THERMAL AGGREGATION OF AQP0 AND PROTECTION BY a CRYSTALLIN

1
THERMAL AGGREGATION OF AQP0 AND PROTECTION BY a
CRYSTALLIN  Students Erin Farr and Shaunte
CookFaculty Advisor Dr. S. Swamy-Mruthinti,
Department of Biology
Effect of heat on AQP0 aggregation
Separation of lens soluble fraction
SUMMARY Aquaporin 0 (AQP0) is a transmembrane
protein, which transports water across the cell
membrane of lens fiber cells. Normal function of
AQP0 is essential to maintain cellular
homeostasis and lens clarity. When thermally
stressed, AQP0 will aggregate and denature,
possibly affecting its water transport function.
a Crystallin is a molecular chaperone, protecting
other proteins when they are undergoing
stress-induced denaturation. The goal of our
summer research is to show the interaction
between a crystallin and AQP0. Thermal
denaturation of calf lens AQP0, in the presence
or absence of a crystallin was studied either in
the native lens membranes or after solubilizing
AQP0 in octyl glucoside. These aggregates were
characterized either by gel permeation high
pressure liquid chromatography (by Ms. Erin Farr)
or by immunochemical analysis (by Ms. Shaunte
Cook). This study clearly demonstrates that
thermal stress aggregates the transmembrane
protein AQP0, and a crystallin prevents the
aggregation of AQP0 by its chaperone-like
activity.
g
b
Aggregate AQP0

• HPLC Analysis Conclusions
• Thermal stress destabilizes AQP0 and a
  crystalline, the molecular chaperone, protects
  AQP0 from thermal aggregation.
• Although the nature of aggregation of AQP0 during
  the thermal stress is far from clear, our data
  suggests that AQP0 aggregation is a multi-step
  process.
• a crystallin appears to follow the trend of AQP0
  aggregation. Though it prevents early
  aggregation, as temperature and time increases
  the chances of the protein staying intact become
  more limited.

a
AQP0 heated at 50C for 12 min
Calf Soluble Fraction
Normal AQP0
g crystallin
AQP0 control (unheated)
b crystallin
a crystallin
Top chromatogram separation of lens soluble
fraction containing a, b and g crystallins Next 3
chromatograms (top to bottom) Rechromatography
of HPLC purified g, b and a crystallins,
respectively.
Top chromatogram AQP0 heated at 50 C. for 12
min (note that the most of the AQP0 is aggregated
upon heating, hence eluted as an aggregated
peak. Bottom Chromatogram AQP0 control
(unheated)
AQP0 with a
AQP0 with a
AQPO
AQP0
AQP0
60 C
60 C
12 min
40 C
12 min
9 min
40 C
9 min
6 min
0 C
6 min
3 min
1 min
Experimental Procedures 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 PBS (Sigma
chemicals, St. Louis, MO), and centrifuged at
12,000 rpm for 10 min at 4 C. 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. 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 4 C. The protein concentration was
adjusted to 1 mg/ml and used in different assays.
  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 20 C using a circulating water
jacket. The same column was used to separate a, b
and g crystallins from calf lenses, excepting the
Immunochemical Characterization The
corresponding proteins that were used in the HPLC
separation were used in the immunochemical
analysis. Two experiments were conducted, one
that tested the aquaporin protein alone. The
other western blot tested the crystallins. These
two blots were then compared, and the
corresponding results were recorded.
3 min
1 min
Time elapsed 1 hr
Thermal aggregation of AQP0 and Proteoliposomes,
protection by a crystallin Octyl glucoside
solubilized AQP0 (1mg/ml) was heated for 1 hr
with heat increase. AQP0 undergoes aggregation
in the absence of a chaperone protein. a
crystallin protected such thermal aggregation,
causing AQP0 to remain in a functional state.
HPLC was used to show the interaction of AQP0 and
a crystallin following thermal-stress..
Thermal aggregation of AQP0 with a crystallin
chaperone Octyl glucoside solubilized AQP0
(1mg/ml) was heated for 12 min in 3 min intervals
at 50 C. AQP0 undergoes aggregation in the
absence of a chaperone protein. a crystallin
protected such thermal aggregation, causing AQP0
to remain in a functional state. HPLC was used to
determine the effects a crystallin has on AQP0 as
a chaperone.
Thermal Aggregation of AQP0 Octyl glucoside
solubilized AQP0 (1mg/ml) was heated for 50 C
(1-12 min). Due to aggregation, AQP0 did not
pass through the column and possibly stuck to the
precolumn hence reduced size of the peaks.
Stained Gel
Western Blot
AQP0 antibody
a A antibody
a B antibody
AQP0
a Crystallin
0 C
40 C
50 C
60 C
70 C
80 C
90 C
Immunochemical analysis In order to visualize
the immunoreactivity in the native confirmation,
we used dot-blot analysis, whereas western-blot
allowed us to characterize the protein at their
monomeric level. Dot Blot About 1 ug of protein
was applied to nitrocellulose filters and allowed
to air-dry. Other sites on the filter were
blocked with 1 non-fat dry milk for 1 hr.
Primary antibodies (anti-AQP0, anti-alpha
crystallin) were diluted 15000) and added and
allowed to react for 1 hr, followed by washing
the filters with Tris buffered saline containing
0.5 Tween 20 (TBST). The filters were incubated
with alkaline phosphatease conjugated secondary
antibody (either raised in rabbit for polyclonal
or raised in mice for monoclonal primary
antibodies) for 1 hr. The filters were washed
with TBST and incubated with alkaline phosphatase
substrate to visualize the antigen-antibody
reactivity. Western blot Western blot analysis
was done similar to dot blot, excepting that the
proteins were first separated on SDS-PAGE,
transferred onto PVDF membrane. These membranes
were blocked with 1 non-fat dry milk, incubated
with primary and secondary antibodies. The
immunoreactivity was visualized by adding the
substrate for alkaline phosphatase.
M 0 C 40 C 50 C 60 C
70 C 80 C 90 C
M 0 C 40 C 50 C 60 C
70 C 80 C 90 C
Immunochemical characterization of alpha
crystallin interaction with AQP0 during
thermal-stress in the lens membranes 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 (0 C - 90 C for 1 hr)
in the presence of calf alpha crystallin.
Following the thermal-stress, the membranes were
centrifuged and the unbound a crystallin (the
supernatant fraction) was removed. The membranes
were then washed 3 times with PBS, solubilized in
SDS-PAGE buffer and resolved (in duplicate) on
4-20 gradient SDS-PAGE. One gel was stained with
coomassie blue and the other gel was blotted onto
PVDF membranes and probed with anti-alpha
crystallin antibody
Conclusions from Immunochemical
Characterization 1. As shown in the dot blot,
neither alpha A nor alpha B crystallins bind to
AQP0 under normal conditions. However, when the
AQP0 was subjected to thermal-stress, there was
an increased level of binding of a crystallin
suggesting its interaction with AQP0. 2. The
western blot analysis also shows increased
binding of alpha crystallin to AQP0 when the
later was subjected to thermal stress.
Acknowledgements These studies were supported by
NSF STEP grant   DUE-0336571