Apolipoprotein D in Lipid Metabolism and Its Functional Implication in Atherosclerosis and Aging

Perdomo, German; Dong, H. Henry

among species (Fig. 1A) . This raises the hypothesis that apoD is regulated at the post-translational level. In support of this hypothesis, we incubated aliquots of sera from normal C57BL/6J mice in the absence and presence of peptide:N-glycosidase F (PNGase F), an amidase that catalyzes the removal of carbohydrate moieties from N-linked glycoproteins. As shown in Fig. 1B , pre-incubation of plasma apoD with PNGase F resulted in apoD de-glycosylation, as evidenced by the production of de-glycosylated apoD with reduced molecular masses. Likewise, serum apoD as well as apoD secreted from axillary glands in humans are also glycosylated [32, 33] . While the physiological signifi- aligned using the ClustalW program. Amino acid residues in box denote two highly conserved N-glycosylation sites in apoD. The consensus N-glycosylation site is Asn-X-Ser/Thr. (B) Plasma apoD is N-glycosylated. Aliquots of plasma (20 µg protein) from C57BL/6J mice were incubated without (-) and with (+) 1,000 U of N-glycosidase F (New England Biolabs) in a total volume of 30 µl at 37°C for 1 hour to remove N-glycan chains from glycopeptides. The reaction mixture was resolved on 4-20% SDS-polyacrylamide gels, followed by immunoblot analysis using anti-apoD. Glycosylated and de-glycosylated forms of apoD are indicated.