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We have analyzed low density lipoproteins (LDL) apolipoprotein (apop) 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. The TN peak was delipidated and redigested with trypsin, and the resulting peptides were purified on two successive HPLC columns. Using this approach, 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 residues within apo B-100 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.
Yang et al. (Sun,) studied this question.