Bakillah A, Jamil H, Hussain M M
Department of Pathology, MCP Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
Biochemistry. 1998 Mar 17;37(11):3727-34. doi: 10.1021/bi972629t.
Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly and secretion of triglyceride-rich lipoproteins. We have presented evidence for a high-affinity interaction between these proteins [Hussain, M. M., et al. (1997) Biochemistry 36, 13060-13067]. In this study, we used chemically modified low-density lipoproteins (LDL) and recombinant human apoB18 to identify amino acid residues in apoB that are critical for its interactions with MTP. Acetoacetylation of 74% of lysine residues and cyclohexanedione modification of 54% of arginine residues completely abolished the interactions between LDL and MTP. Regeneration of lysine and arginine residues by hydroxylamine treatment completely restored the binding of modified LDL to MTP. Carboxyethylation of all the histidine residues decreased, but did not abolish, apoB-MTP interactions. In contrast, glycine methyl ester modifications of aspartic and glutamic acid residues, up to 38-44%, had no effect on LDL-MTP interactions. Furthermore, modification of lysine and arginine, but not the aspartic and glutamic acid, residues in apoB18 also completely abolished its interactions with MTP. These studies indicated that lysine and arginine, but not aspartic and glutamic acid, residues are critical for apoB-MTP interactions, whereas histidine residues are not as critical. Since lysine and arginine residues in apoB are known to interact with the LDL receptors and heparin, we studied the effect of different glycosaminoglycans on apoB-MTP interactions. Glycosaminoglycans had no significant inhibitory effect on apoB-MTP interactions, suggesting that the lysine and arginine residues crucial for apoB-MTP interactions are different from those that interact with the LDL receptor and heparin. The lysine and arginine residues in apoB18 may directly interact with negatively charged residues in the MTP molecule, or they may function to maintain the conformation of the recognition site.
载脂蛋白B（apoB）和微粒体甘油三酯转运蛋白（MTP）对于富含甘油三酯脂蛋白的高效组装和分泌至关重要。我们已经提供了这些蛋白之间存在高亲和力相互作用的证据[侯赛因，M.M.等人（1997年）《生物化学》36卷，13060 - 13067页]。在本研究中，我们使用化学修饰的低密度脂蛋白（LDL）和重组人apoB18来鉴定apoB中对其与MTP相互作用至关重要的氨基酸残基。74%的赖氨酸残基乙酰乙酸化和54%的精氨酸残基环己二酮修饰完全消除了LDL与MTP之间的相互作用。通过羟胺处理使赖氨酸和精氨酸残基再生，完全恢复了修饰后的LDL与MTP的结合。所有组氨酸残基的羧乙基化降低了但并未消除apoB - MTP相互作用。相比之下，高达38 - 44%的天冬氨酸和谷氨酸残基的甘氨酸甲酯修饰对LDL - MTP相互作用没有影响。此外，apoB18中赖氨酸和精氨酸残基的修饰，而非天冬氨酸和谷氨酸残基的修饰，也完全消除了其与MTP的相互作用。这些研究表明，赖氨酸和精氨酸残基而非天冬氨酸和谷氨酸残基对apoB - MTP相互作用至关重要，而组氨酸残基并非如此关键。由于已知apoB中的赖氨酸和精氨酸残基与LDL受体和肝素相互作用，我们研究了不同糖胺聚糖对apoB - MTP相互作用的影响。糖胺聚糖对apoB - MTP相互作用没有显著抑制作用，这表明对apoB - MTP相互作用至关重要的赖氨酸和精氨酸残基与那些与LDL受体和肝素相互作用的残基不同。apoB18中的赖氨酸和精氨酸残基可能直接与MTP分子中的带负电荷残基相互作用，或者它们可能起到维持识别位点构象的作用。
