Osapay K, Tran D, Ladokhin A S, White S H, Henschen A H, Selsted M E
Department of Pathology, University of California, Irvine, California 92697, USA.
J Biol Chem. 2000 Apr 21;275(16):12017-22. doi: 10.1074/jbc.275.16.12017.
Indolicidin is a 13-residue cationic, antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils. The unique composition of indolicidin distinguishes it from alpha-helical and beta-structured cationic peptides, because five of indolicidin's 13 residues are tryptophans: H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH(2). Solid phase synthesis of indolicidin gave rise to a minor byproduct that possessed unusual fluorescence and UV absorbance properties compared with authentic indolicidin. The byproduct was purified by combined ion exchange and reversed phase high pressure liquid chromatography steps and was shown be identical to authentic indolicidin in its microbicidal activity against Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. Mass analysis of the byproduct revealed a 2-atomic mass unit reduction compared with indolicidin, suggesting the deprotonation of two indole side chains to form an intrachain delta(1),delta(1)'-ditryptophan derivative. We confirmed the nature of the cross-linked byproduct, termed X-indolicidin, by absorbance and fluorescence spectroscopy, peptide mapping, and sequence analysis. Edman degradation revealed that Trp-6 and Trp-9 were covalently cross-linked. Compared with indolicidin, X-indolicidin was partially resistant to digestion with trypsin and chymotrypsin, suggesting that the ditryptophan stabilizes a subset of molecular conformations that are protease resistant but that are absent in the native structure.
吲哚杀菌素是一种从牛嗜中性粒细胞胞质颗粒中分离出的由13个残基组成的阳离子抗菌肽酰胺。吲哚杀菌素独特的组成使其区别于α螺旋和β结构的阳离子肽，因为在吲哚杀菌素的13个残基中有5个是色氨酸：H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH₂。吲哚杀菌素的固相合成产生了一种次要副产物，与纯正的吲哚杀菌素相比，该副产物具有异常的荧光和紫外吸收特性。通过离子交换和反相高压液相色谱步骤相结合对该副产物进行纯化，结果表明其对金黄色葡萄球菌、大肠杆菌、白色念珠菌和新型隐球菌的杀菌活性与纯正的吲哚杀菌素相同。对该副产物的质谱分析显示，与吲哚杀菌素相比，其原子质量单位减少了2，这表明两条吲哚侧链去质子化形成了链内δ(1),δ(1)'-二色氨酸衍生物。我们通过吸收光谱和荧光光谱、肽图谱分析及序列分析证实了这种交联副产物（称为X-吲哚杀菌素）的性质。埃德曼降解法显示色氨酸-6和色氨酸-9发生了共价交联。与吲哚杀菌素相比，X-吲哚杀菌素对胰蛋白酶和糜蛋白酶的消化具有部分抗性，这表明二色氨酸稳定了一部分蛋白酶抗性的分子构象，而这些构象在天然结构中并不存在。
