(Received for publication, July 13, 1995; and in revised form, October 13, 1995)
From the
Several lines of transgenic mice were generated with either
active or inactive forms of the human immunodeficiency virus type 1
(HIV-1) protease gene under the control of the mouse lens
A-crystallin promoter. Mice bearing the inactive protease coding
sequence displayed no gross abnormalities in the lens, while mice with
the active protease developed time-dependent bilateral cataracts. One
line, TG
, developed cataracts in utero while the
second line, TG
, developed cataracts postnatally.
TG
mice, homozygous for the transgene, developed severe
microphthalmia and were significantly smaller than the control mice at
postnatal day 30. two-dimensional-polyacrylamide gel electrophoresis
analysis of the protein profiles of TG
and TG
lenses revealed extensive modifications in the lens crystallins.
Proteolysis in the homozygous TG
mouse lenses began at
postnatal day 20 with the disappearance or partial loss of
B1-,
B3-, and
A3-crystallins and the appearance of crystallin
fragments. Protein leakage and the gradual breakdown of cytoskeletal
elements also occurred. In contrast, the opacification of the
homozygous TG
lenses appeared to have been influenced by
differentiation and developmental processes. It apppears that HIV-1
protease expression activates other proteases, and these enzymes, in
concert with the HIV-1 protease, are responsible for the protein
modifications that eventually result in the opacification of the lens.
The pandemic spread of AIDS has promoted intense efforts to
understand the key steps in the life cycle of the human
immunodeficiency virus (HIV). ()HIV primarily targets
T-lymphocytes resulting in defective cell-mediated
immunity(1, 2) . Opportunistic infections,
neurological and neoplastic disorders, and, ultimately, death occurs
because of the reduced immunity.
A transgenic model was developed where mice carried the HIV-1 genome defective only in reverse transcriptase activity. These mice developed cataracts between 3 and 6 months of age(3) . Recently, in a study of over 100 patients infected with HIV, ocular lens opacities were reported in approximately 52% of the patients(4) . These developments have led to interesting questions concerning a possible role of HIV in cataract formation.
The vertebrate lens is primarily composed of proteins
known as crystallins, which account for 90% of its total soluble
protein. In the mammalian lens, these proteins are divided into three
major families: -,
-, and
-crystallins(5) .
-Crystallin is an oligomer of approximately 800 kDa and is
composed primarily of two subunits,
A and
B. The
-crystallins are also oligomers, and they elute in two or more
size classes during gel filtration and range from 50 to 200 kDa. They
are composed of seven different types of subunits,
B1,
B2,
B3,
A1,
A2,
A3, and
A4.
-Crystallins
share sequence homology with the
-crystallins; however, the
-crystallins are exclusively monomeric. It is the organization of
these crystallins that maintains the transparency of the lens.
Denaturation and degradation of the crystallins have been shown to
occur during the formation of several experimental cataracts.
Constructs were made linking only the HIV-1 protease to the lens
A-crystallin promoter. The transgenic animals that were produced
developed cataracts. By examining the progression of lens opacities in
these transgenic mice, the relationship between HIV, specifically the
HIV-1 protease, and the pathways leading to cataractogenesis were
explored.
Transgenic mice were constructed by linking the HIV-1
protease to the lens A-crystallin promoter. This was accomplished
using the pMSG mammalian expression vector (Pharmacia Biotech Inc.),
which was modified by replacing the murine mammary tumor virus long
terminal repeat promoter region with the 412-bp BglII-BamHI mouse lens
A-crystallin promoter
fragment (6) kindly provided by Dr. Joram Piatigorsky (National
Eye Institute, NIH). A single chain, tethered dimeric form of the
active HIV-1 protease gene, termed baa(7, 8) , was modified for mammalian cell
expression by replacing nucleotides 7-90 with
CGTAATAGAAGGAGATATAACCATGGAG and blunt ended and cloned into the SmaI site of the pMSG vector. The protease dimer consisted of
two 99-amino acid subunits linked by Gly-Gly-Ser-Ser-Gly. The mutant
form of this construct was produced by site-directed mutagenesis (GAT
to GGT), resulting in the change of aspartic acid to glycine (25th
residue) in both monomers, thus creating an inactive form of the
protease, designated ba*a*. HindIII digestion of
these plasmids liberated a 2.5-kilobase transgenic construct that
included the
A-crystallin promoter, HIV-1 protease dimer, and SV40
splice and poly-adenylation signals.
The 2.5-kilobase HindIII transgenic constructs containing the active and inactive forms of the HIV-1 protease gene were purified and injected into the pronuclei of 1-day-old fertilized mouse eggs(9, 10) . Mice of the FVB/N line (Charles River Laboratory, Portage, MI or Taconic Farms, Inc., Germantown, NY) were used for the construction and breeding of these transgenic mice(11) . Transgenic mice were identified by standard PCR analysis of purified tail DNA (10) .
Two PCR primers were designed from within the SV40 splice and polyadenylation segment of the transgene: TCGACCTCGAGGGATCTTTG and CCTCTACAAATGTGGTATGGC (positions 41-60 and 646-666 on the pMSG). mRNA expression of the transgene was analyzed using the Perkin Elmer GeneAmp RNA PCR kit and recommended procedures for RT-PCR. Because the SV40 splice site is located within the region amplified by the two oligonucleotide primers, the PCR fragment derived from genomic DNA contaminating the total RNA preparations (or unspliced RNA) is 625 bp long, while the fragment derived from cDNA (and thus spliced mRNA) is 559 bp long.
Strictly adhering to the procedures set forth in the NIH Guidelines for the Care and Use of Laboratory Animals, all transgenic mice were bred for hemizygous and homozygous expression of the HIV-1 protease transgene and compared with sibling control FVB/N mice. Comparison was made on the basis of gross physical characteristics, such as weight and length, and visual examination of the eyes for lens clarity. Pups were euthanized postnatally up to day 30, and the lenses were carefully dissected and stored at -70 °C. The date of birth was considered to be postnatal day 1.
Slot blots were carried out as
described previously (12) for the semi-quantitative analysis of
HIV-1 protease protein content in the lens homogenates.
Affinity-purified recombinant HIV-1 protease (Bachem Bioscience, Inc.)
was used as a standard. In all experiments, the sample volume was 25
µl. Immunoreaction was with rabbit IgG prepared against a synthetic
peptide, GQLKEALLDTGADDTVL, from HIV-1 protease
(underlined and bold is the active site). Immunodetection was with the
Western Light Chemiluminescent Detection System (Tropix, Bedford, MA).
Densitometric analysis was as described previously(13) . For
each condition, quantitation of protein concentration was determined by
analysis of the volume values (pixels/mm) obtained from
three individual runs.
Lens protein patterns were characterized by single and two-dimensional SDS-PAGE using the Pharmacia PhastGel Separation and Development System (14) . Overall protein patterns were obtained by homogenization of the lenses in 8 M urea.
Lenses were also homogenized in 150 µl of 50
mM Tris-HCl, pH 7.4, 0.1 M KCl, 1 mM EDTA,
10 mM -mercaptoethanol, and 0.05% sodium azide. These
homogenates were microfuged at maximum speed for 10 min, and the
supernatant was drawn off and treated as the water-soluble fraction.
The pellet was resuspended in 150 µl of 8 M urea, and the
supernatant was treated as the urea-soluble (water-insoluble) fraction.
The remaining pellet was solubilized in 10 µl of 1% SDS for several
hours and treated as the urea-insoluble fraction. For the application
of each sample to the first-dimension urea-IEF gel, 2 µg of total
protein was loaded into each well. The pI gradient was set up using
Pharmalyte 3-10 (Pharmacia). Transfer of the proteins to the
second dimension was carried out on 12.5% Phastgels.
HIV-1 protease
digests were carried out with affinity-purified recombinant HIV-1
protease (Bachem Bioscience). Column-purified bovine or mouse -,
high (
-) or low (
-) molecular weight
-crystallins, or
-crystallins (4 µg each) were incubated
with HIV-1 protease (0.5 µg) at 37 °C overnight. Crystallins
were also incubated in the presence or absence of 20 mM EDTA
and/or 7 mM CaCl
. After the incubation, an aliquot
(5 µl) of the HIV-1 protease-digested material was mixed with an
equal volume of SDS gel sample buffer. The samples were boiled for 3
min and then loaded onto 12.5% homogeneous Phastgels (Pharmacia).
Following the same procedure, control mouse lens homogenates were also incubated with purified recombinant HIV-1 protease. Homogenates obtained from 10- and 22-day-old control mice were incubated with purified HIV-1 protease in ratios of 10:1 or 30:1 (sample to protease). After incubation, this material was run on both single and two-dimensional-gels.
Vimentin and actin antibodies were obtained
from Sigma and BioDesign, Inc. (Kennebunkport, ME), respectively.
B1-crystallin antibodies were a gift from Dr. Joseph Horwitz
(Jules Stein Institute, UCLA), and antibodies to
A3-crystallin
were obtained from Drs. John N. Hope and J. Fielding Hejtmancik
(National Eye Institute, NIH).
Cataractogenesis was observed in several transgenic lines,
albeit, at different times (Table 1). Those mice containing the
transgene with the mutation in the active site of the protease
exhibited no lens opacities. Two of the lines that developed cataracts
will be reported here. TG mice developed cataracts in
utero. Homozygous expression of the transgene in this line
resulted in severe microphthalmia and cataract formation (Fig. 1a), while hemizygous expression was
characterized only by the formation of a cataract in utero.
Severe microphthalmia was not observed in the lenses from the
hemizygous TG
mice. In the second line, TG
,
homozygous expression of the transgene resulted in the development of
lens opacities 23-24 days postnatally, while in mice hemizygous
for this transgene, cataract formation was delayed to postnatal days
25-30. In the homozygous TG
mice, cataract formation
was observed to begin in the perinuclear region of the lens (Fig. 1b). After liquification of the cortical region,
the cataract appeared to migrate to the back of the lens. The
cataractous lenses from both lines of hemizygous mice appeared similar
in appearance to the lenses of the homozygous TG
mice,
although the lenses of the TG
mice were slightly smaller
(not shown).
Figure 1:
Homozygous mice were
photographed on postnatal day 27. a, TG mouse
eye; b, TG
mouse eye.
For ease of comparison, only the results obtained from
the homozygous mice from the two lines will be discussed unless
otherwise noted. RT-PCR analysis was used to confirm the presence of
HIV-1 protease mRNA in the TG and TG
lines.
As shown in Fig. 2A and Fig. 3, this mRNA was
detected in the lenses of TG
and TG
hemizygous mice. In addition, TG
hemizygous mice
also expressed the mRNA ectopically in other organs tested, such as the
spleen, liver, and kidney (Fig. 3). However, in contrast to the
lens cataract phenotype, there were no gross abnormalities observed in
these organs as a consequence of HIV-1 protease mRNA expression.
Figure 2:
Panel A, RT-PCR analysis of HIV-1
protease mRNA expression in hemizygous TG mice. Lane
1, total RNA extracted from the lenses of hemizygous TG
on postnatal day 20; lane 2, total RNA from cells
transfected with the pMSG vector; lane 3, 123-bp DNA ladder
(Life Technologies, Inc.); lane 4, purified tail DNA harvested
from TG
mice. Panel B, the amount of protease in
each line was examined by slot blot analysis. Lens protein homogenates
(70 µg) were loaded into each slot in replicates of three. Only a
representative sample is shown here. Row a, purified HIV-1
protease controls: 1, 0.05 µg; 2, 0.07 µg; 3, 0.10 µg. Row b, samples of homozygous
TG
homogenates harvested at 10 (1), 20 (2), and 30 (3) days. Row c, homogenates of
homozygous TG
mice harvested on postnatal day 5 (1) and homogenates of control mice harvested on days 10 (2) and 30 (3).
Figure 3:
RT-PCR analysis of the ectopic expression
of HIV-1 protease in different organs of the hemizygous TG
mice. Total RNA was extracted from tissues of 6-week-old mice. Lane
1, 123-bp DNA ladder; lane 2, spleen; lane 3,
kidney; lane 4, liver; lane 5,
eyes.
Slot blot analysis of the lens protein homogenates revealed
extremely low quantities of the protease. It did appear, however, that
in the homozygous mice the HIV-1 protease was expressed early (Fig. 2B). The level of protease found in 10-day-old
TG mice was 0.696 ng of protease/µg total protein.
This level appeared to decrease over the course of the next 20 days.
Protease levels were also detected in 5-day-old TG
mice.
Accurate quantitation of the decrease in the level of protease over
time could not be determined because the amount of protease was only
slightly above background on the blot.
When comparing the size and
weight of the mice, it was observed that the homozygous TG mice were significantly smaller than their control counterparts (Fig. 4). At postnatal day 5, the TG
animals (n = 6) weighed 2.79 ± 0.175 g, while 5-day-old control
mice (n = 6) were 3.21 ± 0.209 g (p < 0.01). By postnatal day 20, the TG
mice weighed
8.49 ± 0.208 g in comparison to the 11.53 ± 0.282 g (p < 0.001) of the controls. The pups were weaned
approximately at day 23, yet by postnatal day 30, the TG
pups weighed only 15.30 ± 0.38 g in comparison to the
17.99 ± 0.695 g weighed by the controls (p < 0.001).
In addition to weight, size was also checked. Mouse length was measured
from snout to rump. At 5 days, the body of the TG
mice was
3.61 ± 0.174 cm in length. In contrast, the length of control
mice was 4.07 ± 0.249 cm. By day 20, the TG
mice
were 5.06 ± 0.450 cm in length, and the controls were 7.12
± 0.512 cm. The weight and length of the hemizygous TG
mice appeared to be only slightly less than the control mice
(data not shown). In contrast to the findings obtained with the
TG
mice, the size and weight of both the homozygous and
hemizygous TG
mice were comparable to the control mice (Fig. 4).
Figure 4: Graphic representation of the differences in weight between the two mouse lines homozygous for the HIV-1 protease transgene.
Preparation of the lens homogenates for gel
analysis revealed differences in the protein concentration of the
lenses. Lenses from four mice were harvested and homogenized
separately. The protein concentrations were determined at each time
point and statistically analyzed. Control lenses harvested at postnatal
day 5 contained 0.256 ± 0.142 mg of protein/lens in the
water-soluble fraction and 0.0069 ± 0.018 mg/lens in the
urea-soluble fraction. As expected, the protein concentration of the
water-soluble fractions increased with age to 1.21 ± 0.056 mg of
protein/lens at postnatal day 24/25. The concentration of the
urea-soluble fractions increased in the same manner (Table 2).
Lenses obtained from TG mice exhibited a similar trend of
protein increase with age until postnatal day 22, when the amount of
protein in the water-soluble fraction began to decrease. By day 25, the
amount of protein in the water-soluble fraction of the lens was 0.619
± 0.089 mg/lens. However, the trend of protein concentration in
the urea-soluble fraction followed that observed with the control
lenses (Table 2). Concentrations for the TG
lenses
were difficult to quantitate because of the small size of the lenses.
At postnatal day 5, the water-soluble fraction contained approximately
0.080 mg of protein/lens, and the urea-soluble fraction had 0.037 mg of
protein/lens. The amount of protein present in both the water- and
urea-soluble fractions decreased with age, making it difficult to
calculate accurate protein concentrations. In addition, by postnatal
day 30, the lens was becoming liquified.
Since single dimension
SDS-PAGE analysis of the two lines revealed that there were strong
differences between their protein profiles, two-dimensional SDS-PAGE
was used to closely monitor these modifications. The homozygous
TG mice were observed from birth to postnatal day 30 so
that the protein modifications leading up to cataract formation could
be followed. The progression of protein changes seen in the
TG
mice is shown in Fig. 5. Protein modifications
developed in the same manner for all of the TG
lenses
examined. Although no visually obvious changes were observed in the
lenses prior to the manifestation of the cataract on day 24,
modifications in the proteins began at postnatal day 20 (Fig. 5C, arrow). By day 24, when the cataract
is present, several new protein spots appeared (Fig. 5D).
Figure 5:
Two-dimensional SDS-PAGE analysis of the
progression of lens protein modifications in TG mice
homozygous for the expression of the HIV-1 protease transgene. Lenses
were harvested postnatally on days 5 (A), 10 (B), 20 (C), and 24 (D) and homogenized in 8 M urea.
Control mouse lenses were harvested postnatally on day 24 (E).
The arrows represent the appearance of new protein spots,
while the circle depicts the disappearance of several
proteins. The major crystallins are identified in panel E. 1,
B-crystallin; 2,
A-crystallin; 3,
B2-crystallin; 4,
B1-,
B3-crystallins; 5,
-crystallin; 6,
A3-crystallin; 7,
A4-crystallin; and 8,
A1-crystallin.
It was important to determine whether
these protein modifications were mainly in the water-soluble or
-insoluble fractions of the homogenates. Two-dimensional analysis
comparing the water-soluble profiles of protein extracts from control
and homozygous TG mice revealed that the trend of protein
changes was similar to that observed in the patterns of the lenses
solubilized in 8 M urea (data not shown). Urea-soluble
(water-insoluble) fractions were analyzed, and the patterns were also
found to be similar to the water-soluble profiles, albeit less protein
was present. In addition, at postnatal day 24, the urea-soluble
fractions showed that the
A-crystallins underwent further
processing, and the amount of
-crystallins seemed to decrease
(data not shown). Comparison with the urea-insoluble protein profiles
revealed a marked increase in the amount of
-crystallin (Fig. 6). Profiles of the hemizygous TG
mice were
similar, except the rate of proteolytic processing was slightly delayed
(data not shown).
Figure 6:
Comparison of the two-dimensional gel
profiles of the urea-insoluble protein fractions of TG and
control lenses harvested postnatally on days 10, 20, and
24.
Since there appeared to be a loss of several of
the crystallins from the lenses of the TG mice, Western
blot analyses were used to determine which proteins were specifically
being degraded (Fig. 7). Lens protein homogenates were extracted
from control mice on postnatal days 5 and 24 and from TG
mice at postnatal day 24. After staining with peptide-specific
crystallin antibodies, it was found that
B1- and
A3-crystallins were degraded from the TG
lenses (Fig. 7, B and C). The
-crystallins also
appeared to be degraded (data not shown), and although the
-crystallins did not disappear, it seemed as though they too were
being modified in some manner (Fig. 7A). In contrast,
B2-crystallin did not appear to be affected (Fig. 7D).
Figure 7:
Western
blot analysis of the fate of some of the lens crystallins in the
cataractous lenses of homozygous TG mice. Proteins were
extracted from control lenses on postnatal days 5 and 24 (lanes 1 and 2, respectively) and from TG
mice on day
24 (lane 3 in all panels). All lanes were loaded with
3 µg total protein. Panel A, lane 4,
column-purified
-crystallin. Blot incubated with antibodies
specific for
-crystallin is shown. Panel B, lane
4, column-purified high molecular weight
-crystallin
(
) fraction. Blot incubated with antibodies specific
to
A3-crystallin is shown. Panel C, lane 4,
column-purified
-crystallin fraction. Blot incubated with
antibodies specific for
B1-crystallin is shown. Panel D, lane 4, column-purified
-crystallin fraction.
Blot incubated with antibodies specific for
B2-crystallin is
shown.
The next issue to be addressed was
whether or not the protease itself could cleave the crystallins.
Purified HIV-1 protease was incubated with column-purified mouse or
bovine crystallins. Gel profiles clearly showed that the protease was
indeed capable of cleaving mouse and bovine - and
-crystallins (data not shown). The protease can also cleave mouse,
but not bovine,
-crystallins. The protease-digested protein
pattern was the same even in the presence of EDTA, which was used to
chelate endogenous Ca
(data not shown). The EDTA was
used to prevent activation of Ca
-dependent proteases
known to be present in the rodent lens. Purified HIV-1 protease, when
incubated with control mouse lens homogenates, yielded a modified
protein pattern (Fig. 8A); however, the two-dimensional
profile exhibited a slightly different pattern than the ones obtained
from the lenses of the homozygous TG
and TG
mice (Fig. 8B). Since it has been shown that some
cytoskeletal proteins can be cleaved by HIV-1 protease (15, 16) , the fate of several of these proteins was
examined by Western analysis of whole lens homogenates of the
TG
line. Vimentin is present in the lens in the elongating
but not mature fiber cells. It is a 56-kDa protein that was observed to
exhibit some lower molecular weight bands in the lens homogenates under
normal conditions. By postnatal day 22, the lower molecular weight
components (26-30 kDa) disappeared, and by day 24, the 56-kDa
band was lost leaving only a band of approximately 22 kDa (Fig. 9). Actin followed a similar trend of degradation with
time (data not shown).
Figure 8: The protein profiles of HIV-protease digests of lens homogenates obtained from control mice. Lenses were harvested on postnatal days 10 (A) and 22 (B). In both panels, lane 1, sample homogenate; lane 2, homogenate + HIV-1 protease; lane 3, homogenate + HIV-1 protease + EDTA. Panel C, the two-dimensional gel profile of the HIV-1 protease digest of the 22-day-old control mouse lens (D) compared with the undigested control homogenate.
Figure 9:
Analysis of the fate of vimentin in
protein extracts obtained from the lenses of homozygous TG mice. Lenses were harvested postnatally at days 10 (1),
15 (2), 20 (3), 21 (4), 22 (5), and
24 (6) and homogenized in 8 M urea. Control lenses
harvested postnatally at days 10 and 24 (lanes 7 and 8) were treated in the same
manner.
Although the protein profiles indicated that
there were some differences in the pattern of modifications between the
two mouse lines, two-dimensional analyses of the TG mice
were less informative because the progression of the protein changes
could not be easily monitored since cataract formation occurred in
utero. Protein profiles of the lenses obtained from TG
mice at postnatal day 5 revealed changes in the number of
A-crystallin fragments when compared to control mouse profiles. In
addition, the spot observed at day 20 in the TG
lens
homogenates was also present in the TG
homogenates
obtained at day 5 (Fig. 10). Very little
-crystallin was
present, and most of it appeared to be present in the urea-soluble
fraction. In contrast to what was observed with the TG
mice,
B1- and
A3-crystallins were only minimally
degraded. In addition, these proteins were present even 30 days after
birth, albeit at lower amounts (data not shown).
Figure 10:
Gel
profiles of the lens homogenates extracted from the lenses of
homozygous TG mice on postnatal day
5.
Several genomic products of retroviruses, such as HIV, are initially expressed as large polypeptide precursors. In the life cycle of HIV-1, one of these products, the HIV-1 protease, can cleave the polypeptide precursors into individual functional proteins. This 11.5-kDa enzyme is an aspartyl protease and is required for both polyprotein processing and virus infectivity(17, 18, 19) . It is believed that the protease first cleaves itself out of the large Gag-Pol fusion precursor resulting in Gag and Pol precursors, which the protease then processes to their final forms.
The functions of the protease appear
to make it an ideal target for therapeutic intervention. In studying
various models for this purpose, transgenic mice containing only the
HIV-1 protease were developed. An earlier transgenic mouse model showed
that cataracts developed in mice containing the entire HIV genome
defective only in reverse transcriptase activity. Because of the large
accumulation of the Gag p24 capsid protein in the lenses, it was
proposed that this protein played a role in the formation of cataracts
at 3-6 months of age(4) . However, the data presented in
this study show that HIV-1 protease alone can cause cataractogenesis.
RT-PCR and slot blot analysis revealed the presence of HIV-1 protease
mRNA and protein in the lenses of both the TG and
TG
mice. The protein levels appeared to decrease with time
probably as a consequence of autolysis or protein leakage. The HIV-1
protease is believed to be the initiating factor of the cataract, since
mice bred with a mutation in the HIV-1 protease active site did not
develop cataracts (Table 1) even though HIV-1 protease mRNA was
present (data not shown). In addition, the cataract phenotype was
either reduced or prevented in TG
and TG
hemizygous mice by treating them with HIV-1 protease-specific
inhibitors, (
)thus supporting a primary role of the protease
in cataract formation.
The mechanisms of cataract formation are not
well understood. However, it is hypothesized that once started, the
pathway to opacification proceeds in certain defined steps regardless
of the initiating factor. During the progression of lens opacification
in the TG mice, protein modifications were observed
approximately at postnatal day 20. The disappearance or partial loss of
B1-,
B3-, and
A3-crystallins seems to be concurrent. In
addition, the appearance of a protein band at this same time point
seems to correspond to a modified
A3/
A1 fragment identified
by David et al.(20) . By day 24, a large band appeared
above the
B-crystallin spot, and based upon its location, it is
suggested that it is comprised of aggregates of
-crystallin
fragments. In addition, between days 20 and 24, the gradual breakdown
of the cytoskeletal elements vimentin and actin occurs. This is in
contrast to the data observed from the control lens homogenates, where
no protein modifications were observed in the water-soluble fractions
up to postnatal day 30 and only the processing of
A-crystallins
was observed in the urea-soluble fractions. We examined the progression
of protein modifications during postnatal cataract formation in another
mouse model. (
)The Philly mouse, a strain with an inherited
cataract, has an abnormal
B2-crystallin resulting from an in-frame
deletion, which leads to a loss of 4 amino acids(21) . The
protein profile obtained prior to the formation of the cataract
(postnatal day 20) was similar to that observed for control mice. At
day 35, when a cataract was present, modifications to lens crystallins
were similar to those observed for the TG
mice, i.e. the loss of the
A3-,
B1-, and
B3-crystallins. The
data appear to support the hypothesis that the progression of
opacification is similar in postnatal cataract development.
The
opacification of the TG lenses appeared to proceed in a
slightly different manner. The
A3/
A1 spot present at day 20
in the TG
lens homogenates was also present in the
TG
mice at day 5. However, very little
-crystallin
was present at this time even in the urea-soluble fraction, which
indicates that some of the crystallins were either not expressed or
were leaking out of the lens. In addition, the
B1-,
B3-,
A3-crystallins all appear to be present in the TG
cataract. This suggests that since cataract formation occurs in utero, the mechanism of opacification proceeds in a
slightly different manner and may be influenced by effects of the
concurrent differentiation and early developmental processes. In
addition, the smaller size of the TG
mice compared to the
TG
or control animals might be attributed to the ectopic
expression of the HIV-1 protease gene and/or a difference in its site
of integration, which would result in an insertional inactivation of an
essential cellular gene.
Analysis of the protein concentration of
the TG lens homogenates revealed that the amount of
protein decreased in the water-soluble fraction with time. However, the
protein concentration of the urea-soluble fraction did not increase
concomitantly. Although there seems to be an increase in the protein
concentration of the urea-insoluble fraction, it is likely that protein
leakage from these lenses is also occurring. Protein leakage has been
shown to occur during cataract formation in humans and in various
cataractous animal
models(22, 23, 24, 25, 26, 27) ,
and leakage could account for the loss of the
-crystallins.
Although it was shown that the HIV-1 protease alone could cleave
purified crystallins, the two-dimensional gel profile of the
protease-digested control lens homogenates did not yield the same
protein pattern as that observed for the TG lenses. This
suggests that the protease may signal the activation of another
enzyme(s) in vivo. One possible candidate for this role is
calpain. The calpains are intracellular, calcium-dependent cysteine
proteases (28) that have been shown to factor in the formation
of nuclear cataracts(29) . There are two forms of calpain.
Calpain I requires micromolar amounts of Ca
, while
calpain II requires millimolar amounts of Ca
and is
the predominant form in the lens(30) . Activated calpain has
been shown to proteolyze a number of lens
crystallins(31, 32, 33) . Calpain II appears
to proteolyze
B1-,
B3-,
A3/A1-, and
A4-crystallins.
B2-crystallin and the
-crystallins resist calpain
proteolysis. Calpains are major proteases in rodent lenses, and recent
work on the selenite cataract suggests that the degradation of the
crystallins by calpain II contributes to light scattering and lens
opacification(34, 35, 36) .
These results indicate that the entire genome of HIV is not necessary to cause cataract formation in mice. Expression of the HIV-1 protease is sufficient, and the level of expression of this protease can be quite low. The data appear to implicate other proteases in the lens for some of the protein modifications occurring during opacification. The specific initiating event in cataractogenesis caused by the HIV-1 protease has not been determined, but lens opacification caused by this protease in concert with other factors in the lens is certain.