Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100

Abstract

Apolipoprotein (apo) B-100, the major protein component in low density lipoprotein (LDL), is the ligand that binds to the LDL receptor. It is important in the metabolism of LDL and elevated plasma levels of LDL-apo B are strongly associated with increased risk of coronary artery disease. Although apo B-100 is of great clinical and biological importance its primary structure has defied chemical elucidation, mainly because of its enormous size, insolubility, and tendency to aggregate. Less than 5% of the apo B-100 sequence has been reported, despite the efforts of many laboratories over the past twenty years. Here we report the complete amino acid sequence of human apo B-100 as deducted by sequence analysis of complementary DNA clones; 2,366 of the 4,536 residues were also confirmed by direct sequencing of apo B-100 tryptic peptides. The distribution of trypsin-accessible and -inaccessible peptides of the protein on LDL is non-random and they can be grouped into 5 hypothetical domains. Of 20 potential N-glycosylation sites identified in the sequence, 13 were found by direct peptide sequencing to be glycosylated, and 4 unglycosylated. Examination of the primary structure of apo B-100 reveals that it contains a large number of long (greater than 70 residues) internal repeats and an even larger number of shorter ones, suggesting that the apo B-100 sequence was derived largely from internal duplications. Finally, using synthetic peptides of a specific region of apo B-100, we have identified a potential LDL receptor-binding domain (residues 3,345-3,381) which can bind to the LDL receptor and suppress 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase activities in cultured human fibroblasts.

Very low density lipoprotein receptor binds apolipoprotein E2/2 as well as apolipoprotein E3/3

“…All of these residues lie on helix 4, one of five helices located in the N-terminal domain of apoE [21]. The regions of human apoB-100 that contain such clusters have been implicated in binding to the LDL receptor (residues 3359-3368, Arg-LeuThr-Arg-Lys-Arg-Gly-Leu-Lys-Leu) [22,23]. Furthermore it has been shown that two such clusters of VTG, residues 493498 (Leu-Lys-Arg-Ile-Leu-Lys) and residues 1079-1084 (Lys-Leu-Lys-Arg-Ile-Leu), mediate binding to the VLDL/ VTG receptor.…”

Primary sequence mapping of human apolipoprotein B‐100 epitopes

“…As defined in the previous study, peptides that eluted at the void volume of the Sephadex G-50 column (peak 1 of Yang et al [5]) were the trypsin inaccessible peptides; those that came off later (peaks T3 -T7) were labeled trypsin-accessible. Sequencing was performed by the manual technique [13], or by a gas-phase sequencer (Applied Biosystems).…”

“…LDL-apoB was digested with trypsin and the tryptic peptides were isolated as described in detail previously [5]. As defined in the previous study, peptides that eluted at the void volume of the Sephadex G-50 column (peak 1 of Yang et al [5]) were the trypsin inaccessible peptides; those that came off later (peaks T3 -T7) were labeled trypsin-accessible.…”

“…The peptides were precipitated with ether and extracted from the resin with trifluoroacetic acid. They were then desalted and purified on reverse-phase high-performance liquid chromatography on a Vydac C4 column (1 x 25 cm) as described previously [5]. The peptides were at least 99% pure by analytical reverse-phase HPLC, had the expected amino acid composition, and their sequences were confirmed by direct manual sequencing [I 31.…”

“…One method is the trypsin digestion of apoB-I 00 on the lipoprotein particles and the sequence analysis and identification of the trypsin-accessible and inaccessible peptides. Based on such an approach, a general model of LDL-apoB conformation has been proposed [5]. Another approach would be the used of monoclonal antibodies (Mabs) that recognize 'exposed' epitopes of LDL apoB and antibodies that do not react well with LDL apoB-100 because their specific apoB-100 epitopes are 'buried'.…”