Proteomic analysis of DC-SIGN on dendritic cells detects tetramers required for ligand binding but no association with CD4.
DC-SIGN (dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin) or CD209 is a type II transmembrane protein and one of several C-type lectin receptors expressed by dendritic cell subsets, which bind to high mannose glycoproteins promoting their endocytosis and potential degradation. DC-SIGN also mediates attachment of HIV to dendritic cells and binding to this receptor can subsequently lead to endocytosis or enhancement of CD4/CCR5-dependent infection. The latter was proposed to be facilitated by an interaction between DC-SIGN and CD4. Endocytosis of HIV virions does not necessarily lead to their complete degradation. A proportion of the virions remain infective and can be later presented to T cells mediating their infection in trans. Previously, the extracellular domain of recombinant DC-SIGN has been shown to assemble as tetramers and in the current study we use a short range covalent cross-linker and show that DC-SIGN exists as tetramers on the surface of immature monocyte-derived dendritic cells. There was no evidence of direct binding between DC-SIGN and CD4 either by cross-linking or by fluorescence resonance energy transfer measurements suggesting that there is no constitutive association of the majority of these proteins in the membrane. Importantly we also show that the tetrameric complexes, in contrast to DC-SIGN monomers, bind with high affinity to high mannose glycoproteins such as mannan or HIV gp120 suggesting that such an assembly is required for high affinity binding of glycoproteins to DC-SIGN, providing the first direct evidence that DC-SIGN tetramers are essential for high affinity interactions with pathogens like HIV.
Bernhard
Oliver K
OK
Centre for Virus Research, Westmead Millennium Institute, Westmead Hospital, Westmead NSW 2145, Sydney, Australia and The University of Sydney, NSW 2006, Australia.
Lai
Joey
J
Wilkinson
John
J
Sheil
Margaret M
MM
Cunningham
Anthony L
AL
D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents; D003713:Dendritic Cells / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D037181:Lectins, C-Type / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008351:Mannans / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D040901:Proteomics; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D015704:CD4 Antigens; D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D015699:HIV Envelope Protein gp120; D037181:Lectins, C-Type; D008024:Ligands; D008351:Mannans; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin