Molecular Immunology 52 (2012) 258–263 Contents lists available at SciVerse ScienceDirect Molecular Immunology jou rn al hom epa ge: www.elsevier.com/locate/molimm Two isoforms of anti-lipopolysaccharide factors identified and characterized from the hemocytes of portunid crabs, Portunus pelagicus and Scylla tranquebarica V.V. Afsal a , Swapna P. Antony a , E.R. Chaithanya a , I.S. Bright Singh b , Rosamma Philip a,∗ a Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, CUSAT, Fine Arts Avenue, Kochi 682016, Kerala, India b National Center for Aquatic Animal Health (NCAAH), CUSAT, Fine Arts Avenue, Kochi 682016, Kerala, India a r t i c l e i n f o Article history: Received 10 May 2012 Received in revised form 4 June 2012 Accepted 4 June 2012 Available online 27 June 2012 Keywords: Antimicrobial peptide Anti-lipopolysaccharide factor Innate immunity Portunus pelagicus Scylla tranquebarica a b s t r a c t Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their deriva- tives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for pre- cursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysac- charide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydropho- bic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Crustaceans live in the aquatic environment where they are exposed to a large number of micro-organisms causing health haz- ards. Since there is no adaptive immunity in crustaceans, the whole burden of anti-pathogen defense falls on the innate immune sys- tem, and the antimicrobial peptides (AMPs) play an important role in invertebrate innate immune defense. Anti-lipopolysaccharide factors (ALFs) are a type of cationic AMPs, that are evolutionarily conserved across a wide range of marine invertebrates, includ- ing the ancient horseshoe crabs and crustaceans and are found to possess broad spectrum activities against gram-positive and gram- negative bacteria, fungi, and even virus (Antony et al., 2011; Liu et al., 2006; Ponprateep et al., 2012). Crustacean ALFs have also been proved to possess cell-penetrating ability and anti-cancer activity. ALFs belong to the group of single domain AMPs with a signal pep- tide at the N-terminal region followed by a conserved LPS-binding domain. The LPS-binding domain, which is the characteristic fea- ture of ALFs, are formed between two conserved cysteine residues ∗ Corresponding author. Tel.: +91 484 2368120; fax: +91 484 2381120. E-mail addresses: rosammap@gmail.com, rose@cusat.ac.in (R. Philip). which form a disulphide loop, and contain a cluster of positively charged residues within it (Hoess et al., 1993). This typical structure makes ALFs capable of binding and neutralizing lipopolysaccha- rides (LPS). The first ALF was isolated from the amoebocytes of the horse- shoe crab Limulus polyphemus (Tanaka et al., 1982) and found to have a strong antibacterial effect on gram-negative R-type bacte- ria. Reports on crustacean ALFs have been increasing in recent years viz., in shrimps (Tharntada et al., 2008); crabs (Afsal et al., 2011, 2012); lobsters (Beale et al., 2008) and crayfishes (Sun et al., 2011). Some decapods have also been reported to express multiple ALF isoforms which vary in length or sequence and display different biological activities. Crab culture is facing constraints in production due to severe health problems resulting in large scale mortality. Under- standing the defense mechanisms of crab may be effective in the development of better disease control strategies in farm- ing. The identification and characterization of immune effectors are believed to be helpful for elucidation of immune defense mechanisms and disease control in crab aquaculture because of their potential use as therapeutic agents and genetic improve- ment as biomarkers on disease-resistant strain selection. Many AMPs have been identified and characterized in crabs till date 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.06.003