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) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cell-derived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one- and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc)(2)-Hex(2)-(NeuAc)(2) being the most complex glycan detected on Thr(194) in both cellular and secreted apoE. Four additional glycans were identified on apoE(283-299), and using beta-elimination/alkylation by methylamine in vitro, we identified Ser(290) as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry.
Lee
Youra
Y
Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney, Australia.
Kockx
Maaike
M
Raftery
Mark J
MJ
Jessup
Wendy
W
Griffith
Renate
R
Kritharides
Leonard
L
D000595:Amino Acid Sequence; D001057:Apolipoproteins E / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D047468:Immunoprecipitation; D008264:Macrophages / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D001057:Apolipoproteins E; D012694:Serine; D019158:N-Acetylneuraminic Acid