Boman H C, Boman I A, Andreu D, Li Z Q, Merrifield R B, Schlenstedt G, Zimmermann R
Department of Microbiology, University of Stockholm, Sweden.
J Biol Chem. 1989 Apr 5;264(10):5852-60.
Radiolabeled preprocecropin B, with an alpha-amidated COOH terminus, and preprocecropin A, extended at the COOH terminus by a glycine residue, were synthesized by solid-phase methods. The respective syntheses were interrupted at intervals to allow the preparation of the predicted procecropins A and B as well as three other truncated derivatives of the cecropin A precursors. All the synthetic peptides were purified to near homogeneity by reverse-phase liquid chromatography and their purity was established by analytical high performance liquid chromatography, gel electrophoresis, and amino acid analysis. A dipeptidyl aminopeptidase was purified about 350 times from the hemolymph of cecropia pupae and characterized by its affinity for different substrates and inhibitors. The synthetic prepro peptides were tested for processing by an extract of dog pancreas microsomes and purified leader peptidase from Escherichia coli, with and without partly purified dipeptidyl aminopeptidase, and the two synthetic proforms were also processed with the dipeptidyl aminopeptidase alone. From these experiments we conclude that the signal/leader peptidase cleaves the peptide bond between Ala-5 and Ala-4. This cleavage site is further substantiated by radio sequencing of procecropin A isolated after synthesis in a coupled system for in vitro transcription, translation, and processing. The two procecropins, which are stable to further digestion by the signal peptidase, are further processed by dipeptidyl aminopeptidase which removes, in two steps, the dipeptides Ala-Pro (residues -4 and -3) and Glu-Pro (residues -2 and -1). Although the synthetic peptide with only one dipeptide (Glu-Pro) preceding the mature cecropin sequence could function as a substrate for dipeptidyl aminopeptidase, it could be demonstrated as an intermediate in the enzymatic reaction with the procecropins. Dipeptidyl aminopoptidase did not cleave the procecropin analog when it was preceded by a single alanine residue, i.e. preprocecropin (-5,38). Antibacterial activity was demonstrated for the mature cecropin A-Gly obtained by processing of the synthetic preproprotein.
通过固相方法合成了具有α-酰胺化羧基末端的放射性标记前肽杀菌肽B，以及在羧基末端通过甘氨酸残基进行延伸的前肽杀菌肽A。各自的合成过程会定期中断，以便制备预测的杀菌肽A和B，以及杀菌肽A前体的其他三种截短衍生物。所有合成肽通过反相液相色谱法纯化至接近均一，其纯度通过分析型高效液相色谱法、凝胶电泳和氨基酸分析确定。从柞蚕蛹的血淋巴中纯化出一种二肽基氨肽酶，纯化倍数约为350倍，并通过其对不同底物和抑制剂的亲和力对其进行表征。用犬胰腺微粒体提取物和从大肠杆菌中纯化的前导肽酶对合成的前肽进行加工测试，有无部分纯化的二肽基氨肽酶参与，并且两种合成的前体形式也单独用二肽基氨肽酶进行加工。从这些实验中我们得出结论，信号/前导肽酶切割Ala-5和Ala-4之间的肽键。在体外转录、翻译和加工的偶联系统中合成后分离得到的杀菌肽A的放射性测序进一步证实了这个切割位点。这两种杀菌肽对信号肽的进一步消化具有稳定性，它们会被二肽基氨肽酶进一步加工，该酶分两步去除二肽Ala-Pro（残基-4和-3）和Glu-Pro（残基-2和-1）。尽管在成熟杀菌肽序列之前仅含有一个二肽（Glu-Pro）的合成肽可以作为二肽基氨肽酶的底物，但它可被证明是与杀菌肽发生酶促反应的中间体。当二肽基氨肽酶之前有一个单一丙氨酸残基，即前肽杀菌肽（-5,38）时，它不会切割杀菌肽类似物。通过对合成前体蛋白进行加工得到的成熟杀菌肽A-甘氨酸表现出抗菌活性。
