Binding of PmClipSP2 to microbial cell wall components and activation of the proPO-activating system in the black tiger shrimp Penaeus monodon.

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. 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. 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, confirming that the pattern sequence at the C-terminus of PmClipSP2 is responsible for binding to microbes, subsequently leading to activation of the proPO cascade.