Fig. 1

Recombinant human DC-SIGN isoforms were expressed, purified, and used to target drug loaded liposomes to fungal glycans. a Full length native human DC-SIGN is composed of a SP (Signal Peptide domain), SD (Signaling Domain), TM (Transmembrane domain), eight neck repeats (NRs), and a carbohydrate recognition domain (CRD). We expressed and purified two truncated recombinant isoforms of human DC-SIGN each with two NRs fused to the CRD (b) DCS78 contains NRs 7 and 8. c DCS12 contains NRs 1 and 2 and each contained a KGK sequence for coupling to a free K residue. Both DC-SIGN isoforms were used to target drug loaded liposomes. d Model of a targeted liposome. DCS12 and DCS78 monomers and predicted homodimers, and/or homotetramers (green irregular structures) have their shapes modeled from crystallographic structures of DC-SIGN monomers and multimers linked to two NRs [23]. They are shown binding to cognate glycans (e.g., red sugar moieties) in the cell walls and exopolysaccharide matrices of fungal cells. DCS12 and DCS78 were coupled to the lipid carrier DSPE-PEG via a lysine amino group (N) in each polypeptide. DHPE-Rhodamine (red star) along with DCS12-PEG-DSPE or DCS78-PEG-DSPE were inserted via their respective lipid moieties, DHPE and DSPE, into the liposomal membrane of amphotericin B loaded pegylated liposomes, AmB-LLs, to make red fluorescent DCS12-AmB-LLs and DCS78-AmB-LL. Each liposome contains approximately 3000 rhodamine molecules, 1500 monomers of a DC-SIGN isoform, and 16,500 AmB molecules [4]. DC-SIGN derived monomers are free to float in the membrane and are predicted to form functional multimers. The various objects in this diagram could not be drawn to the same scale due their extreme size differences. Single letter amino acid coding of particular domains are indicated: MH₆V (hexa-histidine (H) tag for affinity purification), (GS)₂G (flexible linkers to improve accessibility to flanking domains), DDDDK (protease processing site for potential removal of multimeric histidine tag), KGK (contains two lysine (K) residues for potential coupling to lipid carrier). Definitions: DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DHPE (1,2-Dihexadecyl-sn-glycero-3-phosphoethanolamine)