Characterization of Human Apolipoprotein A-I Self-Association at Physiological Concentrations

Human apolipoprotein A-I (apoA-I) is the main protein in high-density lipoprotein (HDL), mediating the transport of cholesterol from peripheral tissues to the liver. The protein is composed of two domains, an N-terminal helix bundle and an unstructured C-terminal domain. The C-terminal domain is responsible for lipid binding and promotes self-association in the lipid-free state. It has been assumed that the protein is monomeric at a protein concentration of 0.1 mg/mL, while at higher concentrations the protein forms oligomers. However, the molecular details of self-association are poorly understood. Therefore, the self-association properties of apoA-I were studied over a wide concentration range using chemical crosslinking, pyrene fluorescence, and size-exclusion chromatography. To determine the extent of apoA-I self-association, wild-type apoA-I was crosslinked with dimethyl suberimidate (DMS) and analyzed by SDS-PAGE and western blots. Crosslinked apoA-I bands were observed between 58-100 kDa in a concentration range between 0.02 and 1.0 mg/mL, indicating the presence of monomers, dimers, trimers, tetramers, and pentamers. Self-association of apoA-I was verified through pyrene fluorescence. A single-cysteine mutation was introduced in the C-terminal domain creating, S201C-apoA-I. The emission fluorescence spectra in a protein concentration range between 0.02 and 0.2 mg/mL displayed strong pyrene excimer peaks, indicating intermolecular interactions in the C-terminal domain at all protein concentrations. Size-exclusion fast protein liquid chromatography (FPLC) was employed to confirm these observations with wild-type apoA-I. Elution profiles of wild-type apoA-I at protein concentrations of > 0.1 mg/mL showed apoA-I oligomers, while at concentrations between 0.02 and 0.05 mg/mL the elution time increased and two protein peaks were observed, likely representing monomers and dimers. The apoA-I dimers observed at physiological concentrations may be an important intermediate when the protein adopts the lipid bound conformation, forming nascent HDL complexes that contain two copies of apoA-I. This research was supported by the CSULB Undergraduate Research Opportunity Program Health Research Peer Group, CSULB Office of Research and Economic Development, and the National Institute of General Medical Sciences of the National Institutes of Health GM089564.