{"doi":"10.3389/fimmu.2023.1244792","title":"Two clip-domain serine protease homologs, cSPH35 and cSPH242, act as a cofactor for prophenoloxidase-1 activation in Drosophila melanogaster","abstract":"Insect phenoloxidases (POs) catalyze phenol oxygenation and o -diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high M r cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila , PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 ( i.e ., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta . In the presence of high M r complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated in vitro . To complement the in vitro analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.","journal":"Frontiers in Immunology","year":2023,"id":346719,"datarank":0.4640094206190288,"base_score":2.5649493574615367,"endowment":2.5649493574615367,"self_citation_contribution":0.38474240361923057,"citation_network_contribution":0.07926701699979821,"self_endowment_contribution":0.38474240361923057,"citer_contribution":0.07926701699979821,"corpus_percentile":null,"corpus_rank":null,"citation_count":12,"citer_count":9,"citers_with_citation_signal":6,"citers_with_endowment":6,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9606,"is_data_producer":true,"deposit_databanks":{"figshare":["10.6084/m9.figshare.23786949"]},"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":553484,"name":"Yang Wang","orcid":"0000-0002-6527-864X","position":1,"is_corresponding":false},{"id":696453,"name":"Haodong Yin","orcid":"0000-0001-6653-6789","position":2,"is_corresponding":false},{"id":212661,"name":"Haobo Jiang","orcid":"0000-0003-1357-1315","position":3,"is_corresponding":false},{"id":212660,"name":"Qiao Jin","orcid":null,"position":0,"is_corresponding":true}],"reference_count":55,"raw_metadata":null,"created_at":"2026-07-19T01:11:53.085356Z","pmid":"37781370","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}