HIV-infected subjects are at increased risk of developing atherosclerosis and cardio-vascular disease. Previous studies conducted by the project team demonstrated that a key mechanism of this co-morbidity is impairment of function of the ATP-binding cassette A1 (ABCA1) cholesterol transporter by the HIV protein Nef. Recently the team demonstrated that the mechanism of ABCA1 inactivation by Nef involves Nef-mediated disruption of ABCA1 interaction with calnexin, a chaperone of endoplasmic reticulum required for maturation of glycosylated proteins. In the absence of calnexin, they observed accumulation at the membrane of ABCA1 species with altered molecular weight, suggesting the expression of ABCA1 protein that is not properly glycosylated.
In this application, Dr. Radoslav's team proposes to study the glycosylation profile of ABCA1 and changes imparted by Nef. The ABCA1 amino acid sequence has been predicted to contain 21 putative N-glycosylation sites according to an in silico analysis of the Uniprot/SwissProt protein database. Two of the 21 sites were so far confirmed experimentally but the role of glycosylation in ABCA1 biology remains uncharacterized. They propose to identify all ABCA1 glycosylation sites, characterize the glycan types present in native ABCA1 and in the context of HIV viral infection, and analyze the functional capacity of ABCA1 constructs carrying mutations in the glycosylation sites. To this end Dr. Radoslav has started a collaboration between Dr. Bukrinsky, George Washington University, and Dr. Goldman, Georgetown University. Dr. Bukrinsky is an expert on the mechanisms of HIV pathophysiology and HIV effects on cholesterol metabolism, while Dr. Goldman provides expertise in glycoproteomics, two complementary aspects of the research needed to address these stimulating questions.
The research team will isolate Flagtagged ABCA1 in the persence and absence of Nef, and will subject it to glycoproteomics using the latest methods of analysis on the QTOF and QTRAP MS analyzers (AB Sciex) at Georgetown University. During their preliminary analysis, they have already identified two new sites which are co-translationally glycosylated. We will also perform functional analysis of ABCA1 carrying mutations in identified glucosylation sites. These results will close a gap in knowledge on the biology of ABCA1 as a key anti-atherogenic factor.