Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0308192 on September 4, 2008

Published online before print September 4, 2008
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(Journal of Leukocyte Biology. 2008;84:1594-1603.)
© 2008 by Society for Leukocyte Biology

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, Jordi B. Torrelles and Larry S. Schlesinger1

The Center for Microbial Interface Biology, Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA

1 Correspondence: Center for Microbial Interface Biology, Department of Medicine, Division of Infectious Diseases, The Ohio State University, 460 West 12th Avenue, Biomedical Research Tower, Room 1004, Columbus, OH, 43210, USA. E-mail: larry.schlesinger{at}osumc.edu

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.

Key Words: C-type lectin • mannose-capped lipoarabinomannan • HIV gp120 • phagosome-lysosome fusion • TGN retention