lens function (58). At 52% RH, the water space between the sample membrane bilayers would be diminished, perhaps imitating the minimized extracellular spaces between the tightly juxtaposed cells of the ocular lens. Second, the sterol-poor component of cataractous lens membranes was more resistant to humidity and temperature modulations than were normal membranes. Over a range of 52 to 92% RH, cataractous membrane periodicity changed by only 25% as compared with 43% for normal membrane periodicity. Similar differences in response to temperature fluctuations were also observed. This may reflect an increase in membrane rigidity associated with these cataracts. The anisotropy or rigidity of lens fiber cell plasma membranes has been shown to increase with aging (59-61) and cataracts (17, 62).
What qualities of the cataractous lens fiber cell membrane would account for more prominent cholesterol domain formation in the presence of diminished levels of cholesterol? One obvious answer would be that these structural changes are a result of extensive modification of lens membrane components, including phospholipids and sterols. For example, membrane lipid oxidation has been shown to be increased in cataract but not normal lens tissue (63), and lipid oxidation products accumulate in the lens as a function of aging (62). If phospholipid-cholesterol interactions are assumed to keep cholesterol domain formation within certain limits, oxidative insult to lens membrane phospholipids may result in reduced phospholipid-cholesterol interaction and increased cholesterol self-association. This hypothesis is supported by experiments conducted in our laboratory showing that oxidative stress independently induces the formation of well defined cholesterol domains in model membrane systems, an effect that is attenuated by treatment with vitamin E.2
Lenticular enrichment of sphingomyelin and its lens-specific derivative, 4,5-dihydrosphingomyelin, may also contribute to maximization of cholesterol domain structures in lens plasma membranes. Sphingophospholipids account for greater than 50% of total lens phospholipids (22) and increase even more substantially with age and cataract development (4, 5, 61, 64). Age-related accumulation of sphingomyelin is also correlated with membrane rigidity (60). The exact role of sphingomyelin and dihydrosphingomyelin in cholesterol domain formation remains to be elucidated, but it can be speculated that their rigid molecular structure may resist strong cholesterol interactions, thus promoting cholesterol clustering.
The biological or functional significance of decreased cholesterol
content and increased cholesterol domain structural organization in
cataracts is not completely understood. Previous studies demonstrating the presence of cholesterol domains in normal lens membranes suggest that cholesterol domains are necessary for transparency or are at least
conductive to transparency at some level (27). Perhaps excessive
cholesterol domain formation contributes to cataractogenesis by
permitting the binding of soluble lens proteins (crystallins) to the
fiber cell plasma membrane. Although a certain basal level of
crystallin association with fiber cell membranes occurs in normal
lenses, massive binding of crystallins to lens membranes occurs in
human cataracts (65). Interestingly, similar crystallin binding effects
were observed for lens membranes isolated from rats treated with
U18666A (66, 67), suggesting that changes in membrane lipid composition
were related to crystallin-membrane binding. Tang et al.
(68) recently demonstrated that increasing the cholesterol content of
model membranes enriched with sphingomyelin attenuated the binding of
-crystallin; however, lens membrane-crystallin binding properties
associated with cholesterol domains have not been examined. These data
suggest a relationship between cataracts, low membrane cholesterol
levels, and the abnormal or excessive association of soluble lens
protein with fiber cell plasma membranes. The structural state of
cholesterol in this process has not yet been identified. Based on
available data, it is hypothesized that recruiting cholesterol out the
phospholipid regions of the membrane bilayer to form larger sterol
domains may result in a net increase in crystallin association with
total lens membrane.
Finally, it should be noted that these findings represent developments
in mature cortical or nuclear cataracts and may not reflect all the
changes that occur in earlier and later stages of cataract progression.
Accurate measurement of changes in membrane structural organization at
later stages of cataract may be complicated by the fact that extensive
membrane vacuolization and disintegration have been reported to occur
in advanced forms of cataract (10, 69). However, structural analyses of
fiber cell membranes derived from younger, healthy lenses as well as
from more progressively diseased lenses will be important in providing
further insights into the pathogenesis of cataracts.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Douglas Borchman and Vittorio Rasi for providing the control and cataractous lenses, respectively, for this study.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant PPG HL22633 (to R. P. M.) and National Institutes of Health Grant EY02568 (to R. J. C.).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.
To whom correspondence should be addressed: Elucida Research LLC,
P. O. Box 7100, Beverly, MA 01915. Tel.: 978-921-4194 (ext. 12); Fax:
978-921-4195; E-mail: rjacob@elucidaresearch.com.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M010077200
2 R. F. Jacob and R. P. Mason, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: C/P, cholesterol/phospholipid; RH, relative humidity.
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REFERENCES |
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