Structure of apolipoprotein B-100 of human low density lipoproteins

We have analyzed low density lipoproteins (LDL) apolipoprotein (apo) B structure by direct sequence analysis of LDL apo B-100 tryptic peptides. Native LDL were digested with trypsin, and the products were fractionated on a Sephadex G-50 column. The partially digested apo B-100 still associated with lipids was recovered in the void volume (designated trypsin-nonreleasable, TN, peptides). The released peptides (designated trypsin-releasable, TR, peptides) in subsequent peaks were repurified on two successive high-performance liquid chromatography (HPLC) columns. Using this appraoch, we sequenced over 88% of LDL apo B-100, extending and refining our previous study (Nature 1986; 323: 738-742) which covered 52% of the protein. TN peptides made up 31%, and the TR peptides, 34% of the apo B-100 sequence; 23.7% were found under both TN and TR categories. Based on its differential trypsin releasability, apo B-100 can be divided into five domains: 1) residues 1 → 1000, largely TR; 2) residues 1001 → 1700, alternating TR and TN; 3) residues 1701 → 3070, largely TN; 4) residues 3071 → 4100, mainly TR and mixed; and 5) residues 4101 → 4536, almost exclusively TN. Domain 1 contained 14 of the 25 Cys residues in apo B. Domain 4 encompassed seven N-glycosylation sites, and contained the putative receptor binding domains. All 19 potential N-glycosylation sites were directly sequenced: 16 were found to be glycosylated and three were not. Three pairs of disulfide bridges were also mapped. Finally, a combination of cDNA sequencing, direct mRNA sequencing, and comparison of published apo B-100 sequences allowed us to identify specific amino acid study provides information on LDL apo B-100 structure that seem to represent bona fide allelic variations. Our study provides information on LDL apo B-100 structure that will be important to our understanding of its conformation and metabolism.