Expression and Characterization of <i>Manduca sexta</i> Stress Responsive Peptide-1; An Inducer of Antimicrobial Peptide Synthesis

Abstract

In response to stress conditions such as wounding or infections in insects, several short peptides are processed to act as cytokines that induce AMP gene expression. To study their structure-activity relationship, immune inducibility, tissue specificity, stress responsiveness, and development relatedness, we chemically synthesized Manduca sexta stress response peptide-1, a 25-residue peptide with one disulfide bond (SRP1: FGVRVGTCPSGYVRRGTFCFPDDDY). Upon injection of the SRP1 into naïve larvae, several antimicrobial peptide genes were expressed at higher levels. The mRNA levels of SRP1 increased significantly in hemocytes and fat body after larvae were challenged with a mixture of bacteria and β-1,3-glucan. The expression patterns of SRP1 and its target genes are somewhat different from SRP2's, suggesting overlapping yet distinct functions. We elucidated the 3D structure of SRP1 in solution by two-dimensional 1 H-1 H NMR spectroscopy. The tertiary structure of SRP1 consists of two short β-strands at Y12−R15 and F18−F20, one type-II β-turn at R15−F18 in its well-defined core and is stabilized by a covalent disulfide bond between C8 and C19. The conformational ensemble of SRP1 from extensive atomistic simulation in explicit solvent (with 3.0 µs total effective sampling) shows high consistency with experimental intramolecular NOEs of the core region. The SRP1 core adopts a fold similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF), suggesting that SRP1 may interact with EGF receptor-like molecules to trigger its biological function.

Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta

“…cDNA synthesis and qRT-PCR analysis of HP5 and antimicrobial peptide mRNA levels were performed using specific primers for them and rpS3. Amplification efficiencies of the cDNA fragments were determined as described before (36). Relative HP5 and antimicrobial peptide mRNA levels were calculated as (1 + E rpS3 ) Ct, rpS3 /(1 + E x ) Ct, x .…”