Session 46Poster Presentations DC-SIGN and Related Molecules Session Day and Time: Thursday 1:30 - 3:30 pm Room: Hall D
370 Attempt to Localize the Binding of DC-SIGN on gp120 J. Ford*, D. Atibalentja, J. Arthos, P. Sun Natl Inst of Allergy and Infectious Diseases, NIH, Rockville, MD
Background: The binding of DC-SIGN to HIV envelope protein gp120 facilitates and enhances HIV infection to T-cells. It is known that DC-SIGN binds to some of the 12 high mannose moieties on gp120 through its carbohydrate recognition domain. Carbohydrates on gp120 can be visualized in 2 sections, one on the silent face with carbohydrates oriented upward with respect to the CD4 binding epitope, and one surrounding the CD4 epitope, pointing downward. In order to localize DC-SIGN binding, we tested the blocking ability of reagents known to bind gp120 in a carbohydrate-dependant manner. 2G12 is a broadly neutralizing antibody that is known to recognize a specific set of high mannose residues on gp120. Cyanovirin-N (CV-N), a small bacterial protein, also binds gp120 via high-mannose glycans. D1D2IgAtp is a dodecameric CD4 construct that exhibits broad neutralization via the CD4 binding epitope. Use of these binding reagents allowed us to exclude some carbohydrate sets as potential binding regions.
Methods: Surface plasmon resonance experiments were performed with a Biacore 3000. DC-SIGNR was immobilized on the surface of amine-coupled sensor chips. The analytes, which consisted of gp120 alone and in combination with inhibitor reagents, were flown in solution over these chips. Inhibition by 2G12, CV-N, and D1D2IgAtp was quantified as the percentage reduction in binding observed when these reagents were incubated with gp120 prior to DC-SIGNR binding. As a control, we used an anti-DC-SIGNR monoclonal antibody that is able to completely block binding of gp120 to DC-SIGNR.
Results: 2G12 bound to gp120 with a KD of about 50nM, and CV-N about 80nM. Inhibition experiments showed that CV-N exhibited a dose-dependent increase in blocking. When incubated together with gp120, CV-N and 2g12 inhibited binding more effectively than CV-N alone. D1D2IgAtp did not inhibit binding of gp120 to DC-SIGNR, even at saturating concentrations.
Conclusions: Lack of blocking by D1D2IgAtp suggests a DC-SIGNR binding site at the silent face, away from the CD4 epitope. Dose-dependent blocking by CV-N suggests DC-SIGN has broad specificity with respect to carbohydrate binding, which decreases as successive carbohydrate sites are occupied by CV-N.
