title: Molecular cloning and characterization of crustin from mud crab Scylla paramamosain.
abstract: Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain.
authors.affiliation: Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. chanprapa.i@chula.ac.th
authors.forename: Chanprapa
authors.initials: C
authors.lastname: Imjongjirak
authors.forename: Piti
authors.initials: P
authors.lastname: Amparyup
authors.forename: Anchalee
authors.initials: A
authors.lastname: Tassanakajon
authors.forename: Siriporn
authors.initials: S
authors.lastname: Sittipraneed
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D033364:Decapoda / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D015723:Gene Library; D016678:Genome / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins