Session 46Poster Presentations DC-SIGN and Related Molecules Session Day and Time: Thursday 1:30 - 3:30 pm Room: Hall D
361 Structure-function Studies of DC-SIGN Interactions with gp120, ICAM-2, and ICAM-3 Stephen V. Su*, Patrick Hong, Karen Flummerfelt, Oscar Negrete, Benhur Lee Geffen Sch of Med at UCLA, Los Angeles, CA
Background: DC-SIGN, a mannose-specific C-type lectin that binds avidly to the gp120 of HIV/SIV, is implicated in the transmission of HIV from dendritic cells (DCs) to permissive T-cells. ICAM-2 and ICAM-3, endogenous ligands for DC-SIGN, mediate transendothelial migration of DCs and T-cell/DC clustering, respectively. To delineate the differential binding determinants of the 3 interactions, we undertook a biochemical, structural, and genetic analysis of DC-SIGN binding to gp120 as compared to ICAM-2 and ICAM-3.
Methods: Recombinant gp120-Fc, ICAM-2-Fc, ICAM-3-Fc, and the soluble extracellular domain of DC-SIGN were used to quantitatively measure their binding affinities in an ELISA based assay. Various sugars were used in a competition assay to map the carbohydrate specificities of these interactions. Circular dichroism was used to investigate conformational changes that might occur during DC-SIGN/ligand interactions. Alanine scanning mutagenesis was performed on all solvent exposed residues in the carbohydrate recognition domain of DC-SIGN. gp120 binding to these mutants was performed using a FACS-based assay.
Results: First, biochemical-binding experiments indicated that DC-SIGN bound to gp120-Fc more than 100- and 40-fold better than ICAM-2-Fc and ICAM-3-Fc, respectively (Kd: gp120 = 2.68 ±0.5 nM; ICAM-2 = 271.6 ±85 nM, ICAM-3 = 129.1 ±35 nM). Also, gp120 binding to DC-SIGN was more sensitive to EndoH treatment than ICAM-2 or ICAM-3. gp120-Fc binding to DC-SIGN showed stereo-specificity as D-mannose competed much more efficiently than L-mannose. Second, circular dichroism experiments showed differences in the percent helicity during soluble DC-SIGN/gp120 interactions, suggesting the occurrence of conformational changes. Third, alanine scanning mutagenesis revealed that amino acids (E354, D366) involved in both calcium chelation and carbohydrate binding had the most dramatic phenotype on gp120-Fc binding. However, mutations of some residues implicated in carbohydrate contacts (D367, S360) had no apparent effect on gp120 binding. Interestingly, DC-SIGN binding to virion associated env was less sensitive to certain mutations.
Conclusions: We have begun to discern the binding characteristics of gp120 to DC-SIGN with respect to ICAM-2 and ICAM-3. These differences and the data herein suggest that DC-SIGN interactions with gp120 can be manipulated to exploit these differences.
