title: Purification and characterization of antibacterial proteins from granular hemocytes of Indian mud crab, Scylla serrata.
abstract: Marine invertebrates depend upon antimicrobial peptides (AMPs) as a major component of innate immunity, as they are rapidly synthesized and diffuse upon pathogen invasion. In this study, we report the identification and characterization of a 11 kDa antimicrobial protein, which we name SSAP (for Scylla serrata antimicrobial protein), from granular hemocytes of the mangrove crab S. serrata. The protein is highly similar to scygonadin, a male-specific AMP isolated from the ejaculatory duct of S. serrata. SSAP was isolated using various chromatographic techniques, viz. ion-exchange, ultra filtration and RP-HPLC, and demonstrated antibacterial activity against Gram positive and Gram negative bacteria. Full length mRNA encoding SSAP was amplified using a combination of RT-PCR and RACE. The nucleotide sequence revealed a full-length ORF of 381 bp coding for a preprotein of 126 amino acids comprising a signal peptide of 24 amino acids and a mature protein of 102 amino acids with a predicted mass of 11435 Da and pI of 5.70. Unlike scygonadin, SSAP is expressed in several tissues of both male and female crabs, as evidenced by RT-PCR, Northern and Western blot analyses. The study suggests that SSAP might be an isoform or a variant of scygonadin and might play an important role in regulating the immunity of the crab upon microbial infection.
authors.affiliation: Department of Molecular Immunology, National Institute for Research in Reproductive Health (ICMR), Parel, Mumbai, India.
authors.forename: Roshan Dinesh
authors.initials: RD
authors.lastname: Yedery
authors.forename: Kudumula Venkata Rami
authors.initials: KV
authors.lastname: Reddy
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification*; D015152:Blotting, Northern; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003445:Crustacea; D005260:Female; D006434:Hemocytes / Q000737:chemistry*; D008297:Male; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000900:Anti-Bacterial Agents; D011506:Proteins; D012333:RNA, Messenger