PROJECT SUMMARY

UPDATE: Patrick graduated in Spring 2008, but he'll be sticking around until mid 2009 to finish up some SWEET HIV-SCIENCE-RELATED STUFF!! UPDATE COMING SOON!!

DC-SIGN, a C-type lectin found on dendritic cells, binds to the envelope glycoprotein of HIV(gp120) with high affinity. It has been hypothesized that DC-SIGN-expressing dendritic cells in the submucosa may facilitate the establishment of a primary HIV infection by binding to HIV and subsequently transferring it to permissive target cells in secondary lymphoid organs. In addition, DC-SIGN can also facilitate infection when present on CD4/coreceptor-expressing cells.

Since DC-SIGN preferentially binds high mannose residues, alanine scanning mutagenesis was performed on the 12 N-linked glycosylation sites in gp120 previously determined to result in high-mannose residues. These mutations were made singly or in combination. In order to facilitate our DC-SIGN/Env binding assay, gp120 from the JRCSF strain was fused to the Fc region of human IgG1. A secondary reagent directed against the human Fc region detected the gp120-Fc binding to DC-SGN expressing cells. Recombinant gp120-Fc fusion proteins were produced by the vaccinia-driven T7 promoter system. Our mutational and biochemical data initially suggested that no single glycosylation mutant resulted in decreased DC-SIGN binding although a subset affected CD4 binding.

We continue to identify the N-glycans that are involved in the optimal gp120/DC-SIGN interaction. We have developed two complementary but fundamentally different assays to discern if DC-SIGN binding to gp120 is flexible and to identify the N-glycans that may give rise to high mannose N-glycans that are more optimally spaced to interact efficiently with DC-SIGN.

Given the putative role of DC-SIGN in HIV transmission and replication, elucidating the exact nature of the interaction between the DC-SIGN lectin and the sugar moieties on gp120 is of great interest for both therapeutic and vaccine-related purposes. Specifically, selectively deglycosylated gp120s deficient in DC-SIGN binding will be tested for their ability to elicit antibodies that will block DC-SIGN/Env interactions.

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