Chemical Synthesis and Enzymic Processing of Precursor Forms of Cecropins A and B

• Published in: The Journal of biological chemistry 1989-04, Vol.264 (10), p.5852-5860
• Main Authors: Boman, H G, Boman, I A, Andreu, D, Li, Z Q, Merrifield, R B, Schlenstedt, G, Zimmermann, R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Anti-Inflammatory Agents, Non-Steroidal; antibacterial agents; Biological and medical sciences; Biotechnology; cecropin B; Chromatography, High Pressure Liquid; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - metabolism; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Indicators and Reagents; Insect Hormones - chemical synthesis; Insect Hormones - genetics; Insect Hormones - isolation & purification; Insect Proteins; Methods. Procedures. Technologies; Miscellaneous; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Moths - enzymology; Protein Precursors - chemical synthesis; Protein Precursors - genetics; Protein Precursors - isolation & purification; Protein Processing, Post-Translational; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)83628-6

Radiolabeled preprocecropin B, with an α-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.