Purification and characterization of antibacterial proteins from granular hemocytes of Indian mud crab, Scylla serrata.
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.
Yedery
Roshan Dinesh
RD
Department of Molecular Immunology, National Institute for Research in Reproductive Health (ICMR), Parel, Mumbai, India.
Reddy
Kudumula Venkata Rami
KV
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
D000900:Anti-Bacterial Agents; D011506:Proteins; D012333:RNA, Messenger