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INTRODUCTION |
The oxysterol-binding protein
(OSBP)1 has been known for
many years and has been fairly well characterized biochemically. It was
originally detected as a cytosolic protein capable of binding 25-hydroxycholesterol (1). OSBP, as its name implies, is capable of
binding a wide variety of oxidized forms of cholesterol with high
affinity (2). It was partially purified and characterized in
mouse-cultured fibroblast cells (3). The cDNA was first isolated in
rabbit (4), then in human (5). OSBP is known to translocate to the
Golgi apparatus after binding to oxysterols (6), and this translocation
is mediated via a pleckstrin homology domain near the N terminus of the
protein (7). OSBP is phosphorylated in the Golgi (8), and this
phosphorylation and Golgi transport seem to be dependent on cholesterol
trafficking and sphingomyelin hydrolysis (9, 10).
Oxysterols accumulate in tissues and can exert potent pharmacological
effects on cellular sterol biosynthesis and uptake. Oxysterols are
oxidized byproducts of cholesterol (11) that cause cytotoxic effects on
a variety of cells. This oxysterol cytotoxicity is exerted via
apoptosis and has been implicated in the pathophysiology of
atherosclerosis (12, 13). Oxysterol cytotoxicity has been demonstrated
in vascular endothelial cells (14), smooth muscle cells (15), and
cultured neuroretinal cells (16). The mechanism by which oxysterols
induce cell death is not fully elucidated but some scientific evidence
suggests that it may involve the oxysterol-binding proteins (17).
Although oxysterols will dramatically reduce the hydroxymethylglutaryl CoA reductase activity and therefore essentially shut down cholesterol synthesis, their induction of apoptosis cannot be rescued by adding excess cholesterol (17). This suggests that OSBP may be playing a
central role in the oxysterol-induced apoptosis.
The oxysterol-binding protein is a member of a family of proteins that
share structural and possibly functional similarities (18). Our
interest in the OSBPs is to investigate their role and that of their
oxysterol ligands in ocular tissues. More specifically we are
interested in determining whether OSBPs and oxysterols are involved in
the pathogenesis of age-related ocular diseases such as macular
degeneration and cataracts. There is existing evidence suggesting that
oxysterol cytotoxicity may be playing a role in the formation of
cataracts (19).
A recent study found an OSBP-like protein expressed exclusively in
metastatic tumor cells (20). The study linked this OSBP-like gene to
tumor dissemination. This publication did not provide a complete
molecular characterization of this OSBP, but alignments performed in
our laboratory indicate that it is likely our novel OSBP. If this is
indeed correct, this makes the identification and characterization of
this gene a very important finding. It may also suggest that oxysterols
could be playing a role not only in atherosclerosis but in cancer as well.
One of the genes presented in our study is the original OSBP and will
be referred to as OSBP1. The novel homolog will be referred to as
OSBP2. We have cloned and characterized the OSBP2 gene as well as
completed the molecular characterization of the OSBP1 gene. A detailed
comparative analysis of OSBP1 and OSBP2 at the molecular and biological
level is presented.
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EXPERIMENTAL PROCEDURES |
Monkey Retinal Tissue--
Fresh eye tissue was obtained from
rhesus monkeys (Macaca mulatta, 2-3 years old) through the
courtesy of the Center for Biologics Research and Testing, U.S. Food
and Drug Administration (Bethesda, MD). Animal studies were conducted
in accordance with the NIH guidelines on the care and use of animals in
research. The monkeys were anesthetized then exsanguinated, and the
enucleation was performed within 3 to 4 min after death. The eyes were
immediately placed on ice or in ice-cold tissue fixative. Macular and
peripheral retinal tissues were obtained using a 5-mm trephine. The
neural retina was separated from the pigment epithelium-choroid and
frozen at
75 °C before further processing. Each retinal sample was
from a pool of ten eyes.
DNA Sequence--
The DNA was sequenced using a PE-Applied
Biosystems Model 377 automated fluorescent sequencer (PE-Applied
Biosystems, Foster City, CA) and/or a Beckman CEQ-2000 capillary
fluorescent sequencer (Beckman Instruments). The sequencing reactions
were performed according to the manufacturer's specifications.
Screening of the Human Retina cDNA Library and Rapid
Amplification of cDNA Ends (RACE)--
A human retina cDNA
library in lambda ZAP (Stratagene, La Jolla, CA) was screened using
standard methodology. OSBP1- and OSBP2-specific PCR probes were
generated from EST sequences. The 5'-RACE was performed using human
retina cDNA synthesized on magnetic Dynabeads® (Dynal Inc., Oslo,
Norway) as previously described (21). The same solid-phase cDNA was
used to perform 3'-RACE using a degenerate oligo(dT)-primer
(NNTTTTTTTTTTTTTTTTTT). The amplified products were either
sequenced directly or cloned using the TOPO-TA cloning kit (InVitrogen,
Carlsbad, CA).
Subcellular Fractionation of Whole Monkey
Retina--
Subcellular fractionation of the retina was performed as
previously described (22). Fresh rhesus monkey (Maccaca
mulatta) retinas (10 retinas) were homogenized in 50 ml of 10 mM HEPES buffer, pH 7.2, containing 5 mM
MgCl2, 4% (w/v) sucrose and Complete Inhibitor (1 tablet/50 ml, Roche Molecular Biochemicals). The homogenate was
subjected to a low speed (300 × g) centrifugation to
separate the nuclei (P1) and the remaining supernatant was centrifuged
at high speed (27,000 × g) to pellet the subcellular organelles (P2). The P2 pellet was subsequently subjected to a 0.5 M NaCl wash (P2-salt) and a 2% Triton X-100 wash
(P2-detergent). The remaining pellet (P2 residue) was not further
processed. The protein amounts for each of the fractions are: S2 (50 ml, 2 mg/ml) 100 mg, P2-salt (15 ml, 0.3 mg/ml) 4.5 mg, and
P2-detergent (15 ml, 0.5 mg/ml) 7.5 mg.
Northern Blot Analysis and Quantification--
Human RNAs were
either purchased from CLONTECH (Palo Alto, CA) or
purified directly from human and monkey tissues using the RNeasy Kit
from Qiagen (Valencia, CA). The Northern blots were probed with
32P-labeled PCR products specific for OSBP1 and OSBP2. The
probes were labeled using the RTS RadPrime DNA labeling system (Life Technologies, Inc.) and Redivue [
-32P]dCTP (Amersham
Pharmacia Biotech). The identities of the PCR probes were confirmed by
sequencing. Human total RNAs (~5 µg) were loaded in a 1%
agarose/formaldehyde gel, separated by electrophoresis and stained with
SYBR Green II (Molecular Probes, Eugene, OR). The relative amounts
of OSBP mRNAs were determined relative to the 28S ribosomal RNA
band using a Storm 860 PhosphorImager (Molecular Dynamics, Inc.,
Sunnyvale, CA) as previously described (23).
Preparation of Antibodies to Human OSBP1 and OSBP2--
An
alignment of the known OSBP peptides was performed using MegAlign
software (DNASTAR Inc., Madison, WI) to identify peptide regions
specific to each OSBP. Two unique peptides were synthesized for OSBP1
(EQYKHQLEETKK and PLGTIHCIFHATGHHYT), and one peptide was synthesized
for OSBP2 (TVITEAKEDSRKAEGS). The synthetic peptides were coupled to
MAP and used to immunize two rabbits each (Princeton Biomolecules, Columbus, OH). The rabbits were exsanguinated, and the
serum collected by centrifugation. The IgG fraction was purified by an
initial precipitation with
(NH4)2SO4 (45%, w/v) followed by
dissolution and dialysis in 20 mM
K2PO4, pH 8.0. The IgG fraction was obtained
after chromatography in a DEAD-AffiGel Blue (Bio-Rad) column.
Antibody Specificity--
Although the peptides used for
immunization were carefully chosen to avoid cross-reactivity to other
proteins, especially other OSBPs, additional tests were performed to
determine their specificity. All immunoreactivity for both antibodies
can be blocked in Western blots and immunohistochemistry by mixing them
with their respective immunizing peptides (OSBP1, EQYKHQLEETKK or
PLGTIHCIFHATGHHYT and OSBP2, TVITEAKEDSRKAEGS). The OSBP1 antibodies
detect a truncated protein in the retina but the correct size peptide
is detected in the P1 nuclear fraction and the P2 residue. The
anti-OSBP2 antibodies react only with a protein at the correct
molecular weight present in the detergent soluble fraction of the
retina. The anti-OSBP2 antibodies react vigorously and specifically
with recombinant OSBP2, but the anti-OSBP1 antibodies do not
cross-react with recombinant OSBP2.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analyses--
Protein samples were mixed with sample buffer at a 1:1
volume ratio and heated at 100 °C for 2 min. Approximately 20 µg
of protein were loaded per well in 10-20% Tricine-SDS gels and run in
1× Tricine-SDS at room temperature for 1-1.5 h at 130-150 V. The
protein electrophoresis reagents and apparatuses were purchased from
Invitrogen/NOVEX (Carlsbad, CA). The gels were transferred overnight
onto a PROTRAN-nitrocellulose membrane (Schleicher & Schuell) using a
Trans-Blot electrophoresis apparatus (Bio-Rad). The transfer was
performed in 25 mM Tris, 192 mM glycine, 20% methanol at 20 V, room temperature overnight. The membrane was equilibrated in 1× Tris-buffered saline (TBS)/Tween 20 for 15 min, and
blocked in 1× TBS, pH 7.4, 5% Carnation nonfat milk, and 0.05% Tween
20, for 2 h. Incubations with the OSBP1- and OSBP2-specific antibodies were performed for 2 h followed by 1-2 h of incubation with anti-rabbit IgG alkaline phosphatase-conjugated secondary antibody
(Sigma). Blots were developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma). Gels were also stained with
Coomassie Blue for 30 min and de-stained with 20% methanol, 10%
acetic acid.
Immunocytochemistry--
Paraffin-embedded monkey retina
sections were de-paraffinized twice each with xylene and chloroform
followed by washes with phosphate-buffered saline/Tween 20. The
sections were blocked using 1:100 diluted goat serum for 15 min. The
tissue sections were then rinsed and incubated with anti-OSBP1 (1:200)
or anti-OSBP2 (1:100) antibody for 2 h followed by additional
washes with TBS/Tween 20. The secondary antibody (mouse anti-rabbit
alkaline phosphatase conjugate, same as for Western blots above) was
applied at 1:500 dilutions for 1 h. After the PBS/Tween 20 washes,
the sections were developed using Fast Red (Fast Red tablets, Roche
Molecular Biochemicals) for 15 min according to the manufacturer's instructions.
Binding of Oxysterols in Retina Subfractions--
The
tritium-labeled cholesterol (1 µCi/µl, 70 Ci/mmol, in ethanol),
7-ketocholesterol (1 µCi/µl, 50 Ci/mmol, in ethanol) and 25-hydroxycholesterol (1 µCi/µl, 78.5 Ci/mmol, in ethanol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO).
The oxysterols and cholesterol were added (2 µCi each) to 100 µl of
the S2 (soluble) and P2-detergent retinal subfractions and binding was
allowed to occur at room temperature for 10 min. The proteins were
separated by high-pressure liquid chromatography (HPLC). The protein
concentrations for the S2 (soluble) and P2-detergent were 2.5 µg/µl
and 0.5 µg/µl of protein, respectively. The protein determinations
were performed using the BCA Protein Assay kit (Pierce).
High-pressure Liquid Chromatography--
HPLC was performed
using a Waters 2790 instrument (Waters Corporation, Milford, MA) with
two attached detectors, a Waters 996 variable wavelength detector and
an IN/US (Tampa, FL)
-RAM radiochromatography detector. The proteins
were separated by size using TSK 4000 and TSK 3000 SW (Superlco,
Bellefonte, PA) columns (7.5 × 30 mm) in tandem. The columns were
eluted at 1 ml/min with 1× TBS, pH 7.4, at room temperature. When
appropriate, fractions (1 ml) were collected for Western blot analyses.
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RESULTS |
Identification and Cloning of cDNAs for OSBP1 and
OSBP2--
We initially cloned the extreme 3'-end portion of the OSBP1
cDNA while mapping human ESTs to 11q13. Using a PCR probe derived from these EST sequences, we screened a human fetal retina cDNA library and isolated a clone that extended the previously cloned OSBP1
cDNA (5) ~2 kb. We were not aware of the identity of our cDNA
until its 5'-end sequence matched the 3'-end portion of the previously
reported OSBP1 cDNA (5), GenBankTM accession number
NM_002556. We then constructed the complete cDNA from the sequence
of our overlapping clones and the ESTs in GenBankTM. The
entire cDNA sequence was also confirmed by sequencing the genomic
clones (see below). The full-length cDNA for OSBP1 is 5083 bp in
length and can be obtained from GenBankTM, accession
number AF185696.
The OSBP2 gene was first identified through a BLASTN (24) search of
GenBankTM using the OSBP1 cDNA sequence. A large
portion of the OSBP1 peptide was represented in matching regions of
chromosome 22 (accession number AC004542). These regions were
sequential but not contiguous, thus defining exon and intron
structures. The genomic sequences and several testes-derived ESTs were
used to construct a partial cDNA sequence for OSBP2, which served
as a basis for designing primers for generating a specific probe.
Solid-phase 5'- and 3'-RACE was performed to complete the cDNA
sequence using human retina cDNA synthesized on magnetic
Dynabeads® (21). Two types of cDNA clones were
isolated by 3'-RACE differing in the length of the 3'-untranslated
sequence. The most abundant OSBP2 cDNA in the retina is 2791 bp in
length. A longer message of 4238 bp is found primarily in testis but
can also be detected in small amounts in retina. The open reading frame
of both messages encodes a peptide of 878 amino acids. Polyadenylation
signals are found at positions 2763-2768 and 4206-4211. The different sizes for OSBP2 seem to be consistent with the use of these two different polyadenylation signals. The cDNA sequence for human OSBP2 is available from GenBankTM (accession number
AF288741). The cDNA sequence and comparison with OSBP1 is
shown in Fig. 1. The similarity between
the OSBP1 and OSBP2 mRNA is 38.7% overall and 52% in the coding
region. At the protein level, OSBP1 and OSBP2 share 63% identity (Fig. 2).

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Fig. 1.
Alignment of the OSBP1 and OSBP2 cDNA
sequence. The full-length OSBP1and OSBP2 sequences were aligned
using MegAlign software (DNASTAR) by the Clustal method.
Uppercase letters indicate the longest open reading frame
and lowercase letters indicate 5'- and 3'-untranslated
regions. The dividers (|) indicate the locations of the introns in
the gene. Polyadenylation signals are underlined
(aataaa). Shaded letters are identity matches
between the two sequences. The two sequences share 38.7% overall
similarity and 52% in the coding region.
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Fig. 2.
Alignment of the OSBP1- and OSBP2-deduced
amino acid sequences. The amino acid sequences were aligned using
MegAlign software (DNASTAR) by the Clustal method. The dividers (|)
indicate the locations of the intron-exon junctions. The oxysterol
signature peptide (EQVHHPP) is boxed. Dark shaded
letters are perfect matches, and lighter shaded letters
represent conservative changes. The pleckstrin homology domain is
underlined. The two peptides share 57% identity by Clustal
and 75% similarity by BLASTP (24).
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Gene Structure and Localization of OSBP1 and OSBP2--
The OSBP1
gene was isolated by PCR screening using Incyte Genomics (St. Louis,
MO). The 3'-end PAC clone, p17641, was isolated using primers 6436 (ATGTGACTTTATTCCACCTGA) and 6437 (GGGCTTTGGATAGAGTGAG). This clone
contained exons 8 to 14 but lacked the 5'-end region of the gene. Two
other PACs (p19144 and p19145) were isolated using primers 2F
(CCTCGCCACAGCCAACATC) and 2R (AATACCCTTCGGACCCTCTCTAGC), which generate
a 2-kb product across exons 2 and 3. These PACs contained the 5'-end
but lacked the middle region containing exons 4-7. Additional attempts
to clone the middle region in the PAC libraries failed. A BAC library
was then screened using primers 3F (TCACCGTGTCCTCCTACTCCAC) and 3R
(CAGGAAATCAAAATCACCAGCAAG), which amplified a 494-bp product across
exons 8 and 9. Three BAC clones (p20714, p20715, and p20716) were
isolated, which contained the 5'-end and middle regions of the gene.
The gene was found to consist of a total of 14 exons. The complete size
of the introns was determined using sequence available from
GenBankTM accession number AP00442 recently submitted
by The Genome Science Center at the University of Kitasato, Japan. We
estimate the size of the whole gene to be around 100 kb.
The sequence of the OSBP1 gene can be obtained from
GenBankTM accession numbers AF185697 (exon 1), AF185698
(exon 2), AF185699 (exon 3), AF185700 (exons 4-7), AF185701 (exon 8 and 9), AF185702 (exon 10), AF185703 (exon 11), AF185704 (exon 12), and
AF185705 (exons 13 and 14).
The OSBP1 gene had been previously localized to 11q13 (5). We have
refined the localization of OSBP1 using the Stanford G3 RH panel
(Research Genetics, Inc., Huntsville, AL). The gene is associated
closely with markers D11S4657 and D11S1368 at 11q12.1. Using sequence
from the ends of our PACs, we were able to further localize the OSBP1
gene to the PAC pDJ606G6 (accession number AC004126) in the contig
constructed by The Eugene McDermott Center for Growth and Development.
The OSBP2 gene sequence is available from GenBankTM because
of the chromosome 22 sequencing efforts of The Sanger Center. A 5'-end
PAC clone (p21781) was isolated by PCR screening (Incyte Genomics)
using primers 36F (TCCAGTGCCCCACTGGCCTTACTGCC) and 37R (GTAGTAAGAGAGCAAACCATTGCCCAGC). Three sequences were joined
together, AC004542, A1022336, and A1079299, and using our PAC and
cDNA sequences, we were able to identify the intron-exon junctions for the human OSBP2 gene. OSBP2 contains 14 exons and spans 217 kb. The
complete annotated gene is available from GenBankTM at
accession number AF288742. The intron-exon junctions for OSBP1
and OSBP2 are shown in Table I.
Expression of OSBP1 and OSBP2 mRNA in Human
Tissues--
The expressions of OSBP1 and OSBP2 were examined by
Northern blot analysis in total RNA from 19 human tissues (Fig.
3). The blots were probed with OSBP1-
(Fig. 3A) or OSBP2- (Fig. 3B) specific PCR
products generated with primers 4F (GCAGCGCCTGGAGGAAAAACA) and 5R
(ACTTCCTCAGGCGGAGAGCTT) for OSBP1 and 29F
(CTACTTCTCAGAGCTGGCCCTGAC) and 41R (GCTGATTTGGGG- CCTGGGTCTCAG)
for OSBP2. OSBP1 is widely expressed in human tissues and a
message of ~5 kb was readily detected. Occasionally a smaller 4-kb
message was also observed. We attempted to characterize the smaller
message by looking for alternative splicing and polyadenylation by
reverse transcription-PCR but were unsuccessful. OSBP2 is expressed in
two forms, a predominant 2.7-kb message and a larger 4.2-kb message.
Retina, pineal gland, and fetal liver are the main sites of expression
for the 2.7-kb message. The 4.2-kb message is also detected in retina
but is most abundant in testis and cerebellum with traces in fetal and whole brain. OSBP2 also has an alternatively spliced variant lacking exon 12 that is detectable in retina only by reverse transcription-PCR (data not shown). This changes the open reading frame after amino acid
692 creating a protein with an aberrant C terminus. We have no evidence
that this alternatively spliced message is translated. The relative
levels of expression of the OSBPs were quantified by normalizing the
SYB Green II (Molecular Probes) stained 28S ribosomal RNA band to the
OSBP1and OSBP2 isotope hybridization signal using a Storm 860 PhosphorImager as previously described (23).

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Fig. 3.
Multitissue Northern blot of OSBP1 and
OSBP2. A, human multitissue Northern blot hybridized
with OSBP1-specific probe. The graph indicates the level of expression
of the 5-kb OSBP1 band relative to the SYBR-stained 28S ribosomal
protein band. B, a different human multitissue Northern blot
hybridized with an OSBP2-specific probe. The dark bars
represent the relative levels of the 2.7-kb message to the 28S
ribosomal band. The light-colored bars represent the
relative levels of the 4.2-kb message. The lanes are labeled
with the name of the tissue. Molecular weight markers (not shown) were
run on the edges of both sides of the gel to determine message sizes.
Positions at 4.4 kb and 28S markers are indicated by arrows;
message sizes are shown on the right side.
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Expression of OSBP1 and OSBP2 mRNA in Monkey Retina--
The
macula is a retinal substructure that among mammals is unique to
primates. This structure is responsible for the exquisite visual acuity
and color vision enjoyed by our species. Because the macula is also an
area responsible for a variety of retinal diseases, we routinely
separate the macula from the peripheral retina to perform molecular and
biochemical analyses. Two punches were made using a 5-mm trephine, one
in the macula, being careful to make the fovea the center, and another
in the peripheral retina. The tissues from the punches were separated
into the neural retina and the corresponding retinal pigment epithelium
(RPE) choroid. Therefore, four different pieces of tissue were
examined, the neural macula (NM), the macular pigment epithelium
choroid, the peripheral neural retina, and the peripheral pigment
epithelium choroid. It should be noted that the RPE is a single cell
layer between the neural retina and the choroid. In young monkeys, the separation of the choroid from the neural retina may lead to some contamination of the neural retina with RPE. However, in our experience most of the RPE cell bodies remain with the choroid. If done carefully, there is no observable contamination of the peripheral pigment epithelium choroid with neural tissue.
A Northern blot was performed using RNA extracted from the four
different tissues and probed with the OSBP1- and OSBP2-specific probes.
OSBP1 showed similar levels of expression in the NM, macular pigment
epithelium choroid, and peripheral neural retina (Fig. 4A) but little or no
expression in the peripheral pigment epithelium choroid. OSBP2 showed
significant levels of expression in both NM and peripheral neural
retina but little or no expression in the macular pigment epithelium
choroid or the peripheral pigment epithelium choroid (Fig.
4B).

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Fig. 4.
Northern blot analysis of OSBP1 and OSBP2 in
monkey retinal tissue. A, Northern blot and relative
levels of expression of OSBP1 in monkey retina tissue; B,
same blot probed with OSBP2. Each sample of RNA represents a pool of 10 monkey retinal samples. Each lane contains ~5 µg of
total RNA. MPEC, macular pigment epithelium/choroid; PNR, peripheral
neural retina; PPEC, peripheral pigment epithelium choroid. This
experiment was repeated twice with different blots, and identical
results were obtained.
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Expression of OSBP1 and OSBP2 Proteins in Monkey Retina--
To
determine whether OSBP1 and OSBP2 proteins were expressed in retina,
antibodies to peptides unique to human OSBP1 and OSBP2 were raised in
rabbits. Tissue punches from different areas of the monkey retina
(macula versus peripheral retina) were used to prepare
protein fractions for SDS-polyacrylamide gel electrophoresis. OSBP1 was
clearly detectable by Western blot analysis in the monkey neural macula
and peripheral retina but not in the RPE/choroid punches, whereas OSBP2
was undetectable in the same samples (data not shown). A subcellular
fractionation was performed in whole monkey retina (including RPE and
choroid) to see whether the proteins differentially fractionated with
the various organelle fractions. Western blots of the subcellular
fractions (Fig. 5) detected OSBP1 in all
fractions (Fig. 5B), whereas OSBP2 was detected only in the
P2-detergent fraction (Fig. 5C). In most of the fractions OSBP1 was detected as a truncated or processed 50-kDa form, but in the
nuclear fraction the full-length peptide (~80 kDa) was detected.
OSBP2 was detected as a 90-kDa protein, and its extraction from the P2
pellet with detergent suggests that it may be associated with
membranes.

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Fig. 5.
Western blot of subfractionated whole monkey
retina. The whole monkey retina (including the pigment epithelium
and choroid) was subfractionated as previously described under
"Experimental Procedures." Each lane was loaded with
~20 µg of protein. Panel A is a Coomassie Blue-stained
gel representative of the two blotted gels. Panels B and
C are Western blots probed with OSBP1 and OSBP2 antibodies,
respectively. An additional lane was included in panel C
containing recombinant thioredoxin OSBP2 as a positive control. The
immunoreactivity in both blots can be completely eliminated by adding
the immunizing peptides to the primary antibodies.
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Immunocytochemical Localization of OSBP1 and OSBP2 in Monkey
Retina--
OSBP1 and OSBP2 were localized in the monkey retina using
the peptide antibodies described above. Representative photographs of
the macula/fovea region and peripheral retina are shown in Fig.
6. The controls were treated identically
except that the primary antibodies were pre-incubated with the
immunizing peptides. The black bar represents 100 µm in
length. In the macula/fovea region, OSBP1 localizes to the ganglion
cell layer, inner plexiform layer, inner nuclear layer (INL), outer
plexiform layer, and the RPE. Little or no labeling was observed in the
outer nuclear layer or the rod outer segments. The OSBP1 labeling in
the ganglion cell layer seems to involve Müller cells, and in the
INL, it seems to occur in a subset of bipolar cells. OSBP2 localizes to the ganglion cell layer but in a different pattern from OSBP1, suggesting preference for a subset of ganglion cells. Like OSBP1, OSBP2
localizes to the inner plexiform layer and INL. In the INL, the OSBP2
was localized to a subset of cells that are adjacent to the inner
plexiform layer, possibly amacrine cells. Diffuse OSBP2 labeling is
observed in other cells of the INL, suggesting some localization to
bipolar, Müller, and/or horizontal cells. OSBP2, unlike OSBP1,
does not seem to be present in the outer plexiform layer, but like
OSBP1, is also absent from the outer nuclear layer and rod outer
segments. The RPE also seems to contain OSBP2. Similar results were
observed for both OSBPs in the peripheral retina. The peripheral retina
contains significantly fewer inner retinal cells than the macula/fovea
region, thus the heavily labeled ganglion and amacrine cells are more
sparse in this area of the retina.

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Fig. 6.
Immunocytochemical localization of OSBP1 and
OSBP2 in monkey retina. The monkey retina tissue preparation and
the immunohistochemical process were performed as described under
"Experimental Procedures." The macula/fovea region was photographed
at half the magnification of that shown for the peripheral retina and
control. The size of the black bar is 100 µm and was
determined using a micrometer. The controls shown are peripheral
retina, which was identically treated except that the primary
anti-OSBP1 and anti-OSBP2 antibodies were pre-treated with the
immunizing peptide.
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Oxysterol Binding Activity of OSBP1 and
OSBP2--
Oxysterol-binding activity was detected in the monkey
retina subfraction by incubating with tritiated oxysterols and
separating the proteins by HPLC. The S2 (soluble) and P2-detergent
fractions were incubated with [3H]cholesterol (CH),
[3H]25-hydroxycholesterol (25HC) and
[3H]7-ketocholesterol (7KC). The proteins were
fractionated by size using a two-column system (TSK-4000, TSK-3000).
The proteins containing bound sterols were detected using an in-line
radiochromatography detector. The free highly hydrophobic oxysterols
and cholesterol bind to the pre-column and are not eluted under these
conditions. Binding activity was found in both the soluble protein
fraction S2 and the P2-detergent fraction (Fig. 8, A and
B). In the S2 soluble fraction (Fig.
7A), CH binding activity was
located mainly in the void volume of the column, suggesting a possible
nonspecific association with macromolecules. Another peak was detected
around 28 min, which bound 25HC preferentially, but also 7KC and CH. The P2-detergent fraction (Fig. 7B) also showed two CH
binding peaks at 11 and 14 min, but the main peak occurred at 30 min, preferentially binding 7KC. The 30-min peak showed no CH binding activity and very little 25HC binding activity. Because OSBP2 was
detected only in the P2-detergent fraction and no 7KC binding activity
was detected in the S2 fraction, we suspected that this activity is
because of OSBP2 binding. Western blot analysis was performed across
the 30-min peak (Fig. 7C) to delineate the elution profile
of OSBP2. The results indicate that the 30-min 7KC binding activity
co-elutes with the OSBP2 immunoreactivity. The results also suggest
that OSBP2 may have preferential binding for 7KC.

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Fig. 7.
Oxysterol binding activity of OSBP2. The
monkey retina subfractions S2 (soluble) and P2-detergent were incubated
with 2 µCi of [3H]cholesterol,
[3H]7-ketocholesterol, and
[3H]25-hydroxycholesterol, and the proteins were
separated by HPLC. The data were collected using
Millennium32 version 3.2 software and exported to Microsoft
Excel. The three chromatograms from each of the two fractions were
superimposed for direct comparison. Panel A, the sterol
binding in the S2 fraction; panel B, the sterol binding in
the P2-detergent fraction; panel C, a Western blot of
fractions 25-35. Fractions (1 ml) were collected and run on
SDS-polyacrylamide gel electrophoresis and blotted. The blot was probed
with a mixture of anti-OSBP1 and anti-OSBP2 antibodies. OSBP2
immunoreactivity was detected in fractions 29, 30, and 31 corresponding
to the 7KC binding activity in panel B. No OSBP1 was
detected in these fractions.
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Several additional controls were performed to demonstrate that the 7KC
binding activity found in the P2-detergent fraction are because of the
presence of OSBP2. 1) Treatment of the P2-detergent fraction with
Proteinase K at room temperature in neutral buffer (conditions that do
not effect the 7KC binding) completely obliterated all binding activity
in a few hours. 2) Pre-extraction of the S2 and P2-detergent fraction
with petroleum ether to remove free and/or bound sterols did not
significantly change the results shown in Fig. 7. 3) Addition of
anti-OSBP2 antibody to the P2-detergent fraction moved the 7KC binding
activity to a larger size peak around 20 min and further addition of
anti-OSBP2 removed all 7KC binding activity. These controls indicate
that the 7KC activity found in the P2-detergent fraction is because of
OSBP2 specifically.
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DISCUSSION |
We are reporting the molecular and biological
characterization of a novel oxysterol-binding protein, OSBP2, and
extend the molecular characterization of the previously reported OSBP1.
We became interested in OSBP2 because of its high and almost unique expression in the retina as well as its striking similarity to OSBP1.
Both genes contain 14 similarly sized exons and nearly identical
intron-exon junctions (Fig. 1, Table I). The genes share a high degree
of similarity both at the nucleotide (Fig. 1) and protein levels (Fig.
2) and may have derived from a gene duplication event. At the peptide
level, OSBP1 and OSBP2 share the oxysterol-binding domain and the
pleckstrin homology domain (7) and are almost identical toward the C
terminus. Recent evidence from other investigators (18) and from
unpublished data within our group indicates that oxysterol-binding
proteins are a large and complex family of genes.
Although OSBP1 and OSBP2 are structurally very similar, they undergo
different transcriptional and posttranslational regulation. Although
OSBP1 mRNA is readily detectable by Northern blot in most human
tissues, OSBP2 mRNA expression is relatively specific to retina,
pineal, testis, and fetal liver (Fig. 3). The expression in fetal liver
may suggest that there is developmental regulation of OSBP2 and may
correlate with earlier observations that implicated oxysterols in
apoptosis control during development (11). The OSBP1 mRNA is more
highly expressed in the macula than in the peripheral retina (Fig.
4A) but the protein seems to be equally distributed. This
may be because of the much higher metabolic activity of the macula,
which may induce a greater OSBP1 turnover. In contrast, OSBP2 mRNA
seems to be readily detectable in both the macula and peripheral neural
retina but the protein is only detectable by Western blot in the
P2-detergent retinal subfraction (Fig. 5C). These Western
blots (Fig. 5) suggest that there is more OSBP1 than OSBP2 protein in
the retina. It should be noted that there is 13 times more total
protein in the S2 fraction than in the P2-detergent fraction. Thus,
unless there is a very large difference in the antibody affinities
(>10-fold), there is probably more OSBP1 than OSBP2 in the retina.
Immunocytochemistry localized OSBP1 and OSBP2 in similar but not
identical regions of the retina (Fig. 6). The localization of OSBP1 and
OSBP2 to the inner retina is complex involving several retinal cell
types and possibly subtypes, and it is difficult to speculate as to its
meaning without further examination by electron and/or confocal
microscopy. However, the localization of these OSBPs to the RPE is of
particular interest for several reasons. The RPE expresses the LDL
receptor (25) and its proximity to the choriocapillaris makes it a
likely target for the uptake of blood LDL. The RPE is also probably
serving as a cholesterol source for other retinal cells such as the
photoreceptors that constantly synthesize rod outer segment membranes.
LDL is one of the major sources of oxidized cholesterol and considering
the cytotoxity demonstrated by these molecules, it is likely that the
RPE has some form of mechanism to bind these oxysterols as they are
released from the LDL complex.
The oxysterol binding activity of OSBP2 was determined by incubating
the detergent-extracted P2 pellet fraction (P2-detergent) with
tritiated oxysterols (25HC and 7KC) and fractionating the proteins by
HPLC (Fig. 7B). The S2 soluble fraction, which lacks OSBP2,
was also treated identically to serve as a control (Fig. 7A). These results suggest that the majority of the 7KC
binding activity is present in the P2-detergent fraction and that this activity is associated with immunoreactivity to OSBP2 (Fig.
7C). Although the experiments in Fig. 7 were not designed to
determine binding constants, the data suggest that OSBP2 preferentially binds 7KC and has little or no affinity for 25HC or CH. The binding observed in the void volume (11 to 12 min) is probably because of
nonspecific association of these sterols with macromolecules such as
hyaluronic acid, which is present in all of our retinal fractions.
There may also be other OSBPs in these fractions that associate with
these macromolecules. Unpublished results from our laboratory indicate
that there are at least 12 different oxysterol-binding protein-like
genes in the human genome and many of these are present in the retina.
Thus, until these OSBPs are better understood, oxysterol-binding
activity in any tissue should be considered to originate from a mixture
of binding proteins. The relatively low oxysterol binding activity
detected in the S2 fraction may be because of the low concentration of
oxysterols used (0.4 nM) or perhaps the narrow choices of
oxysterols used (7KC and 25HC). There are over 80 different forms of
oxidized cholesterol (26), and as can be seen by the relatively high
affinity of OSBP2 for 7KC, different OSBPs may have different
affinities for different oxysterols. The identification of the
oxysterol binding activity of OSBP2 was facilitated by several factors:
OSBP2 fractionated relatively cleanly to the P2-detergent soluble
fraction, the availability of a specific OSBP2 antibody, and the high
affinity of OSBP2 for 7KC, which allowed it to bind 13% of the total
counts at a concentration of 0.4 nM. This allowed the
detection of OSBP2 with very small amounts of tritiated oxysterols.
Although OSBP1 is also present in the P2-detergent fraction, no OSBP1
immunoreactivity was present in this fraction.
OSBPs and their oxysterol ligands may be playing an important role in
the pathogenesis of age-related ocular diseases. A recent study has
shown that there is a gradual accumulation of LDL cholesterol in the
choriocapillaris and Bruch's membrane (27) with aging. This coupled
with the high metabolic rate and cholesterol needs of the retina,
especially the macula, make this area of the eye especially susceptible
to oxysterol accumulation. The functions of the OSBPs and particularly
OSBP2 with its high and almost exclusive expression in the retina could
play an important role in macular degenerations.