ROLE OF CLIP DOMAIN SERINE PROTEINASE 2, PmClipSP2, IN ACTIVATION OF PROPHENOLOXIDASE-ACTIVATING SYSTEM IN THE BLACK TIGER SHRIMP Penaeus monodon

Abstract

Clip domain serine proteinases (ClipSPs) play an important role in the prophenoloxidase-activating (proPO) system. In the shrimp Penaeus monodon, the ClipSP, PmClipSP2, has been previously shown to bind to microbial polysaccharides (LPS and β-1,3-glucan) and likely activates the proPO-system. Moreover, it may play key role in the hemocyte homeostasis by scavenging and neutralizing LPS. To reveal the binding site of the PmClipSP2 protein, the N-terminal clip domain (Clip-PmClipSP) and C-terminal SP domain (SP-PmClipSP2) were separately cloned. The recombinant proteins were then assayed for their binding properties and involvement in proPO activation, antimicrobial activity and LPS neutralization. According to the ELISA-based binding assay, rSP-PmClipSP2, but not rClip-PmClipSP, can bind immobilized LPS and β-1,3-glucan as well as significantly activate PO activity. The binding site at the SP domain is proposed to have a pattern sequence (X-[PFY]-X-[AFILV]-[AFY]-[AITV]-X-[ILV]-X(5)-W-[IL]-X) that is located at the C-terminal region of the SP domain of PmClipSP2. Deletion of the pattern sequence abolished binding to LPS and β-1,3-glucan. Conversely, a recombinant protein containing the pattern sequence (rPT-PmClipSP2-TRX) had the ability to bind to cell wall components but did not exhibit antibacterial activity via radial diffusion assay at 100 µM. Furthermore, rPmClipSP2 cannot directly neutralize toxicity of LPS in S2 cells. These results confirm the function of PmClipSP2 in binding to microbial cell wall components of bacteria and fungi via the pattern sequence at the C-terminus SP domain, subsequently leading to activation of the proPO cascade.