title: In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system.
abstract: Extracellular serine protease cascades mediate immune signaling and responses in insects. In the tobacco hornworm Manduca sexta, nearly 30 serine proteases (SPs) and their homologs (SPHs) are cloned from hemocytes and fat body. Some of them participate in prophenoloxidase (proPO) activation and proSpätzle processing. Here we report the cDNA cloning of hemolymph protease-1b (HP1b), which is 90% identical and 95% similar to HP1a (formerly HP1). The HP1a and HP1b mRNA levels in hemocytes was down- and up-regulated after an immune challenge, respectively. Quantitative real-time polymerase chain reactions revealed their tissue-specific and development-dependent expression, mostly in hemocytes of the feeding larvae. We isolated HP1 precursor (proHP1) from larval hemolymph and observed micro-heterogeneity caused by N-linked glycosylation. Supplementation of the purified proHP1 to plasma samples from naïve larvae or induced ones injected with bacteria caused a small PO activity increase, much lower than those elicited by recombinant proHP1a/b, but no proteolytic cleavage was detected in the zymogens. Incubation of proHP1a/b or their catalytic domains with a cationic detergent, cetylpyridinium chloride, induced an amidase activity that hydrolyzed LDLH-p-nitroanilide. Since LDLH corresponds to the P4-P1 region before the proteolytic activation site of proHP6, we propose that the active but uncleaved proHP1 may cut proHP6 to generate HP6 that in turn activates proPAP1 and proHP8. The catalytic domain of HP1a/b, which by itself does not activate purified proHP6 or hydrolyze LDLH-p-nitroanilide, somehow generated active HP6, HP8, PAP1 and PO in plasma. Together, these results indicate that proHP1 participates in the proPO activation system, although detailed mechanism needs further exploration.
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, United States. Electronic address: haobo.jiang@okstate.edu.
authors.forename: Haobo
authors.initials: H
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D006458:Hemolymph / Q000737:chemistry; D007107:Immune System; D007113:Immunity, Innate; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D018402:Manduca / Q000235:genetics* / Q000254:growth & development / Q000276:immunology; D000073718:Pancreatitis-Associated Proteins; D010858:Pigmentation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome
chemical_list: D018076:DNA, Complementary; D019476:Insect Proteins; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D057057:Serine Proteases
keywords: Clip domain; Enzyme cascade; Hemolymph protein; Insect immunity; Phenoloxidase; Zymogen activation