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Journal of Lipid Research, Vol. 42, 1203-1213, August 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

The OSBP-related protein family in humans

Correspondence to: Vesa M. Olkkonen, To whom correspondence should be addressed., vesa.olkkonen{at}ktl.fi (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Oxysterols are oxygenated derivatives of cholesterol that have a number of biological effects and play a key role in the maintenance of the body cholesterol balance. In this study, we describe the cDNA sequences and genomic structures of the recently identified human oxysterol-binding protein (OSBP)-related protein (ORP) family (Laitinen, S. et al. 1999. J. Lipid Res. 40: 2204–2211). The family now includes 12 genes/proteins, which can be divided into six distinct subfamilies. The ORP have two major structural features: a highly conserved OSBP-type sterol-binding domain in the C-terminal half and a pleckstrin homology domain present in the N-terminal region of most family members. Several ORP genes are present in S. cerevisiae, D. melanogaster, and C. elegans, suggesting that the protein family has functions of fundamental importance in the eukaryotic kingdom. Analysis of ORP mRNA levels in unloaded or acetylated LDL-loaded human macrophages revealed that the expression of ORP genes was not significantly affected by the loading, with the exception of ORP6, which was up-regulated 2-fold.

The present study summarizes the basic characteristics of the OSBP-related gene/protein family in humans, and provides tools for functional analysis of the encoded proteins. — Lehto, M., S. Laitinen, G. Chinetti, M. Johansson, C. Ehnholm, B. Staels, E. Ikonen, and V. M. Olkkonen. The OSBP-related protein family in humans. J. Lipid Res. 2001. 42: 1203;–1213.

Supplementary key words: lipid metabolism, ORP, oxysterol, pleckstrin homology domain, sterol-binding domain

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Oxysterols are 27-carbon products of cholesterol oxidation that display a number of biological activities including cytotoxic effects, perturbation of membrane structure and function of membrane-associated proteins, as well as disturbance of cellular cholesterol homeostasis. Although oxysterols are present at levels several magnitudes lower than cholesterol in human plasma, they are enriched in macrophage foam cells, atherosclerotic plaques, and LDL subfractions that are considered potentially atherogenic. The potential involvement of oxysterols in the development of atherosclerosis has focused considerable interest on this category of sterols (1) (2).

Our understanding of the mechanisms underlying the impact of oxysterols on cholesterol metabolism has greatly increased since the recent elucidation of the functions of nuclear hormone receptors. When activated by oxysterols (3), the liver X receptors (LXR) regulate the expression of several genes in key positions in the maintenance of the body-cholesterol balance. These include genes involved in cholesterol absorption in the gut (4), cholesterol efflux from peripheral cells (4) (5) (6), synthesis of fatty acids (7) (8), remodeling of lipoproteins in the circulation (9), and the bile acid synthetic pathway (10) (11). Furthermore, oxysterols bind to the steroidogenic factor 1 (SF-1), which regulates the expression of a number of genes involved in steroidogenesis and cholesterol metabolism (12) (13) (14) (15).

Oxysterol-binding protein (OSBP) was the first protein identified as a receptor for endogenous oxysterols (16) (17). OSBP is a cytosolic protein that binds 25-hydroxycholesterol with high affinity (18). Upon ligand binding, OSBP translocates to the membranes of the Golgi apparatus (19). This membrane interaction is mediated by a pleckstrin homology (PH) domain located in the N-terminal region of the protein (20) (21). Several studies have implicated OSBP in the regulation of cellular cholesterol and sphingomyelin homeostasis (20) (22) (23) (24). The yeast Saccharomyces cerevisiae has seven OSBP homologues whose functions are currently not well defined (25) (26) (27) (28). There is suggestive evidence for the involvement of these genes in the synthesis of ergosterol, the yeast counterpart of cholesterol (25) (28). In addition, one of the yeast proteins, Kes1p, has been connected to the secretory function of the Golgi apparatus (29).

We recently identified a family of novel human OSBP-related proteins (ORP) at the level of contigs generated from expressed sequence tags (EST) (30). In the present study, we report the cDNA sequences and exon-intron organization of human genes belonging to the OSBP gene family (which at present, consists of 12 members), and summarize data on the tissue expression of the ORP mRNAs. Further, the effect of acetylated LDL (acLDL) loading on ORP gene expression was studied in monocyte-derived human macrophages.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cloning of ORP cDNAs
Total RNA was isolated from the human hepatoma cell-line HUH7 by using the RNeasy kit (Qiagen). One microgram of total RNA was reverse transcribed into cDNA by using gene-specific primers and the Superscript II enzyme (Life Technologies) according to the manufacturer's instructions. In the case of ORP5 and ORP10, cDNA was prepared from human fetal brain total RNA (Stratagene). RT-PCR was carried out in a reaction volume of 20 µl containing one-fifth of the RT-reaction, 16 mM ammonium sulfate, 67 mM Tris-HCl (pH 8.8), 0.01% Tween-20, 200 µM dNTPs, 1.5 mM MgCl₂, 10 pmol of each specific primer, and 0.5 U AmpliTaq DNA-polymerase (Perkin Elmer). Hot-start PCR was initiated with denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60;–64°C for 30 s, extension at 72°C for 30;–180 s, and final extension at 72°C for 10 min. The amplified PCR-products were cloned into pBluescript SK(-) (Stratagene) and transformed into E. coli DH5. Plasmid DNAs were purified with the QIAprep spin column kit (Qiagen). Sequences of the cloned inserts were determined by using a cycle-sequencing kit (BIGDYE) and an automated ABI377 sequencer (Applied Biosystems). In the case of five of the ORP (1, 3, 4, 6, and 8), the cDNA 5' end was determined by using the rapid amplification of cDNA ends (RACE) technique (5' RACE System, version 2.0, Life Technologies). The 3' end of the ORP1 cDNA (fragment 1C, Fig 1) was isolated from a human heart cDNA library in Lambda ZAP II (Stratagene) using the ORP1 EST probe described by Laitinen et al. (30).

View larger version (29K): [in this window] [in a new window]   Figure 1. Cloning strategies of the human ORP cDNAs. The figure illustrates alignments of the cloned ORP cDNA segments, marked with numbers followed by capital letters. The numbers beneath the bars indicate nucleotide positions calculated from the 5' end of each sequence contig. Accession numbers of previously reported cDNA sequences are shown in italics. Gray and white areas of the bars indicate cDNA sequences corresponding to untranslated and translated regions, respectively. The cDNA sequences deduced during this study are indicated with double lines.

Compiling the sequence information
Novel cDNA sequences were constructed by combining sequence information from fragments obtained from the 5' RACE and RT-PCR. Cloning strategies for the ORP cDNAs are illustrated in Fig 1. The Genbank accession numbers for the ORP cDNA sequences cloned during this study are ORP1 (AF323726), ORP2 (AF331963), ORP3 (AF323727), ORP4 (AF323731), ORP5 (AF331964), ORP6 (AF323728), ORP7 (AF323729), and ORP8 (AF323730). In addition to ORP1;–8, screening of human sequence databases resulted in the identification of three novel ORP cDNAs: AK022554 (named here ORP9), AK000370 (ORP10), and AK023226 (ORP11). The 5' ends of the ORP10 and ORP11 cDNAs were determined by RT-PCR and are found under accession numbers AF346291 and AF346292, respectively.

ORP sequences were deduced from the following sequences: ORP1 (AF323726), ORP2 (AF331963, AB018315), ORP3 (AB014604, AF323727), ORP4 (AF323731), ORP5 (AF331964, AB040967), ORP6 (AF323728), ORP7 (AF323729), ORP8 (AF323730, AB040884), ORP9 (AK022554), ORP10 (AF346291, AK000370), and ORP11 (AF346292, AK023226). The exon-intron organization of the ORP genes was determined by comparing the cDNA sequences with genomic nucleotide sequences derived from the nonredundant (NR) and the high-throughput genome sequence (HTGS) GenBank databases.

Computational analysis of DNA and protein sequences
The sequenced DNA fragments were assembled into overlapping contigs and analyzed with the Sequencher-program (Gene Codes Corporation). Both nucleotide and protein similarity searches were performed with BLAST programs (www.ncbi. nlm.nih.gov/BLAST/). Functional domains, as well as secondary structures of the proteins, were determined with the SMART program package (http://smart.embl-heidelberg.de/), the SPLIT35 program (http://pref.etfos.hr/split/), and the PredictProtein software package (http://cubic.bioc.columbia.edu/predictprotein/). The Clustalw-program, Phylip algorithm (http://www2.ebi.ac.uk/clustalw/), was used to construct the ORP phylogenetic tree. The sequences from different organisms used for this were Saccharomyces cerevisiae (http://genome-www.stanford.edu/cgi-bin/SGD/): YAR044w (Osh1p), YDL019c, YOR237w (Hes1p), YPL145c (Kes1p), YHR001w (Yhg1p), YKR003w (Yky3p), and YHR073w (Yhn3p); Drosophila melanogaster (http://fly.ebi.ac.uk: 7081/): AAF47130, AAF56371, AAG22160, and AAF58878; and Caenorhabditis elegans (http://www.wormbase.org/perl/ace/elegans/): ZK1086.1, C32F10.1, F14H8.1, and Y47D3A.17.

Measurement of ORP gene expression by a semiquantitative PCR assay
Mononuclear cells isolated from blood by Ficoll gradient centrifugation were suspended in medium containing 10% human serum. Differentiation into macrophages occurred spontaneously by adhesion to the culture dishes. Mature monocyte-derived macrophages were used for experiments after 10 days of culture. Macrophages were cholesterol loaded by incubation with acLDL (50 µg protein/ml) in RPMI 1640 medium supplemented with 1% Nutridoma (Boehringer Mannheim) for 48 h. At the end of the incubation, intracellular lipids were extracted from aliquots of the cells in hexane/isopropanol, dried under nitrogen, and free cholesterol and total cholesterol were measured by enzymatic assays (Boehringer Mannheim). Esterified cholesterol was measured as the difference between total and free cholesterol (31). For ORP mRNA analysis, the cells were washed with PBS, and total RNA was extracted using Trizol (Life Technologies). ORP gene expression was studied by using the primer-dropping method (32), with the following modifications: 1 µg of total RNA was transcribed into cDNA with random hexamer primers (Amersham Pharmacia Biotech) and Superscript II according to the manufacturer's instructions (Life Technologies). Two microliters (equals 100 ng total RNA transcribed into cDNA) of each RT reaction was used as template for PCR with gene-specific primers (OSBP, ORP1-8; Table 1) as described above. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment was amplified simultaneously in each PCR reaction and used as an internal standard against which the results were normalized. Hot-start PCR was initiated with denaturation at 94°C for 5 min, followed by a specific number of cycles optimized for each gene (Table 1): denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 10 min. Aliquots of the PCR reactions were electrophoresed on 2% agarose gels stained with ethidium bromide, and analyzed using the Kodak EDAS 120 digital camera system and the Kodak 1D Image Analysis software.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ORP cDNA sequences
The ORP cDNA sequences were determined with cDNA cloning techniques and computational analysis (Fig 1). During the cloning process, it became evident that the previously described ORP7 and ORP8 EST (30) are derived from ORP4 and OSBP, respectively. In addition to the six previously discovered ORP (ORP1;–ORP6), two novel ORP gene products closely related to ORP3 or ORP5 were identified. These ORP gene products were named ORP7 and ORP8, respectively. In the case of four cDNAs (ORP1, ORP4, ORP6, and ORP7), the full-length coding sequences were determined with cloning approaches. Partial cDNA sequences of ORP2 (KIAA0772), ORP3 (KIAA0704), ORP5 (KIAA1534), and ORP8 (KIAA1451) have recently been reported in a human cDNA sequencing project (33) (34) (35). Based on the EST sequence data and our RT-PCR results, it became evident that the previously described ORP2 cDNA clone (KIAA0772) contained a 36-bp in-frame deletion at the N-terminus. The 5'-end sequences of the ORP3 and ORP8 cDNAs were determined with the RACE technique. Further, the 5' end of the ORP5 for which the database contained an apparently reorganized cDNA sequence was determined by RT-PCR and sequencing. Upstream stop codons, preceding the putative start codons, were identified for all cDNAs, except for ORP4.

The primary goal of the present study was to determine the full-length cDNA sequences and genomic structures of the previously identified ORP genes (30). However, during the database analysis we discovered three new cDNAs with designated OSBP-like fingerprints (named ORP9;–11). Based on current sequence information, we concluded that ORP9 (AK022554) contains a full-length coding sequence, whereas ORP10 (AK000370) and ORP11 (AK023226) represent partial cDNAs. The 5' ends of the ORP10 and ORP11 cDNAs were deduced by RT-PCR cloning. The basic characteristics of the members in the OSBP-related gene/protein family are summarized in Table 2.

Functional domains and structural relationships of the ORP
The ORP amino acid sequences were deduced from the cDNA sequence information. Computational analysis of the ORP sequences revealed two major structural features ( Fig 2). The most conserved region in all ORP is the C-terminal sterol-binding (SB) domain, consisting of approximately 400 amino acid residues. Within the SB domain, there are a number of highly conserved sequence motifs, of which EQVSHHPP is fully conserved in all members of the family. The other well-conserved sequence motif is a PH domain near the N-terminus of the proteins. PH domains are found in a large number of proteins, most of which are involved in cytoskeletal functions and cell signaling (36) (37). A PH domain of approximately 100 amino acids was identified in nine ORP. ORP2 apparently lacks the N-terminal PH-domain region and can be considered an ORP of a structurally distinct subtype. The ORP9 cDNA available (AK022554) lacks PH domain-encoding sequences, but current EST data suggest that there is also a PH domain-containing splice variant of this protein (data not shown). Potential membrane-spanning segments were identified in the carboxy-terminal ends of ORP5 and ORP8. The protein analysis programs also recognized three ankyrin-like repeats in the amino-terminal region of ORP1 (Fig 2). All of the ORP contain short segments predicted to form coiled-coil structures that could play a role in protein-protein interactions.

View larger version (38K): [in this window] [in a new window]   Figure 2. Alignment of the human ORP. The proteins have been organized into six subfamilies according to amino acid homology: subfamily I (OSBP, ORP4), subfamily II (ORP1, ORP2), subfamily III (ORP3, ORP6, ORP7), subfamily IV (ORP5, ORP8), subfamily V (ORP9), and subfamily VI (ORP10, ORP11). The amino acid positions of the functional domains are indicated with numbers. The PH domain is shown in blue; the SB domain in red. All proteins are aligned according to the highly conserved sequence motif "EQVSHHPP" located in the SB domain (shown in yellow). Putative transmembrane domains and ankyrin-like repeats are shown in green and orange, respectively.

Pairwise comparisons of the ORP amino acid acid sequences were carried out using the PH domain and the SB domain sequences separately ( Fig 3). This comparison revealed clustering of the sequences into groups showing a high degree of identity. Based on the amino acid homology together with similarity observed in the genomic structures (see below), the OSBP-related proteins/genes were divided into six subfamilies: subfamily I (OSBP and ORP4), subfamily II (ORP1 and ORP2), subfamily III (ORP3, ORP6, ORP7), subfamily IV (ORP5 and ORP8), subfamily V (ORP9), and subfamily VI (ORP10, ORP11). The ORP showed >70% amino acid identity in the SB domains within each subfamily. The sequence identity was clearly lower between proteins belonging to different subfamilies (Fig 3).

View larger version (49K): [in this window] [in a new window]   Figure 3. Amino acid homology between the human OSBP. The figure shows the percentage of identical amino acid (aa) residues between the different proteins. The area shaded with gray shows the degree of aa identity in the PH domain (PHD): OSBP (aa 90;–183), ORP4 (184;–276), ORP1 (237;–336), ORP3 (53;–148), ORP6 (88;–183), ORP7 (49;–144), ORP5 (128;–245), ORP8 (108;–225), ORP10 (76;–173), and ORP11 (60;–157). The area without shading shows the aa identity in the SB domain (SBD): OSBP (aa 420;–646), ORP4 (524;–750), ORP1 (550;–775), ORP2 (77;–302), ORP3 (529;–754), ORP6 (572;–797), ORP7 (480;–705), ORP5 (372;–603), ORP8 (366;–597), ORP9 (198;–447), ORP10 (410;–659), and ORP11 (380;–635). This region, comprising approximately 230 aa residues, is the most conserved part of the SB domain.

To illustrate the evolutionary relationships between the proteins, their SB domain sequences were analyzed using the Clustalw software together with seven S. cerevisiae, four D. melanogaster, and four C. elegans proteins ( Fig 4). This analysis divided the proteins into three main evolutionary branches. The first branch contained human ORP subfamilies I (OSBP, ORP4), II (ORP1, ORP2), and III (ORP3, ORP6, ORP7), as well as two predicted Drosophila polypeptides and two yeast proteins. The second branch contained human subfamilies IV (ORP5, ORP8), V (ORP9), and VI (ORP10, ORP11), as well as four yeast, two Drosophila, and two C. elegans proteins. The third branch consisted of two predicted C. elegans proteins and yeast Yhn3p. According to this analysis, the human ORP belong to two major evolutionary lines that are also present in lower eukaryotes.

View larger version (22K): [in this window] [in a new window]   Figure 4. Evolutionary relationships between ORP from different species. The phylogenetic tree was assembled by using the Clustalw software and 27 protein sequences: 12 from humans (Hum), 7 from yeast S. cerevisiae (Sc), 4 from C. elegans (Ce), and 4 from D. melanocaster (Dm). To maximize the reliability of the sequence alignment, we used a highly conserved 220 amino acid-long protein region located in the SB domain for the analysis. The N-terminal end of the selected area contains a highly conserved glycine (corresponding to OSBP G428 found in 25 of 27 proteins). The C-terminal end of the area contained a Gly-X-Trp motif (corresponding to OSBP residues 644;–646), present in all proteins studied.

Exon-intron organization of the ORP genes
The exon-intron organization of the ORP genes was determined by comparing full-length cDNA sequences with nucleotide sequences derived from the NR and the HTGS GenBank databases. For each human ORP gene, accession numbers, as well as chromosomal locations of both finished and unfinished genomic sequences, are summarized in Table 2. Comparison of the genomic structures of the ORP genes revealed some striking similarities. In particular, the length of exons encoding the functional (PH and SB) domains was highly conserved within each ORP subfamily ( Fig 5). This feature of the genomic sequences supported the subfamily division suggested by the protein sequence analysis. Even though the exon-intron structures were conserved within the subfamilies, marked differences in the length of introns were observed that results in extensive variation in the size of the genes; for example, the approximated lengths of the closely related ORP3 and ORP7 genes are 180 kbp and 15 kbp, respectively.

View larger version (72K): [in this window] [in a new window]   Figure 5. Exon-intron structures of the genes coding for human OSBP and ORP. The subfamilies are indicated with Roman numerals. Each box shows the length of the exons in base pairs. The underlined numbers indicate that the total length of the exon is unknown. The untranslated regions are shown in yellow. Exons coding for PH and SB domains are indicated with blue and red boxes, respectively. The diamonds indicate exons identical in length, and those exons containing putative start codons are indicated with red circles. The figure shows only partial genomic structure for the ORP10 gene (indicated with a black arrow).

ORP mRNA expression
To obtain a comprehensive picture of the ORP mRNA tissue-specific expression patterns, data currently available from different sources was compiled in Table 3. The ORP mRNAs are ubiquitously present in human tissues, although they show clear gene- and tissue-specific differences in expression level.

To study whether the expression of the human ORP is influenced by changes in the cellular cholesterol status, we determined whether the ORP mRNA levels are affected by acLDL loading of human macrophages, a treatment known to activate a large number of genes; for example, those responsible for cellular cholesterol efflux (38). In unloaded macrophage specimens, the total, free, and esterified cholesterol concentrations were 30.31 ± 3.50, 10.22 ± 0.32, and 20.09 ± 3.57 µg/mg cell protein, respectively. After acLDL loading, the values were 53.32 ± 8.64, 15.28 ± 0.72, and 38.04 ± 7.96 µg/mg cell protein, respectively. Assessment of the mRNA quantities was carried out by RT-PCR using the endogenous GAPDH mRNA as an internal standard ( Fig 6). The assays were performed by the "primer dropping" method (32), which involves careful optimization of PCR cycle numbers for each target gene (OSBP, ORP1-8). This analysis revealed that the mRNA expression of most of the ORP genes was not significantly affected by the acLDL loading. However, the ORP6 mRNA was up-regulated approximately 2-fold in the acLDL-loaded cells (Fig 6).

View larger version (86K): [in this window] [in a new window]   Figure 6. Effect of macrophage loading with acLDL on ORP mRNA expression. Total RNA isolated from unloaded (white bars) or acLDL-loaded (black bars) human macrophages was reverse transcribed by using random priming. Specific OSBP or ORP1;–8 cDNA fragments were amplified simultaneously with GAPDH, which was used as an internal control. A: Analysis of the PCR products in 2% agarose gels. Std, Roche DNA molecular weight marker VI; O, OSBP; 1;–8, the corresponding ORP. The mobility of the ORP and GAPDH PCR fragments is indicated on the right. B: Quantitation of the ORP signals relative to GAPDH. The mean signal in unloaded cells was set to 1. The results represent mean ± SEM from 4;–6 independent experiments. The asterisk indicates a significant difference between the unloaded and acLDL-loaded specimens (t-test, P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we employed a powerful combination of bioinformatics and cloning approaches to deduce the full-length cDNA sequences and genomic structures of the recently identified human ORP genes (30) (39). In addition to the seven previously reported genes, we identified five novel ORP genes. The hallmark of the ORP amino acid sequences is the SB domain located in the C-terminal half of the proteins. This is the most conserved part of the proteins, which can be used to identify members of the gene family. In particular, the EQVSHHPP sequence, denoted as the OSBP fingerprint, is most useful for this purpose. Another important sequence feature is a PH domain near the N-terminus of the proteins. A major biological function of PH domains is interaction with the headgroups of phosphoinositides (37). The PH domain of OSBP is necessary for the biological activities of the protein (20) and has been reported to bind phosphatidyl inositol-4,5-bis-phosphate or a related lipid (21). As in four of the yeast ORP (Kes1p, Hes1p, Yhg1p, and Yky3p), the human ORP2 lacks a PH domain, indicating that it may be functionally distinct from the PH domain-containing members of the protein family. Regarding association with cellular membranes, subfamily IV has a unique property. Both ORP5 and ORP8 have a C-terminal hydrophobic segment that is predicted to act as a transmembrane anchor. Three ankyrin repeats were identified close to the N-terminus of ORP1. This feature seems to be unique to human ORP1 and yeast Osh1p/Swh1p (26) and YDL019c (27) (28). Ankyrin repeats have been identified in more than 170 human proteins, and are known to mediate protein-protein interactions (40).

The ORP genes are widely distributed in different human chromosomes, which implies that they have not arisen as a result of recent gene duplications within a single chromosome, but are of considerably older descent. The number of genes identified in humans, 12, is significantly higher than that found in lower eukaryotes (S. cerevisiae, D. melanogaster, and C. elegans), which most probably reflects functional diversification of the gene family in vertebrates or mammals. Based on similarity in amino acid sequence and gene structure, the human OSBP-related proteins/genes can be divided into six distinct subfamilies. The subfamilies belong to two major evolutionary branches, both of which also contain members from the lower eukaryotes. This implies that members of both branches are involved in functions of fundamental importance in the eukaryotic kingdom. The lower eukaryotic organisms, which are easily amenable to genetic manipulation, provide useful model systems for elucidating the functions of the OSBP family.

The current ORP mRNA expression data (Table 3) suggest that the ORP genes, although present ubiquitously, are subject to tissue-specific transcriptional regulation. A good example is provided by members of subfamily III: ORP6 expression level is highest in the skeletal muscle and the brain, whereas ORP3 is most abundant in the kidney, and ORP7 in the gastrointestinal tract. It is noteworthy that a majority of the ORP show a high expression level in the brain, the most cholesterol-rich organ.

ORP4 is identical to the recently reported Hela metastatic gene that was isolated by a differential display approach. Interestingly, increased ORP4 mRNA levels were found in circulating tumor cells in peripheral blood samples (41). Whether altered ORP gene expression plays a role in human cancer remains to be determined. During the cloning process, we observed that some of the ORP mRNAs may have undergone differential splicing (data not shown). In support of this, a recent database release described an alternatively spliced ORP4 variant (XM000963), which was significantly different from the ORP4 cDNA reported here. This new ORP4 variant had a nearly identical SB domain, but lacked the PH domain and contained 8 exons not present in the cDNA reported here. It is therefore possible that functionally distinct forms of the ORP may arise from the same gene due to differential mRNA splicing. That such alternative splicing of ORP messages can occur in a tissue-specific manner is implied by our previous Northern blotting data (30).

OSBP has previously been shown to exert distinct regulatory effects on the biosynthesis of cholesterol and sphingomyelin (20) (23) (24). However, there is evidence that regulation of sterol and sphingomyelin biosynthesis is probably not the sole function of the OSBP gene family. ORP genes are present in Drosophila, which is unable to synthesize the sterol backbone (42). Further, the yeast ORP gene KES1 has been shown to play an important role in secretory transport through the Golgi complex (29), suggesting a function of the protein family in vesicle transport. On the other hand, the ORP could bind and introduce sterols to enzymes derivatizing them for purposes such as steroid hormone or bile acid synthesis, or modulate the transactivation potency of transcription factors (e.g., LXR, LXRß, or SF-1). Furthermore, ORP could be involved in the synthesis or transport of oxysterols, which are suggested to provide a means of cholesterol efflux from peripheral cells (43) (44) (45).

Analysis of ORP mRNA expression in unloaded and acLDL-loaded human macrophages did not reveal marked changes in the message levels, indicating that the ORP genes are not subject to major cholesterol-induced transcriptional regulation. The only mRNA displaying significant acLDL-induced up-regulation was that of ORP6 (2-fold induction), whereas most of the messages displayed a mild decrease in the acLDL-treated cells.

The present study summarizes hallmarks of the OSBP-related gene family in humans and provides tools for functional analysis of the encoded proteins. Further insight into the role of the ORP can now be obtained by using cDNA expression approaches to determine the impact of the proteins on the biosynthesis and trafficking of lipids, on sterol-regulated transcriptional responses, as well as on protein trafficking through the vesicle transport routes.

	FOOTNOTES

Abbreviations: acLDL, acetylated LDL; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LXR, liver X receptor; OSBP, oxysterol-binding protein; ORP, OSBP-related protein; PH, pleckstrin homology; RACE, rapid amplification of cDNA ends; SB, sterol-binding; SF-1, steroidogenic factor-1.

	ACKNOWLEDGMENTS

We thank Seija Puomilahti, Liisa Ikävalko, and Pirjo Ranta for skillful technical assistance. The KIAA0704, KIAA0772, KIAA1451, and KIAA1534 cDNAs were kindly provided by Dr. Takahiro Nagase, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan. This work was supported by the Academy of Finland (grants 42163, 45817, and 50641 to V.M.O; 43184, 43668, and 49987 to E.I.), the Sigrid Juselius Foundation (V.M.O. and E.I.), and the Finnish Cultural Foundation (S.L.).

Manuscript received February 13, 2001; and in revised form April 23, 2001

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