title: Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus.
abstract: Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab.
authors.affiliation: EMBL, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China. zhxcui@yahoo.com.cn
authors.forename: Zhaoxia
authors.initials: Z
authors.lastname: Cui
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Danhua
authors.initials: D
authors.lastname: Wu
authors.forename: Weisha
authors.initials: W
authors.lastname: Luan
authors.forename: Shuangyan
authors.initials: S
authors.lastname: Wang
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Li
authors.forename: Chengwen
authors.initials: C
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D004156:Catechol Oxidase / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics* / Q000276:immunology*; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000235:genetics* / Q000276:immunology*; D014733:Vibrio / Q000276:immunology
chemical_list: D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D057057:Serine Proteases