Bakillah A, Nayak N, Saxena U, Medford R M, Hussain M M
Department of Pathology, School of Medicine, MCP Hahnemann University, Philadelphia, Pennsylvania 19129, USA.
Biochemistry. 2000 Apr 25;39(16):4892-9. doi: 10.1021/bi9924009.
Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly of triglyceride-rich lipoproteins. Evidence has been presented for physical interactions between these proteins. To study the importance of apoB-MTP binding in apoB secretion, we have identified a compound, AGI-S17, that inhibited (60-70% at 40 microM) the binding of various apoB peptides to MTP but not to an anti-apoB monoclonal antibody, 1D1, whose epitope overlaps with an MTP binding site in apoB. AGI-S17 had no significant effect on the lipid transfer activity of the purified MTP. In contrast, another antagonist, BMS-200150, did not affect apoB-MTP binding but inhibited MTP's lipid transfer activity. The differential effects of these inhibitors suggest two functionally independent, apoB binding and lipid transfer, domains in MTP. AGI-S17 was then used to study its effect on the lipid transfer and apoB binding activities of MTP in HepG2 cells. AGI-S17 had no effect on cellular lipid transfer activities, but it inhibited coimmunoprecipitation of apoB with MTP. These studies indicate that AGI-S17 inhibits apoB-MTP binding but has no effect on MTP's lipid transfer activity. Experiments were then performed to study the effect of inhibition of apoB-MTP binding on apoB secretion in HepG2 cells. AGI-S17 (40 microM) did not affect cell protein levels but decreased the total mass of apoB secreted by 70-85%. Similarly, AGI-S17 inhibited the secretion of nascent apoB by 60-80%, but did not affect albumin secretion. These studies indicate that AGI-S17 decreases apoB secretion most likely by inhibiting apoB-MTP interactions. Thus, the binding of MTP to apoB may be important for the assembly and secretion of apoB-containing lipoproteins and can be a potential target for the development of lipid-lowering drugs. It is proposed that the apoB binding may represent MTP's chaperone activity that assists in the transfer from the membrane to the lumen of the endoplasmic reticulum and in the net lipidation of nascent apoB, and may be essential for lipoprotein assembly and secretion.
载脂蛋白B（apoB）和微粒体甘油三酯转移蛋白（MTP）对于富含甘油三酯的脂蛋白的有效组装至关重要。已有证据表明这些蛋白质之间存在物理相互作用。为了研究apoB-MTP结合在apoB分泌中的重要性，我们鉴定出一种化合物AGI-S17，它能抑制（40微摩尔时为60 - 70%）各种apoB肽与MTP的结合，但不抑制与抗apoB单克隆抗体1D1的结合，1D1的表位与apoB中MTP的结合位点重叠。AGI-S17对纯化的MTP的脂质转移活性没有显著影响。相反，另一种拮抗剂BMS-200150不影响apoB-MTP结合，但抑制MTP的脂质转移活性。这些抑制剂的不同作用表明MTP中存在两个功能独立的结构域，即apoB结合结构域和脂质转移结构域。然后使用AGI-S17来研究其对HepG2细胞中MTP的脂质转移和apoB结合活性的影响。AGI-S17对细胞脂质转移活性没有影响，但它抑制了apoB与MTP的共免疫沉淀。这些研究表明AGI-S17抑制apoB-MTP结合，但对MTP的脂质转移活性没有影响。然后进行实验以研究抑制apoB-MTP结合对HepG2细胞中apoB分泌的影响。AGI-S17（40微摩尔）不影响细胞蛋白质水平，但使分泌的apoB总量降低了70 - 85%。同样，AGI-S17抑制新生apoB的分泌60 - 80%，但不影响白蛋白分泌。这些研究表明AGI-S17最有可能通过抑制apoB-MTP相互作用来降低apoB分泌。因此，MTP与apoB的结合对于含apoB脂蛋白的组装和分泌可能很重要，并且可能成为开发降脂药物的潜在靶点。有人提出apoB结合可能代表MTP的伴侣活性，它有助于从内质网的膜向腔转移以及新生apoB的净脂化，并且可能对脂蛋白的组装和分泌至关重要。
