We have determined the primary structure of human apolipoprotein D (apoD) by aligning peptides derived from digestions by cyanogen bromide, trypsin, and chymotrypsin. Our results confirm the primary structure derived from cDNA [Drayna et al. (1986) J. Biol. Chem. 261, 16535-16539]. ApoD consists of 169 amino acid residues, including 5 cysteines. Tryptic peptide analysis indicated that Cys41 and Cys16 are joined by a disulfide bridge. Using a combination of manual Edman degradations and mass spectrometric analysis on a purified cluster of chymotryptic fragments, we identified an intramolecular disulfide bridge between Cys8 and Cys114 and an intermolecular bridge between Cys116 of apoD and Cys6 of apoA-II. In addition, sites of N-glycosylation were found at Asn45 and Asn78. Because apoD contains two intramolecular disulfide linkages and has a high content of proline to disrupt α-helical structures, formation of the amphipathic helical regions that characterize the other soluble apolipoproteins is unlikely. We conclude that apoD binds to lipoprotein surfaces through structures other than α-helices, such as disulfide links.
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