Prophenoloxidase (proPO) activation in Manduca sexta: an analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a cofactor.
Proteolytic activation of prophenoloxidase (proPO) is an integral part of the insect immune system against pathogen and parasite infection. This reaction is mediated by a proPO-activating proteinase (PAP) and its cofactor in the tobacco hornworm, Manduca sexta (Proc. Natl. Acad. Sci. USA 95 (1998) 12220; J. Biol. Chem. 278 (2003) 3552; Insect Biochem. Mol. Biol. 33 (2003) 1049). The cofactor consists of two serine proteinase homologs (SPHs), which associate with immulectin-2, a calcium-dependent lectin that binds to lipopolysaccharide (Insect Biochem. Mol. Biol. 33 (2003) 197). In order to understand the auxiliary effect of SPH-1 and SPH-2 in proPO activation, we started to investigate the molecular interactions among proPO, PAP-3, and the proteinase-like proteins. M. sexta SPH-1 and SPH-2 were purified from hemolymph of prepupae by hydroxylapatite, gel filtration, lectin-affinity, and ion exchange chromatography. They existed as non-covalent oligomers with an average molecular mass of about 790 kDa. MALDI-TOF mass fingerprint analysis revealed a new cleavage site in SPH-1 before Asp85. The PAP cofactor did not significantly alter Michaelis constant (KM) or kcat of PAP-3 towards a synthetic substrate, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide, but greatly enhanced proPO activation by PAP-3. The apparent KM for proPO was determined to be about 9.4 microg/ml, close to its estimated concentration in larval hemolymph. In the presence of excess proPO and a set amount of PAP-3, increasing levels of phenoloxidase (PO) activity were detected as more SPHs were added. Half of the maximum proPO activation occurred when the molar ratio of PAP-3 to SPH was 1:1.4. Gel filtration experiments suggested that proPO, PAP-3, and the cofactor formed a ternary complex.
Wang
Yang
Y
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater 74078, USA.
Jiang
Haobo
H
D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D003067:Coenzymes / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006458:Hemolymph / Q000737:chemistry / Q000201:enzymology; D007700:Kinetics; D018402:Manduca / Q000737:chemistry / Q000201:enzymology*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
D003067:Coenzymes; D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D012697:Serine Endopeptidases; C115269:pro-phenol oxidase activating proteinase