AVS 58th Annual International Symposium and Exhibition
    Biomaterial Interfaces Division Tuesday Sessions
       Session BI-TuA

Paper BI-TuA11
Binding of C-reactive Protein to Lipoprotein Nanoparticle Mimics: A Gel Electrophoresis Study

Tuesday, November 1, 2011, 5:20 pm, Room 105

Session: Protein-Membrane Interactions
Presenter: Min Wang, University of Colorado Denver
Authors: M.S. Wang, University of Colorado Denver
S.M. Reed, University of Colorado Denver
Correspondent: Click to Email
C-reactive protein (CRP) is an acute phase serum protein involved in inflammation that recognizes pathogenic agents, and activates complement. Because CRP levels can increase by over 1000-fold from basal levels within 72 hr in response to inflammation, it has been used as a biomarker to predict the risk for cardiovascular disease (CVD). In addition, CRP has been shown to bind oxidized low density lipoprotein (oxLDL) through the phosphatidylcholine (PC) headgroup that is exposed on the surface of oxLDL and has been found to co-localize with atherosclerotic lesions. It is suggested that CRP was deposited in these lesions when engulfed by macrophage while it is still bound to oxLDL. Because LDL and the other lipoprotein particles (LPPs) have diameters (d) in the nanometer range, they can be considered as biological nanoparticles. In fact, LPPs are classified into four major categories according to their size and density: high density lipoprotein (d=8-13nm), LDL (d=20-30nm), intermediate density lipoprotein (d=30-40nm), and very low density lipoprotein (d>40nm). Moreover, the physiological functions of LPPs are greatly influenced by the size. For example, the presence of sdLDLs (d<25.5nm) are associated with increased risk of coronary artery disease (CAD) and diabetes; while increased levels of HDL are considered to be atheroprotective. Therefore, LPP profiling has emerged as a tool to more accurately assess the development of metabolic risk factors such as CAD. While the PC moieties on oxLDL are likely ligands for CRP recognition, it is still unclear why CRP binds only oxLDL although PC is expressed on native LDL and on all cell membrane. In this work, we correlated the effects of particle size (i.e. membrane curvature) to CRP binding using LPP mimics. To this end, we engineered LPP mimics using lipid-coated gold nanoparticles (PC-AuNPs) to explore the influence of LPP size on CRP binding. Binding analysis of CRP to the mimics was performed using gel electrophoresis (GE). The migration of CRP and PC-AuNP was directly visualized after electrophoresis, and the presence of CRP was confirmed using Western blots. The overlapping bands from the gel and Western blot confirmed that CRP bound to the PC-AuNPs and co-migrated during GE. Together, we demonstrated that 1) PC-AuNPs are size-separable, 2) the lipid layer around the PC-AuNP remained intact during GE, and 3) CRP binds to a relevant LDL-sized PC-AuNP mimic. This is, to our knowledge the first use of GE to separate lipid-coated nanoparticle and to evaluate non-covalent binding of protein-nanoparticle interactions.