Proceedings of The National Conference
On Undergraduate Research (NCUR) 2006
The University of North Carolina at Ashville
Ashville, North Carolina
April 6-8, 2006
Aquaporin 0 and alpha-crystallin interaction during thermal stress
Joseph Fachini and Ajay Pillai
Laboratory of Ocular Proteomics
Department of Biology
University of West Georgia
Carrollton, GA 30118, USA
Faculty Advisor: Dr. S. Swamy-Mruthinti
Abstract
Aquaporin 0 (AQP0) is a transmembrane protein. Its function is to transport water across the
cell membrane of lens fiber cells. Normal function of AQP0 is essential to maintain cellular
homeostasis and lens clarity. -crystallin is a molecular chaperone, protecting other proteins
during stress-induced denaturation. Recent studies from our mentor’s lab have shown that crystallin protects AQP0 from heat-induced aggregation. Although AQP0 is the predominant
transmembrane protein of the lens fiber cells, several studies demonstrated that it is not a binding
site for  crystallin under normal conditions. However, nothing is known about the potential
binding of a crystalline under metabolic or environmental stress conditions. This study aims to
show whether -crystallin specifically binds to AQP0 under thermal stress, possibly to protect
the latter from heat-induced denaturation. We used chromatographic and immunochemical
analyses to study such interaction. HPLC analysis showed temperature and time-dependent loss
of monomeric AQP0 when incubated alone. Initially there was an increase in the aggregated
AQP0, however, at higher temperatures and at longer times intervals, this aggregate peak also
diminished, possibly due to the formation of super-aggregates that could not pass through the
HPLC column. Presence of-crystallin during the thermal stress did not change the loss of
monomeric AQP0, however, it prevented the formation super-aggregates. There was an increase
in the HPLC peak representing the -crystallin-AQP0 complex. Immunochemical analysis
confirmed the temperature and time-dependent increase in the amount of -crystallin bound to
AQP0. Interestingly, neither  nor  crystallins show any such interactions. These data suggest
that the binding of -crystallin to AQP0 and the resultant protection of the transmembrane
protein are a direct result of the chaperone-like function of -crystallin. The degeneration of the
chaperone-like activity of -crystallin during aging may be directly associated with the
aggregation of AQP0 and the subsequent formation of cataracts.
Keywords: Aquaporin 0, Alpha crystallin, Thermal Stress, Chaperones, Cataracts
1. Introduction:
The sole function of vertebrate eye lens is to transmit the incident light onto the retina. The
orderly network of lens fiber cells with an intercellular space less than a wavelength of light is
essential for the transmittance of the light. Much of the function of the eye lens is dependent on
the circulating water currents that bring in nutrients and eliminate the metabolic wastes (Mathias
et al., 1999). In an avascular tissue like lens, such water currents are essential to maintain lens
clarity. Cataracts, the cloudy-formations found within the eye lens, are the leading cause of
blindness worldwide with the major risk factors of cataract development being aging and
diabetes. After the age of 65, the risk and incidence of cataracts increases exponentially. This
coupled with the increasing longevity of the average American makes cataracts a major
healthcare issue in the United States.
The lens fiber cells of the vertebrate eye lens contain crystallins in the cytoplasm and
Aquaporin 0 in the cellular membrane. Of the three major crystallins, , , and -crystallins, crystallin is the most prevalent protein in the adult mammalian lens. This alone belongs to small
heat shock proteins that were shown to protect other protein from stress-induced denaturation
and aggregation, UV radiation and exposure to free oxygen radicals. -crystallin is a
heteropolymeric protein composed of the subunits A(HspB4) and B(HspB5) in a ratio of 3:1
respectively (Bhat et. al., 1989). In the lens, it consists of 30-40 of these subunits and has a
molecular weight of 600-800 kda (Bhat et. al., 1989). It is theorized that -crystallin prevents
protein denaturation by a molecular chaperone-like function (Horwitz et al., 1992).
Aquaporin 0 is exclusively expressed in the lens and transports water across the lens fiber cell
membranes (Meizel et al., 1985). It belongs to a family of Aquaporins, a group of proteins
transporting water and glycerol in cells from bacteria to humans. AQP0 provides the mechanism
for the circulating water currents described above. In order to maintain lens fiber cell
homeostasis, it is highly essential that the structural integrity of AQP0 be maintained. It has
been shown that AQP0 undergoes aggregation when subjected to thermal stress (SwamyMruthinti et. al., 2002). It is expected that aggregated AQP0 may cause a loss of transparency in
the lens and development of cataracts. This study is aimed to show the interaction of -crystallin
with AQP0 when the latter is subjected to thermal stress.
2. Material and Methods:
2.1 Lenses:
Calf lenses were purchased from Pelfreeze, (Little Rock, AK) and stored at –80 C until use.
Decapsulated calf lenses were homogenized in ice-cold phosphate buffered saline (PBS from
Sigma chemicals, St. Louis, MO), and centrifuged at 12,000 rpm for 10 min at 40C. The pellet
was washed sequentially twice with PBS, twice with 7M urea (prepared in PBS), twice with 0.1
N NaOH and again twice with PBS. The pellet was recovered by centrifugation at 12,000 rpm
for 10 min at 4 C. The membrane pellet was suspended in PBS and used in this assay.
2.2 Solubilization of AQP0:
The lens transmembrane protein, AQP0 was selectively solubilized in non-ionic detergent octyl
b-D-glucopyranoside (octylglucoside, from Calbiochem, La Jolla, CA). Dry powder of
octylglucoside was added to the membranes, sonicated for 30 sec in a bath type sonicator and
allowed to stand on ice for at least 1 hr, followed by centrifugation at 12,000 rpm for 10 min at
40C. The protein concentration was adjusted to 1 mg/ml and used in different assays.
2. 3 Thermal denaturation of AQP0 and the effect of -crystallin.
Octyl glucoside solubilized AQP0 was incubated either alone or with -crystallin (1:1 protein
weight ratio), at different temperatures (20 – 500C for 1 hr) or for different time intervals (0-24
min) at 500C. The proteins were placed on ice, till further analysis.
Similarly, isolated lens membranes were incubated at different temperatures (20-90 C) either
alone or with -crytallin (1:1 protein weight ratio). The incubated membranes were washed with
ice-cold PBS for 4 times and recovered by centrifugation at 12,000 rpm for 10 min at 40C. The
washed membranes were solubilized in 2% octyl glucoside in PBS and used in further studies.
2.4 HPLC Characterization:
AQP0 was solubilized in octyl glucoside and separated on SEC 3000 SW (60 mm) column with
a flow rate of 1 ml/min and the absorbance was monitored at 280 nm and at 0.05 AUF. The
mobile phase was 2% octyl glucoside in PBS. The temperature of the column and the mobile
phase was kept at 200C using a circulating water jacket.
2.5 Immunochemical Analysis:
In order to test and visualize the reactivity between the crystallins and the AQP0, dot-blot
analysis was instituted. The Western-Blot method allowed for the characterization of the protein
at the monomeric level.
2.5.1 Dot-blot Method:
Nitrocellulose paper filters are prepared by incubation with alkaline phosphatase conjugated
secondary antibody. The antibodies are raised in rabbits for polyclonal expression or in mice for
monoclonal expression. Approximately 1 ug of protein was applied to nitrocellulose filters and
allowed to air-dry. The blots are placed in a reaction tube. The remaining sites on the filter were
blocked by adding 1% non-fat dry milk and incubated with shaking for one hour, after which the
blocking buffer is removed. Primary antibodies, anti-AQP0 and anti-alpha crystallin, are diluted
in a 1:5000 ratio and poured into the tube. The tube is placed on a shaker in an incubator. The
reaction takes place over the course of one hour. The blots then wash in TBST buffer, composed
of Tris buffered saline containing 0.5% Tween 20. The blots wash for fifteen minutes and then
the old TBST buffer is removed and new buffer is added. This is done until the blots have been
washed three times. The blots then undergo reaction with the secondary antibody for one hour.
Afterwards, the antibodies are removed and the blot is washed again. The blots are incubated
with alkaline phosphatase substrate, which allows for the visualization of the relative reaction of
the different blots. They are then rinsed with water and removed for analysis.
2.5.2 Western Blot Method:
The proteins are separated by SDS-PAGE gel electrophoresis and transferred onto PVDF
membranes. These membranes are then blocked by incubation in 1% non-fat dry milk for one
hour. After the blocking buffer is removed, The membranes were probed with primary antibody
for one hour, washing, reaction with the secondary antibody for one hour, and then an additional
washing. Incubation with alkaline phosphatase substrate allows for the visualization of the blot.
3. Results
3.1 HPLC characterization of -crystalline interaction with AQP0 during thermal-stress.
AQP0 aggregate
0 min at 50 C
AQP0
+ AQP0 complex
AQP0
0 min at 50 C
3 min at 50 C
3 min at 50 C
6 min at 50 C
6 min at 50 C
9 min at 50 C
9 min at 50 C
12 min at 50 C
12 min at 50 C
12 min at 50 C
Fig. 1 HPLC characterization of thermal aggregation of AQP0
Figure 1 (left side chromatographs) – Octyl glucoside solubilized AQP0 was subjected to
thermal stress for different time intervals at 500C either alone, or mixed with a-crystallin at 1:1
protein weight ratio. The first peak is the AQP0 aggregate (about 1 million da molecular
weight). The AQP0 is eluted at110 da (equivalent to AQP0 tetramers). The last peak is excess
octyl glucoside (used as internal control). Right side chromatographs – -crystallin was added to
the AQP0 prior to incubation. The -crystallin is eluted at 600-800 da (equivalent to functional
oloigomer of 40 monomers). The peak at about 900 kda is equivalent to -crystallin + AQP0
complex.
As shown in figure 1, the normal AQP0 eluted as 110 kda peak, which is equivalent to
tetrameric composition of normal AQP0. Upon incubation for 3 min at 50 C, there was increase
in the AQP0 aggregate peak with a concomitant decrease in normal 110 kda peak. Interestingly,
further increase in the time of incubation resulted in the decrease in both aggregated and normal
AQP0 (fig. 1 left-side chromatographs). Addition of -crystallin during incubation resulted in
an increase in the peak representing the -crytallin and AQP0 complex. Increase in the time of
incubation resulted in the loss of normal 110 kda peak with a concomitant increase in the size of
peak of -crystallin and AQP0 complex.
3.2 Immunochemical characterization of -crystallin interaction with AQP0 during
thermal-stress.
In order to show whether there is any nonspecific association of a-crystallins to the membranes
or artifacts during membrane preparations, as well as to check the specificity of the antibodies,
normal membrane preparations and HPLC purified a-crystallin samples were applied the
nitrocellulose membranes and probed with AQP0 and A- and B-crystallin antibodies. As
shown in figure 2, only the fist dot reacted with AQP0 and only the second dot reacted with A
and B-crystallin antibodies.
Figure 2 Specificity of antibodies used in the study – dot blot analysis
Figure 2 showing the specificity of antibodies. About 5 ug of lens membranes and equal quantify
of alpha crystalline were applied to nitrocellulose filter and probed with respective antibodies.
0C
40 C
50 C
60 C
70 C
80 C
AQP0
-crystallin
Figure 3 Time and temperature dependent interaction of a-crystallin and AQP0 during thermal
stress.
Figure 3 showing the interaction of a-crystallin with AQP0 when the alter is undergoing thermal
stress. There is a time and temperature dependent increase in the interaction.
90 C
In order to show whether thermal-stress induced interaction occurs between AQP0 and alpha
crystallin in the native lens membranes, the membranes were subjected to thermal-stress (either
for different time intervals at 500C or different temperatures 0 – 900C) in the presence of calf crystallin at a weight ratio of 1:1. Following the thermal-stress, the membranes were washed
with ice-cold PBS, solubilized and analyzed by dot-blot analysis, using AQP0 and -crystallin
antibodies.
4. Discussion and Conclusions
This study clearly shows that -crystallin interacts with AQP0 during thermal-denaturation.
HPLC analysis showed temperature and time-dependent loss of monomeric AQP0 when
incubated alone. Initially there was an increase in the aggregated AQP0, however, at higher
temperatures and at longer times intervals, this aggregate peak also diminished, possibly due to
the formation of super-aggregates that could not pass through the HPLC column. Presence ofcrystallin during the thermal stress did not change the loss of monomeric AQP0, however, it
prevented the formation super-aggregates. There was an increase in the HPLC peak representing
the -crystallin-AQP0 complex. Similarly, immunochemical analysis confirmed the temperature
and time-dependent increase in the amount of -crystallin binding to AQP0. Interestingly,
neither  nor  crystallins show any such interactions. These data suggest that the binding of crystallin to AQP0 and the resultant protection of the transmembrane protein are a direct result of
the chaperone-like function of -crystallin. The degeneration of the chaperone-like activity of
-crystallin during aging may be directly associated with the aggregation of AQP0 and the
subsequent formation of cataracts.
5. Acknowledgements
The authors wish to express their sincere thanks to Dr. John Hansen for his help with the
chaperone studies. The authors also appreciation the help of Ms. Patricia Onuegbu, Ms. Erin
Farr and Ms. Shaunte Cook with the HPLC and immunochemical studies. This study has been
funded by grants from NSF-STEP program and intramural grants from University of West
Georgia (to Dr. SSM).
6. References
Harry Maisel, The Ocular Lens: Structure, Function, and Pathology (New York, New York,
Marcel Dekker, INC., 1985), Ch. 5, Biochemistry of Lens Plasma Membranes and Cytoskeleton
(Jose Alcala and Harry Maisel, Wayne State University School of Medicine, Detroit, Michigan).
Bhat, S.P., and Nagineni, C.N. (1989) Alpha B subunit of lens specific protein alpha-crystallin is
present in other ocular and non-ocular tissues. Biochem. Biophys. Res. Commun. 158, 319325.
Swamy-Mruthinti, S., Srinivas, V., Hansen, J.E. and Mohan Rao Ch. (2002) Prevention of
Thermal aggregation of AQP0 by α crystallin, Exp. Eye Res., 72 (suppl. 2), p40.
Horwitz J, Alpha Crystallin can Function as a Molecular Chaperone, (Proc Natl Acad Sci USA
1992), 89: 104499-53
Mathias, R. T. and Rae, J. L. Transport Properties of the Lens (1985), Am. J. Physiol. 249,
C181-90