LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction (original) (raw)
LBP circulates in association with apoB-containing lipoproteins in healthy persons. The distribution of LBP among lipoproteins in human serum derived from healthy volunteers was evaluated using agarose gel electrophoresis in combination with Western blot analysis. Agarose gel electrophoresis avoids the harsh conditions of several other techniques for separation of lipoproteins, such as high salt concentrations and high gravitational forces encountered in ultracentrifugation or perturbation from polyanion precipitation, which cause loss of associated proteins (22). Accordingly, LBP was not detectable by ELISA (detection limit, 200 pg/ml) in the lipoprotein fractions separated by ultracentrifugation. In addition, this technique enables the study of interactions of serum constituents proportional to serum levels.
Surprisingly, Western blot analysis with an LBP-specific antibody shows that in all subjects investigated, LBP is located only at the β (LDL/VLDL) mobility region (Figure 1). As a control, LBP-depleted serum derived by selective-affinity immunosorption was used, which lacked reaction with the anti-LBP antibody (Figure 1). Immunoblotting for apoB demonstrates colocalization of LBP with apoB (Figure 1). LBP was not detected in the α mobility region and did not colocalize with apo A-I (Figure 1).
Distribution profile of LBP among lipoproteins in serum of healthy persons. Serum from three healthy persons (lanes 1–3) and LBP-depleted serum (lane 4) were subjected to agarose gel electrophoresis, blotted, and probed with specific antibodies to human LBP, apoA-I, and apoB. LBP colocalizes with apoB in the β mobility region and not with apoA-I.
Free LBP, purified from human plasma and devoid of lipoproteins, migrates between the β and α regions on agarose gel electrophoresis (Figure 2). We studied whether the β mobility of LBP in serum is due to association of LBP with lipoproteins. To this end, lipoproteins were fractionated by ultracentrifugation. The isolated lipoproteins were preincubated at approximately 50% of their plasma concentration overnight at 37°C with LBP (30 μg/ml) and subjected to electrophoresis, and Western blot analysis of LBP was performed. Preincubation of purified LBP with VLDL or LDL restored the β mobility of LBP (Figure 2). This conversion in electrophoretic migration did not occur when LBP was preincubated with HDL. These results show that in serum, the association of LBP with LDL and VLDL is responsible for its β-electrophoretic mobility.
Effect of LDL and VLDL on the electrophoretic mobility of purified LBP. Purified LBP was preincubated with PBS and isolated LBP-free HDL, VLDL, or LDL (0.1, 0.035, and 0.5 mg cholesterol/ml respectively). Agarose gel electrophoresis of purified LBP and the preincubated fractions was followed by Western blot analysis of LBP using a specific antibody. Purified LBP migrates in between the β and α mobility regions. Preincubation of LBP with isolated VLDL or LDL shifts the migration of LBP to the β mobility region.
Further evidence for the association between LBP and apoB-containing lipoproteins in human serum was found using an assay that specifically and quantitatively detects complexes of LBP and apoB-containing lipoproteins in serum. In this assay, LBP-containing lipoproteins are captured from serum by solid-phase anti-LBP antibodies, followed by detection of apoB in the captured lipoproteins by peroxidase-labeled anti-apoB antibodies. Consistent with our prior experiments, this assay demonstrates the association of LBP with apoB-containing lipoproteins in serum (Figure 3).
Presence of apoB in LBP-containing lipoproteins. LBP-containing lipoproteins were captured from serum by applying serum to 96-well plates coated with an mAb against LBP (diamonds) followed by extensive washing of the plates. As a control serum was also applied to noncoated plates (squares) or plates coated with an aspecific antibody (rat anti-murine TNF-R75) (triangles). Presence of apoB in the captured lipoproteins was detected by addition of a peroxidase-labeled mAb against apoB and is expressed as mean ± SD of the OD 450 nm of four wells.
To characterize the association between LBP and the different lipoproteins in a more quantitative manner, a binding assay was used. Binding of purified LBP to isolated lipoproteins standardized for cholesterol concentration was studied using this assay. Consistent with the results already described here, a concentration-dependent binding of LBP to LDL and VLDL is observed (Figure 4). In contrast, the association of purified LBP with HDL is only minor.
Association of LBP with different lipoprotein classes. Lipoproteins isolated from human serum by ultracentrifugation, free of LBP, and standardized for cholesterol concentration were immobilized to 96-well plates and incubated with biotinylated LBP. Bound LBP was detected by peroxidase-conjugated streptavidin and TMB. Binding of LBP to the lipoproteins is expressed as mean ± SD of the OD 450 nm of four wells after correction for background. LDL and VLDL display high LBP-binding capacity in contrast to HDL.
In conclusion, we now demonstrate that isolated LDL and VLDL display higher LBP-binding capacity compared with HDL. This is fully in line with our observation that LBP circulates predominantly in association with apoB-containing lipoproteins.
LPS interacts predominantly with apoB-containing lipoproteins. LBP binds LPS and catalyzes the transfer of LPS from micelles into lipoproteins (12). In the context of this function of LBP and our data demonstrating that LBP circulates as a complex with apoB-containing lipoproteins, we studied the relation between LBP-lipoprotein interaction and LPS binding to lipoproteins. To this end, biotin-labeled LPS was preincubated with human serum from healthy volunteers, followed by separation of serum lipoproteins on agarose gels and Western blot analysis. Figure 5 reveals that LPS incubated in serum is recovered in the β mobility region. This pattern is consistent with a predominant interaction of LBP with apoB-containing lipoproteins. To test whether LPS binding to apoB-containing lipoproteins is LBP dependent, biotin-labeled LPS was incubated with LBP-depleted serum. Figure 5 demonstrates that LPS also binds to β lipoprotein in the absence of LBP.
Distribution profile of LPS among lipoproteins in serum of healthy persons. Serum of three healthy donors and LBP-depleted serum were preincubated with biotinylated LPS. Agarose gel electrophoresis of the sera was followed by Western blotting. Biotinylated LPS was detected using peroxidase-conjugated streptavidin and a chemiluminescent substrate. LPS incubated with serum is recovered in the β mobility region.
To study whether non–lipoprotein-associated serum factors are responsible for the interaction of LPS with LDL and VLDL, serum lipoproteins were separated by agarose gel electrophoresis followed by Western blot analysis. Biotinylated LPS was allowed to associate with the (apo)lipoproteins on the blot membrane. The distribution profile obtained was similar to the profile obtained when LPS was preincubated with serum (data not shown). This suggests that β-lipoprotein–associated factors account for the LPS binding and that other serum factors are not essentially affecting the distribution.
The binding of LPS to lipoproteins was further characterized using an assay similar to that just described for studying LBP-lipoprotein interaction. Binding of LPS to isolated lipoproteins standardized for cholesterol concentration was studied. Also in this assay and fully in line with the results found by Western blot analysis, LDL and VLDL display high LPS-binding capacity under LBP-free conditions, whereas LPS binding to purified HDL is minor (Figure 6).
Association of LPS with different lipoprotein classes. Lipoproteins isolated from human serum by ultracentrifugation, free of LBP, and standardized for cholesterol concentration were immobilized to 96-well plates followed by incubation with biotinylated LPS. Binding of LPS to the lipoproteins was detected by peroxidase-conjugated streptavidin and TMB and expressed as mean ± SD of the OD 450 nm of four wells after correction for background. LDL and to a lesser extent VLDL display high LPS-binding capacity in contrast to HDL.
LBP bound to lipoproteins enhances the interaction of LPS with lipoproteins. To elucidate whether LBP associated with LDL and VLDL is functionally active in transferring LPS to lipoproteins, we compared LPS binding to lipoproteins with binding of LPS to LBP-lipoprotein complexes. To this end, LDL and VLDL were immobilized onto 96-well plates, and a concentration range of LBP was allowed to bind to the lipoproteins for 18 hours at 37°C. Plates were washed to remove unbound LBP before biotinylated LPS was added to the preformed LBP-lipoprotein complexes. LBP associated with LDL and VLDL enhanced the binding of LPS to these lipoproteins dose-dependently (Figure 7). These data strongly indicate that LBP bound to LDL and VLDL displays functional properties and enhances the LPS-binding capacity of LDL and VLDL.
LBP associated with LDL and VLDL enhances the interaction of LPS with lipoproteins. Plates were coated with isolated LDL (2 mg cholesterol/ml) or VLDL (14 mg cholesterol/ml). The immobilized lipoproteins were preincubated with a concentration range of LBP overnight at 37°C. Unbound LBP was removed by washing the plates, and biotinylated LPS was added to the LBP-lipoprotein complexes. Plates were washed to remove unbound LPS and bound LPS was detected using peroxidase-conjugated streptavidin and TMB. Binding of LPS to the lipoproteins is expressed as mean ± SD of the OD 450 nm of four wells after correction for background. LBP associated with LDL and VLDL enhances the binding of LPS dose-dependently.
Both LBP and LPS associate with apolipoprotein B. To elucidate whether binding of LBP and LPS to LDL and VLDL depends on interaction with apoB, present in both LDL and VLDL, we studied the binding of LBP and LPS to purified apoB. Contemporaneously, binding of LBP and LPS to purified apoA-I, the predominant apolipoprotein in HDL, was evaluated. Both LBP and LPS bind to apoB dose-dependently (Figure 8, a and b). Binding of LPS to apoA-I was, however, not significant (Figure 8a), which is in accordance with the minor binding of LPS to HDL. Surprisingly, LBP forms a complex with purified apoA-I (Figure 8b).
Association of LBP and LPS with apoB and apoA-I. (a) Binding of biotin-labeled LPS to apolipoproteins was evaluated. To this end, plates were coated with a concentration range of apoB and apoA-I, and biotin-labeled LPS was added. Bound LPS was detected by peroxidase-conjugated streptavidin and TMB and expressed as mean ± SD of the OD 450 nm of four wells after correction for background. (b) Binding of biotin-labeled LBP to immobilized apoB (25 nM) or apoA-I (100 nM) was detected by peroxidase-conjugated streptavidin and TMB and expressed as mean ± SD of the OD 450 nm of four wells after correction for background. (c) The relative affinities of LBP for apoB and apoA-I were evaluated. Biotinylated LBP was added to plates coated with apoB (25 nM). Inhibition of this interaction of LBP with apoB by apoA-I and apoB was studied by adding concentration ranges of apoA-I and apoB together with LBP. Bound LBP was detected by peroxidase-conjugated streptavidin and TMB and expressed as mean ± SD of the OD 450 nm of three wells after correction for background.
Because circulating LBP is predominantly associated with lipoproteins containing apoB and not apoA-I (Figure 1), we compared the relative affinities of LBP for apoB and apoA-I. To this end, plates were coated with apoB, and a dilution series of apoA-I and apoB was added to the plates together with biotinylated LBP (Figure 8c). The concentration necessary for 50% reduction of the signal was 50 nM for apoB and 500 nM for apoA-I. These results can be due either to a difference in affinity for LBP or to the possibility that apoA-I and apoB have affinity for a different site of LBP. However, when plates were coated with apoA-I, comparable results were found (data not shown): apoA-I concentrations necessary for 50% reduction of the signal were tenfold higher than apoB concentrations. These data strongly suggest that apoA-I and apoB compete for the same binding site on LBP and that the affinity of LBP for apoB is tenfold higher than for apoA-I.
Because lipoprotein-associated LBP enhances the binding of LPS to apoB-containing lipoproteins, we evaluated whether LBP also catalyzes the binding of LPS to the purified apolipoproteins. To this end, plates were coated with apoB and apoA-I, and biotinylated LPS was added to the immobilized apolipoproteins together with 1.7 nM LBP. This LBP concentration was demonstrated to enhance the binding of LPS to LDL and VLDL (Figure 7). LBP enhanced the binding of LPS to apoA-I, whereas it decreased the binding of LPS to apoB (Figure 9). These results suggest that the interaction of LBP with apoA-I enables the binding of LPS to this apolipoprotein, whereas LBP competes with LPS for binding to apoB.
LBP enhances binding of LPS to apoA-I and reduces binding of LPS to apoB. Plates were coated with apoA-I (25 nM) or apoB (100 nM). A total of 1.7 nM of LBP was added together with biotinylated LPS, followed by peroxidase-conjugated streptavidin and TMB. Binding of LBP to the apolipoproteins is expressed as mean ± SD of the OD 450 nm of four wells after correction for background. The binding of LPS to apoA-I is markedly enhanced by LBP, whereas LBP reduces the binding of LPS to apoB.
Overall, these data suggest that apoB contains a binding site for both LBP and LPS, and it appears that this binding site accounts at least in part for the association of LBP and LPS with LDL and VLDL in serum.
LBP and LPS associate with LDL and VLDL in serum of septic patients. Lipoprotein metabolism, as well as the composition of lipoproteins, is altered during the acute-phase response. In addition, serum levels of LBP rise dramatically during sepsis (15). Because the data so far indicate that the association of LBP with lipoproteins affects the functional properties of LBP and lipoproteins, which is of utmost interest during endotoxemia, we evaluated in which form the plenitude of LBP produced during sepsis circulates. Serum derived from four septic patients (LBP serum concentration: 121–589 μg/ml compared with 13–16 μg/ml in the healthy controls) were subjected to agarose gel electrophoresis followed by Western blot analysis. As observed for LBP in serum from healthy persons, LBP circulates during septicemia predominantly associated with LDL and VLDL (Figure 10). However, an additional LBP band is observed between the β and the α regions, indicative for the presence of free LBP. As expected, apoA-I levels dropped markedly in the septic patients (Figure 10). Also, no association of LBP with HDL was observed in septic patients.
Distribution profile of LBP and LPS in serum of septic patients. Agarose gel electrophorese of serum from four septic patients (lanes 1–4) was followed by Western blot analyses using specific antibodies for LBP, and apoB. Agarose gel electrophorese of serum from three septic patients (lanes 1–3) and a serum pool of healthy persons (lane 4) was followed by Western blot analyses using specific antibodies for apoA-I. LBP predominantly colocalizes with apoB. An additional band compared with normal serum is observed in all subjects between the β and α regions, and is most explicit in subject 3. Preincubation of LPS with the sera followed by Western blot analyses demonstrates the distribution of LPS among lipoproteins during an acute-phase response. LPS colocalizes with LBP and apoB in the β region under these conditions.
In addition, we investigated whether the alterations in the composition of lipoproteins during infection and the presence of free LBP affected the distribution of LPS among lipoproteins. Biotinylated-LPS preincubated with serum from septic patients was found to migrate with β-electrophoretic mobility and to comigrate with apoB. These findings are in accordance with the data obtained from healthy persons and suggest that also during an acute-phase response, LPS binds predominantly to LDL and VLDL (Figure 10).