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, 84 (6), 1594-603

Mutation in the DC-SIGN Cytoplasmic Triacidic Cluster Motif Markedly Attenuates Receptor Activity for Phagocytosis and Endocytosis of Mannose-Containing Ligands by Human Myeloid Cells

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Mutation in the DC-SIGN Cytoplasmic Triacidic Cluster Motif Markedly Attenuates Receptor Activity for Phagocytosis and Endocytosis of Mannose-Containing Ligands by Human Myeloid Cells

Abul K Azad et al. J Leukoc Biol.

Abstract

The transmembrane C-type lectin, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), has three conserved cytoplasmic tail motifs: the tyrosine (Y)-based, dileucine (LL), and triacidic cluster (EEE), which are believed to regulate ligand binding, uptake, and trafficking. We mutated each of these motifs by alanine substitution and tested their roles in phagocytosis and receptor-mediated endocytosis of the highly mannosylated ligands, Mycobacterium tuberculosis mannose-capped lipoarabinomannan (ManLAM) and HIV-1 surface glycoprotein gp120, respectively, in transfected human myeloid K-562 cells. Compared with wild-type and other mutants, the EEE mutant of DC-SIGN showed a reduced cell-surface expression, near abolishment in the phagocytosis of ManLAM-coated beads (90.5+/-0.4%), and a marked reduction in the endocytosis of soluble gp120 (79.3+/-0.7%). Although, the Y mutant of DC-SIGN did not exhibit any effect on phagocytosis and intracellular trafficking to the phagolysosome, the LL mutant caused the majority of the receptor and/or ligands to remain bound to the cell surface, indicating a role for the LL motif as an internalization signal. The majority of the EEE mutant protein was found to be retained by the intracellular trans-Golgi network and not by the late endosomal/lysosomal compartment of transfected K-562 cells. Collectively, our data indicate a dual role for the EEE motif as a sorting signal in the secretory pathway and a lysosomal targeting signal in the endocytic pathway.

Figures

Fig. 1.
Fig. 1.
(A) Structures of the transmembrane C-type lectins: the MR, DEC-205, and DC-SIGN with their conserved cytoplasmic tail motifs. The oval-shaped, extracellular domains represent the CRDs for each lectin. The conserved motifs are shown in the cytoplasmic tail. (B) Amino acid sequence of the cytoplamic tail of each of the lectins showing their conserved motifs in boxes. The sequences shown take into account that the MR and DEC-205 are type 1 transmembrane proteins, and DC-SIGN is a type 2 transmembrane protein.
Fig. 2.
Fig. 2.
Surface expression of DC-SIGN and its cytoplasmic tail mutant proteins in transfected K-562 cells. (A) K-562 cells were transfected with empty vector, DC-SIGN WT, or mutant construct DNA and after 18–20 h, stained with PE-conjugated DC-SIGN Ab. The surface expression was assessed by flow cytometry and expressed as MFI. The light gray line in the histogram represents empty vector control and the dark gray line, the expression of WT or mutant forms of DC-SIGN. Shown is a representative experiment (n=6). (B) A bar graph plotted from the MFI values derived from A, showing the levels of surface expression of WT and mutant DC-SIGN proteins relative to the vector control in K-562 cells. FL2-H, Fluorescence 2-height; a.u., arbitrary units.
Fig. 3.
Fig. 3.
Western blot analysis of K-562 WT and cytoplasmic tail mutants for total DC-SIGN protein. K-562 cells transfected with DC-SIGN empty vector (expression-negative control), WT, or mutant construct DNA were lysed, and total soluble proteins from lysates were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with Abs against DC-SIGN and GAPDH (housekeeping control protein) and then with HRP-conjugated secondary Ab. Western blot shows the presence of equivalent amounts of DC-SIGN protein in WT and all mutant cell lines. Shown is a representative experiment (n=2).
Fig. 4.
Fig. 4.
Cell association and P-L fusion of ManLAM-coated beads in K-562 transfectants expressing DC-SIGN WT and mutant proteins. K-562 cells were transfected, adhered to polylysine-coated coverslips, and then incubated with ManLAM-coated fluorescent beads (MOI 1:100) for 6 h. Cell monolayers were fixed, permeabilized, stained with primary (DC-SIGN and CD63) Abs followed by secondary (fluorophore-conjugated IgG) Abs, and analyzed by confocal microscopy. (A) Representative confocal images of DC-SIGN WT and mutant receptor (blue)-expressing K-562 cells showing cell-associated, ManLAM-coated beads (green and yellow in the case of P-L fusion) and the intracellular lysosomal marker CD63 (red). Cell-associated beads were counted from an average of 25–30 cells/coverslip for each sample in duplicate in each experiment. The arrow (in the vicinity of the Y mutant) indicates the lysosomal staining of a neighboring, untransfected cell. Bar graphs represent the mean data ± sem of three independent experiments and display the levels of total cell association of ManLAM-coated beads (B) and P-L fusion (C) by the K-562 transfectants. Statistical analysis for significance was done by one-way ANOVA followed by Post-Tukey test (***, P<0.0001).
Fig. 5.
Fig. 5.
Mannan-inhibitable binding of ManLAM-coated beads to K-562 surface-expressed DC-SIGN WT and mutant receptors. K-562 cells were transfected, adhered to polylysine-coated coverslips, and then preincubated with or without mannan for 30 min before incubation with ManLAM-coated beads (MOI 1:50) for 6 h. Cell monolayers were fixed, permeabilized, stained with primary DC-SIGN Abs followed by secondary (fluorophore-conjugated IgG) Abs, and analyzed by confocal microscopy. Cell-associated beads were counted from an average of 25–30 cells/coverslip for each sample in duplicate in each experiment. Bar graphs represent the mean data ± sem of two independent experiments and show the extent of mannan inhibition of association of beads with K-562 transfectants, which are presented in bead association per cell (A) and percentage of mannan inhibition of bead association (B).
Fig. 6.
Fig. 6.
Receptor-mediated endocytosis of the soluble ligand gp120 by K-562 transfectants expressing DC-SIGN WT and LL and EEE mutant proteins. K-562 cells were transfected, adhered to polylysine-coated coverslips, and then preincubated with or without mannan for 30 min before incubation with HIV-1 gp120 for 15 min. Cell monolayers were fixed, permeabilized, stained with primary (DC-SIGN and gp120) Abs and then with secondary (fluorophore-conjugated IgG) Abs, and examined by confocal microscopy. (A, – Mannan) Representative confocal images of the endocytosed gp120 (green spots on left and yellow spots on right panels) by K-562 transfectants expressing the receptor (red on right panels) in the absence of mannan. Right panels represent the phase-contrast images. Arrows indicate representative untransfected cells without DC-SIGN expression. (A, + Mannan) Representative confocal images of the endocytosed gp120 by K-562 transfectants expressing the receptor (red on right panels) in the presence of mannan. Right panels represent the phase-contrast images. (B) Bar graph represents the mean data ± sem of two independent endocytosis experiments without mannan, where total associated gp120 was measured in MFI units from 20 to 25 cells/coverslip for each sample in duplicate in each experiment. Statistical analysis was by one-way ANOVA followed by Post-Tukey test (*, P<0.05).
Fig. 7.
Fig. 7.
Cell association of phagocytic (ManLAM beads) and endocytic (gp120) ligands by K-562 WT and EEE mutant transfectants having equivalent levels of DC-SIGN expression. K-562 cells were transfected, adhered to polylysine-coated coverslips, and then incubated with green fluorescent ManLAM beads for 5 h to allow for phagocytosis (A) or with gp120 for 15 min to allow for receptor-mediated endocytosis (B). Cell monolayers were fixed (without permeabilization), then stained with DC-SIGN (A) or with DC-SIGN and gp120 (B) Abs, followed by staining with fluorophore-conjugated secondary Abs, and examined by confocal microscopy. Expressed DC-SIGN protein and cell-associated gp120 were measured in MFI units, and cell-associated fluorescent beads were enumerated as mean (±sd) beads/cell. (A, left) Bar graphs show the mean data (MFI units or beads) ± sd of 13 selected K-562 cells from each group of WT and EEE mutant transfectants having equivalent surface expression of DC-SIGN but differential cell association of ManLAM beads. (A, right) Representative confocal images of DC-SIGN WT and EEE mutant transfectants from the phagocytosis assay. (B, left) Bar graphs show the mean data (MFI units) ± sd of 10 selected K-562 cells from each group of WT and EEE mutant transfectants having equivalent surface expression of DC-SIGN but differential cell association of gp120. (B, right) Representative confocal images of DC-SIGN WT and EEE mutant transfectants from the receptor-mediated endocytosis assay.
Fig. 8.
Fig. 8.
Comparative intracellular retention of DC-SIGN WT and mutant proteins in the TGN of the K-562-transfected cells. K-562 cells were transfected, adhered to polylysine-coated coverslips, and incubated for 18–20 h to allow for protein expression. Cell monolayers were fixed, permeabilized, stained with DC-SIGN and γ-adaptin Abs and counterstained with fluorophore-conjugated secondary Abs, and examined by confocal microscopy. (A) Representative confocal images of DC-SIGN WT and mutant receptor (green)-expressing K-562 cells showing the colocalization (yellow) between the TGN marker γ-adaptin (red) and the receptor protein. An average of 25–30 cells/coverslip in duplicate from each sample was examined for colocalization in each experiment. The arrow (in the vicinity of the YEEE mutant) indicates the TGN staining of a neighboring, untransfected cell. (B) The bar graph represents the mean data ± sem of two independent experiments and shows the percentage of each category of transfectants positive for colocalization. Statistical analysis for significance was performed by one-way ANOVA followed by Post-Tukey test (**, P<0.01).
Fig. 9.
Fig. 9.
Comparative intracellular trafficking of DC-SIGN WT and mutant proteins to the late endosomal/lysosomal compartment in the K-562-transfected cells, which were transfected, adhered to polylysine-coated coverslips, and incubated for 18 h, followed by a 30-min incubation with the ligand mannan to allow for endocytic trafficking of the DC-SIGN protein. Cell monolayers were fixed, permeabilized, stained with DC-SIGN and CD63 Abs and counterstained with fluorophore-conjugated secondary Abs, and examined by confocal microscopy. (A) Representative confocal images of DC-SIGN WT and mutant receptor (green)-expressing K-562 cells showing the colocalization (yellow) of the late endosomal/lysosomal marker CD63 (red) with DC-SIGN. An average of 20–25 cells/coverslip in duplicate from each sample was examined for colocalization in each experiment. The arrow (in the vicinity of the Y mutant) indicates the lysosomal staining of a neighboring, untransfected cell. (B) The bar graph represents the mean ± sem of two independent experiments and shows the percentage of transfectants positive for colocalization. Statistical analysis for significance was performed by one-way ANOVA followed by Post-Tukey test (**, P<0.01).

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