A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain

Autor: Tongtong Kong, Weisong Wan, Ming Zhang, Yi Gong, Jude Juventus Aweya, Daimeng Zhang, Shengkang Li
Rok vydání: 2018
Předmět:
Lipopolysaccharides
0301 basic medicine
Signal peptide
Proteases
Brachyura
Scylla paramamosain
Aquatic Science
Arthropod Proteins
Serine
Random Allocation
03 medical and health sciences
Anti-Infective Agents
Hemolymph
Animals
Environmental Chemistry
Amino Acid Sequence
Blood Coagulation
Phylogeny
Serine protease
Enzyme Precursors
Innate immune system
Base Sequence
biology
Gene Expression Profiling
04 agricultural and veterinary sciences
General Medicine
Prophenoloxidase
biology.organism_classification
Blood Coagulation Factors
Recombinant Proteins
Open reading frame
Poly I-C
030104 developmental biology
Gene Expression Regulation
Biochemistry
040102 fisheries
biology.protein
0401 agriculture
forestry
and fisheries
RNA Interference
Vibrio parahaemolyticus
Serine Proteases
Sequence Alignment
Catechol Oxidase
Zdroj: Fish & Shellfish Immunology. 79:52-64
ISSN: 1050-4648
DOI: 10.1016/j.fsi.2018.05.013
Popis: The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5′ untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3′ UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His156, Asp226, Ser321). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain.
Databáze: OpenAIRE
Externí odkaz: