Ember J A, Johansen N L, Hugli T E
Research Institute of Scripps Clinic, La Jolla, California 92037.
Biochemistry. 1991 Apr 16;30(15):3603-12. doi: 10.1021/bi00229a003.
An extensive structure-activity study of synthetic analogues of the C3a anaphylatoxin was conducted. Our goal was to map C3a-C3a receptor interactions by designing synthetic analogue molecules having maximal biologic potency. Nonspecific binding of the polycationic C3a to polyanionic molecules on cellular surfaces often obscures specific binding to the receptor. Less cationic synthetic C3a analogues would be useful tools in identifying and characterizing the various cell types having C3a receptors. These factors should also be useful as pharmacologic probes for mechanism studies, as high-affinity ligands for target cell identification, and for receptor isolation. Attachment of amino-terminal hydrophobic groups such as Fmoc to C3a analogues [as orginally introduced by Gerardy-Schahn et al. (1988) Biochem. J. 255, 209] markedly enhanced the potency of synthetic C3a peptides. The enhancement effect on potency from introducing hydrophobic groups to C3a analogues was interpreted as possibly being nonspecific. Our systematic search for an optimal peptide length, composition, and N-terminal hydrophobic unit resulted in several superpotent C3a analogues having 200-1500% the potency of natural C3a. One particularly potent C3a peptide was designed by incorporating two tryptophanyl residues at the N-terminal end of a 15-residue C3a analogue. The superpotent peptide W-W-G-K-K-Y-R-A-S-K-L-G-L-A-R has several residues differing (underlined) from the sequence corresponding to positions 63-77 in human C3a, a region that contains the essential functional site of the molecule. This 15-residue model peptide exhibited the greatest biological potency of all peptides tested, being 12-15 times more active than natural C3a. Since an optimal distance was found to exist between the N-terminal hydrophobic unit (W-W) and the C-terminal primary binding site (LGLAR), we concluded that the hydrophobic unit interacts specifically with a secondary binding site on the C3a receptor. The presence of both a primary (effector) and secondary (hydrophobic) binding site on these linear synthetic ligands, which can interact cooperatively with the C3a receptor, presumably accounts for the high relative potency of the analogues. Our design of superpotent analogues of C3a demonstrates the feasibility for constructing small synthetic peptides to mimic natural biologic factors that depend on secondary or tertiary structure for their activity. These synthetic peptide studies demonstrate that a linear array of amino acids (e.g., W-W) can successfully substitute for a conformation-dependent binding site on a bioactive factor.
我们对C3a过敏毒素的合成类似物进行了广泛的构效关系研究。我们的目标是通过设计具有最大生物活性的合成类似物分子来描绘C3a与C3a受体的相互作用。多阳离子的C3a与细胞表面的多阴离子分子的非特异性结合常常掩盖了其与受体的特异性结合。阳离子性较弱的合成C3a类似物将是鉴定和表征具有C3a受体的各种细胞类型的有用工具。这些因素也可用作机制研究的药理学探针、用于靶细胞鉴定的高亲和力配体以及用于受体分离。将氨基末端疏水基团（如Fmoc）连接到C3a类似物上[如Gerardy-Schahn等人（1988年）最初引入的，《生物化学杂志》255卷，209页]显著增强了合成C3a肽的活性。将疏水基团引入C3a类似物对活性的增强作用被解释为可能是非特异性的。我们对最佳肽长度、组成和N末端疏水单元进行系统搜索，得到了几种超活性C3a类似物，其活性是天然C3a的200 - 1500%。一种特别有效的C3a肽是通过在一个15个残基的C3a类似物的N末端掺入两个色氨酸残基而设计的。超活性肽W-W-G-K-K-Y-R-A-S-K-L-G-L-A-R有几个残基（下划线部分）与人类C3a中对应于63 - 77位的序列不同，该区域包含分子的关键功能位点。这个15个残基的模型肽在所有测试肽中表现出最大的生物活性，比天然C3a活性高12 - 15倍。由于发现N末端疏水单元（W-W）与C末端主要结合位点（LGLAR）之间存在最佳距离，我们得出结论，疏水单元与C3a受体上的一个二级结合位点特异性相互作用。这些线性合成配体上存在一个主要（效应）和二级（疏水）结合位点，它们可以与C3a受体协同相互作用，这大概解释了类似物的高相对活性。我们设计的C3a超活性类似物证明了构建小的合成肽以模拟依赖二级或三级结构发挥活性的天然生物因子的可行性。这些合成肽研究表明，氨基酸的线性排列（如W-W）可以成功替代生物活性因子上依赖构象的结合位点。
