Chen P F, Marcel Y L, Yang C Y, Gotto A M, Milne R W, Sparrow J T, Chan L
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Eur J Biochem. 1988 Jul 15;175(1):111-8. doi: 10.1111/j.1432-1033.1988.tb14172.x.
Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.
差异胰蛋白酶可及性和针对人载脂蛋白（apo）B - 100的单克隆抗体（Mabs）都是探测低密度脂蛋白（LDL）上apoB结构和构象的重要工具。在本研究中，我们通过胰蛋白酶消化绘制了LDL apoB - 100 C末端区域（720个残基）超过80%的图谱。我们的结果扩展了我们之前的数据[Yang等人（1986年），《自然》（伦敦）323卷，738 - 742页]，证实apoB - 100约420个残基的C末端区域在很大程度上对胰蛋白酶不可及，而在此区域之前的部分有穿插的对胰蛋白酶可及和不可及的肽段。我们确定了包含四个不同单克隆抗体识别的表位的特定apoB - 100肽段的氨基酸序列：两个表位已定位在20个残基范围内，一个定位在36个残基，最后一个定位在80个残基。我们使用多克隆抗血清鉴定了从克隆于表达载体λgt11中的apoB - 100的C末端延伸的不同长度的16个重叠的apoB - 100 cDNA克隆。然后针对各个单克隆抗体对这些克隆进行测试。通过对显示对不同单克隆抗体有差异反应性的重叠克隆进行核苷酸序列分析，我们已将每个单克隆抗体的单个表位定位到从DNA序列预测的约50 - 150个氨基酸残基范围内。通过使用部分纯化的融合蛋白和化学合成的寡肽竞争LDL结合来完成确认和进一步的精细定位。两个表位（单克隆抗体7和22）定位到apoB - 100的C末端20个氨基酸，一个（单克隆抗体16）定位到残基4154 - 4189，另一个（单克隆抗体20）定位到残基3926 - 4005。单克隆抗体16沉淀超过80%的LDL颗粒。单克隆抗体20仅沉淀变性的apoB，而不沉淀天然LDL apoB[Milne等人（1987年），《分子免疫学》24卷，435页]。单克隆抗体7和22的独特之处在于它们沉淀储存修饰的LDL apoB比新鲜分离的LDL - apoB要好得多。尽管表位表达和胰蛋白酶可及性是研究蛋白质构象的两个有用探针，但对于我们所检测的抗体，当应用于LDL apoB时，这两个参数之间没有明显的相关性。
