Thesis (M.Sc.)--Chulalongkorn University, 2013

Antimicrobial peptides (AMPs) are considered to play an important role in humoral defense against pathogen infection. CrustinPm1 and crustinPm7 are two of most abundant crustin identified from the hemocytes of the black tiger shrimp, Penaeus monodon. This research aims to study (1) activities and binding properties of the recombinant crustinPm1 (rcrustinPm1) and recombinant crustinPm7 (rcrustinPm7), (2) the secondary structure of both crustins, (3) crystallization of the rcrustinPm1, and (4) the regulation of crustinPm1 and crustinPm7 gene expressions. In this study, crustinPm1 and crustinPm7 were overexpressed in Pichia pastoris. Antimicrobial assays demonstrated that rcrustinPm1 was active against Gram-positive bacteria only but rcrustinPm7 inhibited both Gram-positive and Gram-negative bacteria. The rcrustinPm1 and rcrustinPm7 showed no proteinase inhibitory activity against the commercial proteinases and proteinase from Bacillus subtilis. Study of lipid-protein interaction showed that both crustins could specifically bind phosphatidic acid (PA). Enzyme-linked immunosorbent assay suggested that crustins bind to PA with positive cooperativity of Hill slope (H) > 2. This indicates that at least two molecules of crustins interact with one PA molecule. In addition, circular dichroism spectroscopy was used to determine secondary structure of crustinPms. CrustinPm1 contained 40.81% of alpha-helix and 22.34% of beta-sheet, whereas crustinPm7 contained 32.86% of alpha-helix and 27.53% of beta-sheet. Crystallization of rcrustinPm1 was performed and crystals were obtained in some crystallization conditions. However, these crystals gave no diffraction. Therefore, crystallization conditions will be optimized for further crystal growth. Regulatory pathways of crustinPm1 and crustinPm7 were investigated by PmRelish and PmMyD88 knockdown. It is likely that crustinPm1 was mediated through Toll signaling pathway, while crustinPm7 was related with both Toll and Imd pathways.