Insect Biochemistry and Molecular Biology 33 (2003) 1049–1060 www.elsevier.com/locate/ibmb Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs Haobo Jiang a,* , Yang Wang a , Xiao-Qiang Yu b , Yifei Zhu c , Michael Kanost c a Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA b Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri–Kansas City, Kansas City, MO 64110, USA c Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA Received 21 March 2003; accepted 16 June 2003 Abstract Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg 51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS- stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.  2003 Elsevier Ltd. All rights reserved. Keywords: Insect immunity; Melanization; Phenoloxidase; Serine proteinase; Clip domain; Serpin; Serine proteinase homolog; Tobacco hornworm 1. Introduction Insect phenoloxidase (PO) catalyzes the production of quinones that are intermediates for cuticle sclerotization * Corresponding author. Tel.: +1-405-744-9400; fax: +1-405-744- 6039. E-mail address: haobo@okstate.edu (H. Jiang). Abbreviations: PO, phenoloxidase; proPO, prophenoloxidase; PAP, prophenoloxidase-activating proteinase; PPAE or ppA, prophenoloxi- dase-activating enzyme; PPAF, prophenoloxidase-activating factor; proPAP-3, prophenoloxidase-activating proteinase-3 precursor; SPH, serine proteinase homolog; SDS, sodium dodecyl sulfate; PAGE, poly- acrylamide gel electrophoresis; DFP, diisopropyl fluorophosphate; IEARpNa, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide; IEARase, IEARpNa- hydrolyzing enzyme; Con A, concanavalin A; ELISA, enzyme-linked immunosorbent assay; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; RT-PCR, reverse transcriptase-polymerase chain reaction 0965-1748/$ - see front matter  2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0965-1748(03)00123-1 and melanin synthesis (Hopkins and Kramer, 1992; Sug- umaran, 1996; Nappi and Vass, 2001). Quinones may also participate in host defense responses such as wound healing and killing of entrapped parasites or pathogens. To minimize detrimental effects of the reactive inter- mediates to host tissues and cells, the conversion of pro- phenoloxidase (proPO) to PO is regulated as a local, transient reaction against nonself. A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation (Ashida and Brey, 1998; Kanost et al., 2001). Recognition of foreign organ- isms or aberrant surfaces triggers the pathway, leading to the activation of prophenoloxidase-activating proteinase (PAP). PAP, also known as prophenoloxidase activating enzyme (PPAE), is the terminal proteinase that converts proPO to PO. We have isolated PAP-1 and PAP-2 from the tobacco hornworm, Manduca sexta (Jiang et al.,