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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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The transfected 293T cells were expressing pEGFP and SR-PSOX proteins detected by confocal microscopy. 293T cells were transiently transfected with pEGFP-N3 empty vector or pEGFP-N3-SR-PSOX construct (wild-type SR-PSOX cloned into pEGFP-N3 vector) for 24 hours respectively. The fluorescence images of the cells were observed under confocal microscope after the cells were immunostained with APC-labeled rat anti-human SR-PSOX antibody. Top panel (vector), the cells were transfected with pEGFP-N3 empty vector alone. Bottom panel (wild type), the cells were transfected with pEGFP-N3-SR-PSOX construct.
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Figure 2: The transfected 293T cells were expressing pEGFP and SR-PSOX proteins detected by confocal microscopy. 293T cells were transiently transfected with pEGFP-N3 empty vector or pEGFP-N3-SR-PSOX construct (wild-type SR-PSOX cloned into pEGFP-N3 vector) for 24 hours respectively. The fluorescence images of the cells were observed under confocal microscope after the cells were immunostained with APC-labeled rat anti-human SR-PSOX antibody. Top panel (vector), the cells were transfected with pEGFP-N3 empty vector alone. Bottom panel (wild type), the cells were transfected with pEGFP-N3-SR-PSOX construct.

Mentions: The scavenger receptor SR-PSOX is a 30 kDa type I transmembrane glycoprotein. The coding sequence of SR-PSOX (837 bp) was subcloned into pEGFP-N3 vector (Figure 1). All the SR-PSOX mutants were constructed as described in materials and methods, and confirmed by sequencing. To verify if recombinant plasmid pEGFP-N3-SR-PSOX could be normally expressed on the surface of cells, a pEGFP-N3 empty vector and pEGFP-N3-SR-PSOX were transfected into 293T cells respectively. After 24 hours transfection, the cells were stained with anti-SR-PSOX antibody, counter stained with a Cy3-conjugated secondary antibody, and the images were then directly viewed under a fluorescent microscope. As shown in Figure 2, EGFP was well expressed in the cells transfected with either of the recombinant plasmids. As expected, SR-PSOX was expressed on the plasma membranes.


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

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

The transfected 293T cells were expressing pEGFP and SR-PSOX proteins detected by confocal microscopy. 293T cells were transiently transfected with pEGFP-N3 empty vector or pEGFP-N3-SR-PSOX construct (wild-type SR-PSOX cloned into pEGFP-N3 vector) for 24 hours respectively. The fluorescence images of the cells were observed under confocal microscope after the cells were immunostained with APC-labeled rat anti-human SR-PSOX antibody. Top panel (vector), the cells were transfected with pEGFP-N3 empty vector alone. Bottom panel (wild type), the cells were transfected with pEGFP-N3-SR-PSOX construct.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The transfected 293T cells were expressing pEGFP and SR-PSOX proteins detected by confocal microscopy. 293T cells were transiently transfected with pEGFP-N3 empty vector or pEGFP-N3-SR-PSOX construct (wild-type SR-PSOX cloned into pEGFP-N3 vector) for 24 hours respectively. The fluorescence images of the cells were observed under confocal microscope after the cells were immunostained with APC-labeled rat anti-human SR-PSOX antibody. Top panel (vector), the cells were transfected with pEGFP-N3 empty vector alone. Bottom panel (wild type), the cells were transfected with pEGFP-N3-SR-PSOX construct.
Mentions: The scavenger receptor SR-PSOX is a 30 kDa type I transmembrane glycoprotein. The coding sequence of SR-PSOX (837 bp) was subcloned into pEGFP-N3 vector (Figure 1). All the SR-PSOX mutants were constructed as described in materials and methods, and confirmed by sequencing. To verify if recombinant plasmid pEGFP-N3-SR-PSOX could be normally expressed on the surface of cells, a pEGFP-N3 empty vector and pEGFP-N3-SR-PSOX were transfected into 293T cells respectively. After 24 hours transfection, the cells were stained with anti-SR-PSOX antibody, counter stained with a Cy3-conjugated secondary antibody, and the images were then directly viewed under a fluorescent microscope. As shown in Figure 2, EGFP was well expressed in the cells transfected with either of the recombinant plasmids. As expected, SR-PSOX was expressed on the plasma membranes.

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