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Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.

Cambi A, de Lange F, van Maarseveen NM, Nijhuis M, Joosten B, van Dijk EM, de Bakker BI, Fransen JA, Bovee-Geurts PH, van Leeuwen FN, Van Hulst NF, Figdor CG - J. Cell Biol. (2004)

Bottom Line: The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood.During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm.Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, Netherlands.

ABSTRACT
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

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DC-SIGN is organized in microdomains on the cell surface of K-DC-SIGN. (A) The expression levels of DC-SIGN on untransfected K562 and on K-DC-SIGN were assessed by FACS® analysis. The open histogram represents the isotype control, and shaded histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). (B) DC-SIGN expressed by K562 transfectants strongly binds to ICAM-3 and gp120. The adhesion was determined using 1-μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). Blocking exerted by EGTA indicates that DC-SIGN binds to the ligands in a Ca2+-dependent manner. The average of five independent experiments is shown (P < 0.001). (C) K-DC-SIGN cells were specifically labeled with 10-nm gold particles, as described in Materials and methods. Cells were allowed to adhere onto poly-l-lysine–coated formvar film and photographed in an electron microscope. Gold particles were detected on the periphery on the thinner less electron dense parts of cells, where good contrast could be achieved. One representative picture is shown. Bar, 200 nm.
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fig1: DC-SIGN is organized in microdomains on the cell surface of K-DC-SIGN. (A) The expression levels of DC-SIGN on untransfected K562 and on K-DC-SIGN were assessed by FACS® analysis. The open histogram represents the isotype control, and shaded histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). (B) DC-SIGN expressed by K562 transfectants strongly binds to ICAM-3 and gp120. The adhesion was determined using 1-μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). Blocking exerted by EGTA indicates that DC-SIGN binds to the ligands in a Ca2+-dependent manner. The average of five independent experiments is shown (P < 0.001). (C) K-DC-SIGN cells were specifically labeled with 10-nm gold particles, as described in Materials and methods. Cells were allowed to adhere onto poly-l-lysine–coated formvar film and photographed in an electron microscope. Gold particles were detected on the periphery on the thinner less electron dense parts of cells, where good contrast could be achieved. One representative picture is shown. Bar, 200 nm.

Mentions: To study the correlation between functional state and cell surface organization of DC-SIGN, we used K562 transfectants stably expressing DC-SIGN. K-DC-SIGN expressed high levels of DC-SIGN (Fig. 1 A) and bound strongly to 1-μm fluorescent beads coated with ICAM-3 or GP120 (Fig. 1 B). The binding of K-DC-SIGN to these ligands was specific, as shown by the inhibition exerted by anti–DC-SIGN mAbs. Moreover, the lack of adhesion in presence of EGTA confirmed that DC-SIGN bound to these ligands in a Ca2+-dependent manner, typical of C-type lectin-like receptors.


Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.

Cambi A, de Lange F, van Maarseveen NM, Nijhuis M, Joosten B, van Dijk EM, de Bakker BI, Fransen JA, Bovee-Geurts PH, van Leeuwen FN, Van Hulst NF, Figdor CG - J. Cell Biol. (2004)

DC-SIGN is organized in microdomains on the cell surface of K-DC-SIGN. (A) The expression levels of DC-SIGN on untransfected K562 and on K-DC-SIGN were assessed by FACS® analysis. The open histogram represents the isotype control, and shaded histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). (B) DC-SIGN expressed by K562 transfectants strongly binds to ICAM-3 and gp120. The adhesion was determined using 1-μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). Blocking exerted by EGTA indicates that DC-SIGN binds to the ligands in a Ca2+-dependent manner. The average of five independent experiments is shown (P < 0.001). (C) K-DC-SIGN cells were specifically labeled with 10-nm gold particles, as described in Materials and methods. Cells were allowed to adhere onto poly-l-lysine–coated formvar film and photographed in an electron microscope. Gold particles were detected on the periphery on the thinner less electron dense parts of cells, where good contrast could be achieved. One representative picture is shown. Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171967&req=5

fig1: DC-SIGN is organized in microdomains on the cell surface of K-DC-SIGN. (A) The expression levels of DC-SIGN on untransfected K562 and on K-DC-SIGN were assessed by FACS® analysis. The open histogram represents the isotype control, and shaded histogram indicates the specific staining with anti–DC-SIGN (AZN-D1). (B) DC-SIGN expressed by K562 transfectants strongly binds to ICAM-3 and gp120. The adhesion was determined using 1-μm ligand-coated fluorescent beads. Specificity was determined by measuring binding in presence of AZN-D1. No blocking was observed in presence of isotype control (not depicted). Blocking exerted by EGTA indicates that DC-SIGN binds to the ligands in a Ca2+-dependent manner. The average of five independent experiments is shown (P < 0.001). (C) K-DC-SIGN cells were specifically labeled with 10-nm gold particles, as described in Materials and methods. Cells were allowed to adhere onto poly-l-lysine–coated formvar film and photographed in an electron microscope. Gold particles were detected on the periphery on the thinner less electron dense parts of cells, where good contrast could be achieved. One representative picture is shown. Bar, 200 nm.
Mentions: To study the correlation between functional state and cell surface organization of DC-SIGN, we used K562 transfectants stably expressing DC-SIGN. K-DC-SIGN expressed high levels of DC-SIGN (Fig. 1 A) and bound strongly to 1-μm fluorescent beads coated with ICAM-3 or GP120 (Fig. 1 B). The binding of K-DC-SIGN to these ligands was specific, as shown by the inhibition exerted by anti–DC-SIGN mAbs. Moreover, the lack of adhesion in presence of EGTA confirmed that DC-SIGN bound to these ligands in a Ca2+-dependent manner, typical of C-type lectin-like receptors.

Bottom Line: The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood.During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm.Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, Netherlands.

ABSTRACT
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

Show MeSH
Related in: MedlinePlus