Functional Characterization of Newly-Discovered Mutations in Human SR-BI

In rodents, SR-BI has been firmly established as a physiologically relevant HDL receptor that mediates removal of HDL-cholesteryl esters (CE). However, its role in human lipoprotein metabolism is less defined. Recently, two unique point mutations in human SR-BI — S112F or T175A — were identified in subjects with high HDL-cholesterol (HDL-C) levels. We hypothesized that mutation of these conserved residues would compromise the cholesterol-transport functions of SR-BI. To test this hypothesis, S112F- and T175A-SR-BI were generated by site-directed mutagenesis. Cell surface expression was confirmed for both mutant receptors in COS-7 cells upon transient transfection, albeit at lower levels for T175A-SR-BI. Both mutant receptors displayed defective HDL binding, selective uptake of HDL-CE and release of free cholesterol (FC) from cells to HDL. Mutant receptors were also unable to re-organize plasma membrane pools of FC. While these impaired functions were independent of receptor oligomerization, inability of T175A-SR-BI to mediate cholesterol-transport functions could be related to altered N-linked glycosylation status. In conclusion, high HDL-C levels observed in carriers of S112F- or T175A-SR-BI mutant receptors are consistent with the inability of these SR-BI receptors to mediate efficient selective uptake of HDL-CE, and suggest that increased plasma HDL concentrations in these settings may not be associated with lower risk of cardiovascular disease.


Introduction
The reverse cholesterol transport (RCT) pathway, whereby high density lipoproteins (HDL) transport cholesterol from peripheral tissues to the liver for catabolism [1], is critical for combating coronary artery disease since it prevents the accumulation of excess cholesterol, lipids, and cellular debris, and thus the formation of atherosclerotic plaques on arterial walls [2]. In the last step of RCT, HDL binds to scavenger receptor class B type I (SR-BI), an 82 kDa glycosylated cell-surface receptor that mediates selective uptake of HDL-cholesteryl esters (CE) into the liver [3], without holo-particle uptake [4][5][6]. SR-BI also stimulates free cholesterol (FC) efflux from peripheral tissues to HDL [7], although its role in stimulating this early step of RCT to decrease macrophage foam cell formation remains controversial [8,9].
The selective uptake of HDL-CE is only achieved if HDL and SR-BI form a ''productive complex'', where the receptor and ligand precisely align to allow CE transfer to occur [10]. Previous studies have validated the importance of SR-BI's extracellular domain in the selective uptake process through use of chimeric receptors [11,12], epitope tag insertion [13], mutagenesis [14,15], and blocking antibody directed against the extracellular domain [16]. Further, our recent work demonstrates that hydrophobicity of the N-terminal half of the extracellular domain of SR-BI [15], as well as a specific conformation of the C-terminal half of the extracellular domain held together by disulfide bonds [17], are critical for receptor function.
Transgenic overexpression [18][19][20] or hepatic adenoviralmediated [21,22] SR-BI cDNA transfer in mice decreased plasma HDL-cholesterol (HDL-C) levels and increased cholesterol catabolism and excretion. On the other hand, reduced SR-BI expression or whole-body knock-out of the SR-BI gene resulted in increased plasma HDL-C levels [23]. Although these studies have firmly established SR-BI as a physiologically relevant HDL receptor in rodents, its role in human lipoprotein metabolism is less defined. The human homologue of SR-BI, also known as CLA-1 (CD36 and LIMPII analogous-1), serves as a receptor for HDL and mediates HDL-CE selective uptake [24]. Similar to rodents, it is also most abundantly expressed in the liver and steroidogenic tissues [24]. Human SR-BI variants are associated with changes in HDL-cholesterol [25,26] and protein levels in peripheral tissues [27]. Most recently, two point mutations in human SR-BI -at Ser112 (to Phe) or at Thr175 (to Ala) -were identified in two subjects with high HDL-C levels [28]. As these are highly conserved residues across species, we predicted that SR-BI's function would be compromised.
In this study, we characterized the functionality of these SR-BI variants in COS-7 cells to test our hypothesis that high HDLcholesterol levels in subjects harboring mutations at Ser112 or Thr175 result from the inability of SR-BI to mediate efficient HDL-CE selective uptake.

Cell lysis
Transiently-transfected COS-7 cells were washed twice with cold PBS (pH 7.4) and lysed with 1% NP-40 cell lysis buffer containing protease inhibitors. Protein concentrations were determined by the Lowry method as previously described [30].

Cell surface receptor expression
Cell surface biotinylation of SR-BI in transiently-transfected COS-7 cells was performed as previously described [15]. In separate experiments, cell surface expression of SR-BI was verified by flow cytometry as described using FACS Calibur (Flow Cytometry Core, Medical College of Wisconsin) [17].  [29]. Statistical analysis was determined by one-way ANOVA.

Free cholesterol efflux and cholesterol oxidase sensitivity
Cells transiently transfected with cDNA encoding empty vector, wild-type or mutant human SR-BI were pre-labeled with [ 3 H]cholesterol and assayed for FC release from cells to HDL, or for sensitivity to exogenous cholesterol oxidase at 72-hours or 48-hours post-transfection, respectively, as previously described [13]. Statistical analysis was determined by one-way ANOVA.

SR-BI oligomerization
Cells transiently expressing wild-type and mutant SR-BI were lysed in PBS containing protease inhibitors. SR-BI oligomerization was assessed by 6% PFO-PAGE as previously described [15].

Variable cell surface expression of mutant SR-BI receptors
Ser112 and Thr175 are evolutionarily conserved residues. Using site-directed mutagenesis, we created S112F and T175A point mutations in human SR-BI to test their ability to mediate cholesterol-transport functions. We chose to study SR-BI function in transfected COS-7 cells since they do not have endogenous SR-BI expression (as confirmed by immunoblot and flow cytometry analyses; [31] and data not shown). We used two different methods to assess cell surface expression of both SR-BI mutants. In the first method, biotinylation and immunoblot analysis of COS-7 cell lysates revealed that wild-type and S112F-SR-BI expressed at approximately similar levels at the cell surface. However, T175A-SR-BI expressed more than 50% less than wild-type SR-BI based on band densitometry ( Figure 1A, top panel). Total cell expression of all SR-BI constructs are shown in Figure 1A (middle panel). These observations were mirrored in parallel experiments where flow cytometry analyses also revealed that T175A-SR-BI expressed at much lower levels at the cell surface than wild-type or S112F-SR-BI ( Figure 1B).

Mutant receptors displayed an impaired ability to bind HDL and mediate selective uptake of HDL-COE
To determine if mutant SR-BI receptors shared similar functional properties as wild-type SR-BI, we assessed the ability of all SR-BI receptors to bind HDL and mediate the selective uptake of HDL-COE by transiently transfecting COS-7 cells with vectors encoding mutant SR-BI proteins. As shown in Figure 2A, S112F-and T175A-SR-BI displayed significantly lower levels of HDL binding as compared to wild-type SR-BI, even after normalization to cell surface expression based on flow cytometry analyses ( Figure 2C) from parallel wells from the same experiments. As expected, the inability of both mutants to properly bind HDL translated to an impaired ability to mediate efficient selective uptake of HDL-COE ( Figures 2B, D).

Mutant receptors display decreased efflux of free cholesterol to HDL
In addition to its role in selective uptake of HDL-CE, SR-BI also plays a role in stimulating the transfer of FC from peripheral cells to acceptor particles such as HDL [3]. We measured the ability of wild-type, S112F-and T175A-SR-BI receptors to efflux FC from pre-labeled COS-7 cells to HDL acceptors. As shown in Figure 3, both the S112F-and T175A-SR-BI mutant receptors displayed significant decreases in FC efflux as compared to wildtype SR-BI. As expected [32][33][34][35], little to no wild-type-or mutant SR-BI-mediated FC efflux was observed when lipid-free apoA-I was used as an acceptor (data not shown), thus suggesting that mutant SR-BI receptors affect intracellular and/or membrane pools of FC only in the presence of HDL.
Mutant SR-BI receptors are unable to re-organize plasma membrane free cholesterol Native SR-BI plays a unique role in re-organizing the pools of plasma membrane FC as judged by the enhanced conversion of cholesterol to cholestenone in the presence of exogenous cholesterol oxidase [36]. Quantification of membrane-associated cholestenone following incubation with exogenous cholesterol oxidase revealed that both the S112F-and T175A-SR-BI mutant receptors displayed significantly decreased abilities to re-distribute plasma membrane content of FC as compared to wild-type SR-BI ( Figure 4).
Defective cholesterol transport functions of SR-BI mutants are not due to changes in their oligomeric status SR-BI exists as dimers and higher-order oligomers at the plasma membrane [37][38][39]. We and others suggest that SR-BI oligomerization is required for efficient selective uptake of HDL-CE [17,39,40], lending support to the notion that SR-BI oligomers form a hydrophobic channel to allow transfer of CE from HDL into the plasma membrane [41]. To determine whether the inability of the S112F-and T175A-SR-BI mutant receptors to mediate proper cholesterol-transport functions was due to their inability to form higher-order oligomers, we assessed their oligomeric status by performing native gel electrophoresis in the presence of perfluorooctanoic acid (PFO), a detergent known to stabilize interactions between oligomers [15,17,42]. Based on our analyses, both the S112F-and T175A-mutant receptors were able to form higher order oligomers similar to wild-type SR-BI ( Figure 5). These data suggest that the impaired function exhibited by the human SR-BI mutations is independent of the oligomeric status of the receptors.
T175A-SR-BI may be impaired due to altered glycosylation patterns Residue 175 of human SR-BI is part of an N-linked glycosylation sequence (NXS/T) [43,44] that is highly conserved among species ( Figure 6A). Mutation of Thr175 to Ala disrupts proper glycosylation of Asn173 as evidenced by a faster mobility on SDS-PAGE as compared to wild-type SR-BI or the S112F-SR-BI mutant receptor ( Figure 6B). This data suggests that the lower cell surface expression and impaired function of T175A-SR-BI may be the result of an altered glycosylation status of the receptor.

Discussion
The recent identification of two novel missense mutations, S112F and T175A in human SR-BI, prompted us to characterize the functionality of these two variants of SR-BI that underlie elevated HDL-cholesterol levels in humans. Our data revealed that both mutant SR-BI receptors had significantly impaired abilities to bind HDL and mediate the selective uptake of HDL-COE. In addition to displaying defective efflux of FC to HDL, both mutants were also unable to redistribute the plasma membrane pools of FC. The impaired functions of these mutant receptors were not due to changes in oligomeric status. However, decreased cell surface expression and defective functions of T175A-SR-BI could be explained, in part, by an altered glycosylation status as compared to wild-type SR-BI.
The accumulation of HDL-cholesterol levels upon deletion of the SR-BI gene in mice [23] strongly suggests that the atheroprotective role of SR-BI [45][46][47] is primarily due to its ability to mediate the selective uptake of CE from circulating HDL particles [23,48,49]. Based on our in vitro studies, we suggest that inefficient clearance of HDL-C from circulation may explain the 37% increases in HDL-C (with no significant differences in triglycerides, LDL-cholesterol or body mass index) in the two individuals heterozygous for mutations at Ser112 or Thr175 compared to family member controls [28]. As carriers of P297S-SR-BI, the first human SR-BI mutation, displayed similar increases in HDL-C levels [50], our data lend support to the notion that human SR-BI does indeed play a role in hepatic HDL-CE uptake.
The athero-protective effects of SR-BI can also be explained, in part, by its role in mediating FC efflux from lipid-laden macrophages to HDL, although this remains controversial [8,[51][52][53][54]. The reduced ability of S112F-and T175A-SR-BI mutant receptors to efflux FC to HDL mirrors similar trends observed with the P297S-SR-BI mutant receptor in mice [50]. At least in the case of T175A-SR-BI, lower levels of FC efflux may correlate with its lower levels of expression at the cell surface [7,55]. The inability of both mutant receptors to efficiently mediate cholesterol-transport functions may be associated, in part, with their inability to mediate plasma membrane reorganization of cholesterol, as we have previously observed with other SR-BI receptors harboring mutations in this region of the extracellular domain [15]. More in-depth cholesterol-transport and membrane re-organization studies in macrophage or hepatocyte/hepatoma cell lines warrant further investigation and can provide sharper insight as to how these mutant SR-BI receptors behave when all efflux machinery (i.e. ABCA1 and ABCG1) is present.
Mutation of Thr175 to alanine appears to disrupt N-linked glycosylation at the evolutionarily-conserved Asn173 residue as judged by its faster migration as compared to the wild-type or S112F-SR-BI receptors following SDS-PAGE. It was previously demonstrated that N-linked oligosaccharide chains at Asn173 are a critical determinant of cell surface expression and efficient lipid uptake [43]. In support of these observations, reduced cell surface expression of T175-SR-BI similarly correlates with reduced selective HDL-CE uptake efficiency. Although oligosaccharides on glycoproteins have been shown to participate in protein-protein interactions in other systems [56,57], our analyses indicated that SR-BI oligomerization is not affected by altered glycosylation status. Rather, it is likely that altered glycosylation in T175A-SR-BI and subsequent changes in cell surface expression result from either improper folding in the ER and consequent transport through the Golgi, or decreased receptor stability [43].
Mutation of Ser112 to Phe did not affect cell surface receptor expression, although it did significantly affect HDL-CE selective uptake efficiency. Interestingly, Ser112 lies adjacent to another Nlinked glycosylation site (Asn108) that is critical for cell surface expression and function [43]. It is certainly possible that introduction of a large amino acid like phenylalanine could impede the addition of oligosaccharides at Asn108, however an altered glycosylation status S112F-SR-BI was not evident from our immunoblot analyses. Alternatively, proper receptor-ligand interactions may be compromised by (i) altered secondary or tertiary structure of the area surrounding Ser112, (ii) the large nature of the phenyl side chain [58], or (iii) the inability of phenylalanine to Human SR-BI Mutations PLOS ONE | www.plosone.org participate in potential hydrogen bonding with residues on HDL proteins due to the lack of a hydroxyl group [59][60][61]. Remarkably, both the S112F and T175A point mutations occur in conserved regions of hydrophobicity in the N-terminal half of the extracellular domain of SR-BI. We have previously shown that the hydrophobicity of this region is critical for SR-BI-mediated cholesterol transport function [15] and hypothesized that this region may interact with the plasma membrane as has been reported for other integral membrane proteins [62,63]. While mutation of Thr to Ala does not impose major effects on hydrophobicity, mutation of Ser to Phe (hydrophobic index of 20.8 and 2.8, respectively [64]) significantly increases the hydrophobicity of the region surrounding residue 112. This leads us to speculate that the region surrounding the Phe residue becomes less solvent-exposed, and perhaps even more 'buried' in the membrane, thus preventing key conformational changes that may be required to support efficient selective uptake of HDL-CE. These ideas are currently being investigated.
Although human and rodent SR-BI share similar expression patterns, tissue abundance and in vitro receptor activities [3,24,38,65], the role of human SR-BI in hepatic clearance of HDL-CE and its relevance to human physiology has remained a    The evolutionarily-conserved consensus sequence for Nlinked glycosylation is shaded in gray for rabbit (AY_283277), mouse (NM_016741), rat (NP_113729.1), hamster (A53920) and human (NM_005505) SR-BI. The Thr-to-Ala mutation at residue 175 (in bold) disrupts this consensus sequence. (B) COS-7 cell lysates transiently expressing wild-type or mutant SR-BI were separated by 12% SDS-PAGE. SR-BI was detected by immunoblot analysis using an antibody directed against the C-terminal cytoplasmic domain of the receptor. Data is representative of 7 independent experiments. doi:10.1371/journal.pone.0045660.g006 mystery until recently. Not surprisingly, our functional characterization of these two newest human SR-BI mutations raises new questions. Due to inefficient clearance of plasma HDL particles, do carriers of the S112F or T175A mutations possess larger-sized HDL particles, similar to carriers of the P297S mutation [50], and SR-BI knock-out mice [23,50]? Has the oxidation status and efflux capacity of circulating HDL (i.e. HDL 'functionality') been altered? Importantly, carriers of SR-BI mutations at Ser112, Thr175 or Pro297 do not currently show clinical signs of atherosclerosis. Therefore, identification and investigation of additional subjects with the same or new SR-BI mutations will be invaluable as we address the on-going debate as to whether higher HDL-C levels are truly athero-protective [66][67][68][69]. Answers to these lingering questions are particularly relevant in light of current efforts directed at developing HDL-raising therapeutics to lower cardiovascular risk [70,71].