Glycosylation and Sialylation of Macrophage-derived Human Apolipoprotein E Analyzed by SDS-PAGE and Mass Spectrometry EVIDENCE FOR A NOVEL SITE OF GLYCOSYLATION ON SER290

Apolipoprotein E (apoE)1 is a 34-kDa glycosylated apolipoprotein of 299 amino acids. ApoE is synthesized and secreted by most cells including hepatocytes, smooth muscle cells, neuronal cells, and macrophages (1–3) and demonstrates extraordinary functional diversity. It has important roles in remnant lipoprotein clearance, the immune response, Alzheimer disease, cell proliferation, and lymphocyte activation (4, 5). More recent studies suggest that elevated plasma apoE precedes elevation of C-reactive protein and confers increased risk of cardiovascular death in the elderly (6). Proteomics-based approaches have identified elevated high density lipoprotein (HDL)-apoE as being associated with coronary disease (7). In contrast, macrophage-specific expression of apoE protects against atherosclerosis in mice (8, 9). The mechanisms by which macrophage apoE is antiatherogenic may include stimulating the removal of excess cholesterol from macrophage foam cells as well as anti-inflammatory, antiproliferative, and immunomodulatory properties (4, 5, 10–12). An accurate understanding of the structure of apoE secreted from macrophages is important for our understanding of its properties and its role in the atherosclerotic process. Structural studies on apoE have provided important insights into its biological properties (13). Crystallography has demonstrated that the N-terminal domain is structured in a globular four-helix bundle with the helices orientated in an antiparallel alignment (14). The structure of the C terminus has not been resolved by crystallography, but circular dichroism spectroscopy indicates it to be highly α-helical (14). Recently, NMR studies of monomeric, full-length human apoE indicated that the C-terminal domain in the intact protein adopts a more defined structure than it does as an isolated fragment (15). Lipid binding occurs at the C terminus (residues 244–272), resulting in unfolding of the molecule into a helical hairpin with the binding region for the low density lipoprotein (LDL) receptor contained within the N terminus at its apex (16). Mucin-type O-glycosylation is a particularly common, complex, and important post-translational modification of secreted and cell surface glycoproteins (17, 18) that is difficult to accurately characterize; however, several recent reports have facilitated analysis (19, 20). Cellular apoE and plasma apoE exist as multiple glycoforms, which vary in charge because of variable sialylation. The initial analysis of the carbohydrate content of plasma very low density lipoprotein (VLDL)-apoE by colorimetric methods and gas chromatography demonstrated that the major unmodified hexose in apoE was galactose and that N-acetylglucosamine, N-acetylgalactosamine, and sialic acid were present (21, 22). Two-dimensional gel electrophoresis (2-DE) identified up to six sialylated apoE (Es) glycoforms in cells for any given genotype and fewer sialylated glycoforms in plasma (22). ApoE does not contain the consensus sequence (NX(T/S/C)) required for N-linked glycans, and carbohydrate residues are attached to apoE via an O-linkage to residue Thr194 (23–25). More recent studies using 2-DE and MALDI-TOF/TOF (23) confirmed previous results and identified five glycosylated glycoforms of apoE in plasma VLDL with the most complex sugar structures containing two sialic acid residues (HexNAc-Hex-NeuAc-NeuAc). There were more negatively charged glycoforms present on 2-DE than were distinguished by MALDI-TOF/TOF, raising the possibility that complex structures containing more than two sialic acid residues may be inherently unstable during MS analysis. Importantly, this recent study did not analyze apoE glycoforms in, or secreted from, cells. The purpose of this study was to undertake the first detailed characterization of the glycan structures of apoE from primary human macrophages by 1-DE, 2-DE, and mass spectrometry. We found that cellular and secreted apoE in human macrophages has at least eight different glycoforms with (HexNAc)2-Hex2-(NeuAc)2 being the most complex glycan identified. We extend previous studies by the identification of a novel site of glycan attachment on Ser290 near the functionally important apoE C terminus in addition to glycosylation of Thr194 and show that a major glycoform is present in each of the spots separated by 2-DE.