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Function modification of SR-PSOX by point mutations of basic amino acids.

Liu W, Yin L, Chen C, Dai Y - Lipids Health Dis (2011)

Bottom Line: A cell model to study the functions of SR-PSOX was successfully established.Functional studies showed that the mutants with H80A, H85A, and K105A significantly increased the activities of oxLDL uptake and bacterial phagocytosis compared with the wild-type SR-PSOX.In addition, we have also found that mutagenesis of either of those amino acids strongly reduced the adhesive activity of SR-PSOX by using a highly non-overlapping set of basic amino acid residues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Tongji University School of Medicine, 1239 Siping Road, Shanghai, 200092, China.

ABSTRACT

Background: Atherosclerosis (AS) is a common cardiovascular disease. Transformation of macrophages to form foam cells by internalizing modified low density-lipoprotein (LDL) via scavenger receptor (SR) is a key pathogenic process in the onset of AS. It has been demonstrated that SR-PSOX functions as either a scavenger receptor for uptake of atherogenic lipoproteins and bacteria or a membrane-anchored chemokine for adhesion of macrophages and T-cells to the endothelium. Therefore, SR-PSOX plays an important role in the development of AS. In this study the key basic amino acids in the chemokine domain of SR-PSOX have been identified for its functions.

Results: A cell model to study the functions of SR-PSOX was successfully established. Based on the cell model, a series of mutants of human SR-PSOX were constructed by replacing the single basic amino acid residue in the non-conservative region of the chemokine domain (arginine 62, arginine 78, histidine 80, arginine 82, histidine 85, lysine 105, lysine 119, histidine 123) with alanine (designated as R62A, R78A, H80A, R82A, H85A, K105A, K119A and H123A, respectively). Functional studies showed that the mutants with H80A, H85A, and K105A significantly increased the activities of oxLDL uptake and bacterial phagocytosis compared with the wild-type SR-PSOX. In addition, we have also found that mutagenesis of either of those amino acids strongly reduced the adhesive activity of SR-PSOX by using a highly non-overlapping set of basic amino acid residues.

Conclusion: Our study demonstrates that basic amino acid residues in the non-conservative region of the chemokine domain of SR-PSOX are critical for its functions. Mutation of H80, H85, and K105 is responsible for increasing SR-PSOX binding with oxLDL and bacteria. All the basic amino acids in this region are important in the cells adhesion via SR-PSOX. These findings suggest that mutagenesis of the basic amino acids in the chemokine domain of SR-PSOX may contribute to atherogenesis.

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Uptake of DiI-oxLDL by the transfected 293T cells. 293T cells were transiently transfected with pEGFP-N3 empty vector, wild-type SR-PSOX and different mutant constructs respectively. After 24 hours transfection, cells were incubated with DiI-oxLDL for 4 hours at 37°C. The relative intensity of red fluorescence represents the ability of DiI-oxLDL uptaked by the cells. The results show the mean ± SEM of the intensity of red fluorescence in the EGFP-positive 293T cells from three independent experiments. *: P < 0.05, **: P < 0.01, each compared to the wild-type SR-PSOX (WT).
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Figure 4: Uptake of DiI-oxLDL by the transfected 293T cells. 293T cells were transiently transfected with pEGFP-N3 empty vector, wild-type SR-PSOX and different mutant constructs respectively. After 24 hours transfection, cells were incubated with DiI-oxLDL for 4 hours at 37°C. The relative intensity of red fluorescence represents the ability of DiI-oxLDL uptaked by the cells. The results show the mean ± SEM of the intensity of red fluorescence in the EGFP-positive 293T cells from three independent experiments. *: P < 0.05, **: P < 0.01, each compared to the wild-type SR-PSOX (WT).

Mentions: Since oxLDL is a ligand for SR-PSOX, replacement of certain amino acid in the non-conservative region of the chemokine domain of SR-PSOX may result in altering its capability to oxLDL uptake. To test the hypothesis, 293T cells were transfected with SR-PSOX wide-type and mutant plasmids. After transfection for 24 hours, the cells were incubated with Dil-oxLDL and the uptake of DiI-oxLDL in the cells was analysed by FACS (Additional file 2: Figure S2). As shown in Figure 4, the uptake of DiI-oxLDL by H80A, H85A and K105A mutant-expressed cells was increased by 58%, 96% and 72% respectively compared with that of wild-type SR-PSOX, and the enhancements were statistically significant. However, the cells expressing other mutants only slightly increased the uptake of DiI-oxLDL, suggesting that mutation of H80, H85 and K105 is very important for the cell to absorb oxLDL. These results implicate that mutagenesis of the basic amino acid in the non-conservative region of the chemokine domain may contribute to the atherogenic process.


Function modification of SR-PSOX by point mutations of basic amino acids.

Liu W, Yin L, Chen C, Dai Y - Lipids Health Dis (2011)

Uptake of DiI-oxLDL by the transfected 293T cells. 293T cells were transiently transfected with pEGFP-N3 empty vector, wild-type SR-PSOX and different mutant constructs respectively. After 24 hours transfection, cells were incubated with DiI-oxLDL for 4 hours at 37°C. The relative intensity of red fluorescence represents the ability of DiI-oxLDL uptaked by the cells. The results show the mean ± SEM of the intensity of red fluorescence in the EGFP-positive 293T cells from three independent experiments. *: P < 0.05, **: P < 0.01, each compared to the wild-type SR-PSOX (WT).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102630&req=5

Figure 4: Uptake of DiI-oxLDL by the transfected 293T cells. 293T cells were transiently transfected with pEGFP-N3 empty vector, wild-type SR-PSOX and different mutant constructs respectively. After 24 hours transfection, cells were incubated with DiI-oxLDL for 4 hours at 37°C. The relative intensity of red fluorescence represents the ability of DiI-oxLDL uptaked by the cells. The results show the mean ± SEM of the intensity of red fluorescence in the EGFP-positive 293T cells from three independent experiments. *: P < 0.05, **: P < 0.01, each compared to the wild-type SR-PSOX (WT).
Mentions: Since oxLDL is a ligand for SR-PSOX, replacement of certain amino acid in the non-conservative region of the chemokine domain of SR-PSOX may result in altering its capability to oxLDL uptake. To test the hypothesis, 293T cells were transfected with SR-PSOX wide-type and mutant plasmids. After transfection for 24 hours, the cells were incubated with Dil-oxLDL and the uptake of DiI-oxLDL in the cells was analysed by FACS (Additional file 2: Figure S2). As shown in Figure 4, the uptake of DiI-oxLDL by H80A, H85A and K105A mutant-expressed cells was increased by 58%, 96% and 72% respectively compared with that of wild-type SR-PSOX, and the enhancements were statistically significant. However, the cells expressing other mutants only slightly increased the uptake of DiI-oxLDL, suggesting that mutation of H80, H85 and K105 is very important for the cell to absorb oxLDL. These results implicate that mutagenesis of the basic amino acid in the non-conservative region of the chemokine domain may contribute to the atherogenic process.

Bottom Line: A cell model to study the functions of SR-PSOX was successfully established.Functional studies showed that the mutants with H80A, H85A, and K105A significantly increased the activities of oxLDL uptake and bacterial phagocytosis compared with the wild-type SR-PSOX.In addition, we have also found that mutagenesis of either of those amino acids strongly reduced the adhesive activity of SR-PSOX by using a highly non-overlapping set of basic amino acid residues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Tongji University School of Medicine, 1239 Siping Road, Shanghai, 200092, China.

ABSTRACT

Background: Atherosclerosis (AS) is a common cardiovascular disease. Transformation of macrophages to form foam cells by internalizing modified low density-lipoprotein (LDL) via scavenger receptor (SR) is a key pathogenic process in the onset of AS. It has been demonstrated that SR-PSOX functions as either a scavenger receptor for uptake of atherogenic lipoproteins and bacteria or a membrane-anchored chemokine for adhesion of macrophages and T-cells to the endothelium. Therefore, SR-PSOX plays an important role in the development of AS. In this study the key basic amino acids in the chemokine domain of SR-PSOX have been identified for its functions.

Results: A cell model to study the functions of SR-PSOX was successfully established. Based on the cell model, a series of mutants of human SR-PSOX were constructed by replacing the single basic amino acid residue in the non-conservative region of the chemokine domain (arginine 62, arginine 78, histidine 80, arginine 82, histidine 85, lysine 105, lysine 119, histidine 123) with alanine (designated as R62A, R78A, H80A, R82A, H85A, K105A, K119A and H123A, respectively). Functional studies showed that the mutants with H80A, H85A, and K105A significantly increased the activities of oxLDL uptake and bacterial phagocytosis compared with the wild-type SR-PSOX. In addition, we have also found that mutagenesis of either of those amino acids strongly reduced the adhesive activity of SR-PSOX by using a highly non-overlapping set of basic amino acid residues.

Conclusion: Our study demonstrates that basic amino acid residues in the non-conservative region of the chemokine domain of SR-PSOX are critical for its functions. Mutation of H80, H85, and K105 is responsible for increasing SR-PSOX binding with oxLDL and bacteria. All the basic amino acids in this region are important in the cells adhesion via SR-PSOX. These findings suggest that mutagenesis of the basic amino acids in the chemokine domain of SR-PSOX may contribute to atherogenesis.

Show MeSH
Related in: MedlinePlus