Characterization and gene organization of anti-lipopolysaccharide factor in the black shrimp Penaeus monodon

Abstract

Different isoforms of the ALF homologues (ALFPm1 to 5) have been previously identified from Penaeus monodon expressed sequence tag (EST) database (http://pmonodon.biotec.or.th). The nucleotide and amino acid sequences of the P. monodon ALF homologues were analyzed and categorized into two groups, ALFPm1 and 2 in group A and ALFPm3 to 5 in group B. The genomic sequences of the two ALF gene groups were obtained by using the PCR and genome walking techniques. The ALF group A gene consisted of three exons interrupted by two introns whereas the ALF group B gene contained four exons interrupted by three introns. The alignment of genomic sequences with the ALF cDNA sequences revealed that different transcripts in both groups were generated by alternative RNA splicing of the pre-mRNA transcripts. The 5´ upstream sequences of the two ALF groups contained the putative cis-regulatory elements, including the activator protein 1, the Octamer, the GATA, the nuclear factor-kappaB, and the GAAA motifs, which possibly promoted transcription in response to infection as in other antimicrobial peptide genes. The RT-PCR analysis revealed that although all ALF isoforms were expressed in individual shrimp, the ALFPm2 and 3 were the major or authentic ALFs in the hemocytes. The expression of ALFPm2 and 3 were increased in response to Vibrio harveyi infection indicating the important function of the ALFs against bacterial invasion. To further characterize the activity of ALFPm2, the recombinant ALFPm2 protein (rALFPm2) was produced in the Pichia pastoris and then purified using cation exchange chromatography. The purified rALFPm2 showed antibacterial activity against Escherichia coli 363 and Bacillus megaterium. Moreover, 20 μM of rALFPm3 but not rALFPm2 exhibited the antiviral activity against the white spot syndrome virus (WSSV) by inhibiting the propagation of WSSV in hematopoietic stem cells of Pacifastacus leniusculus. Analysis of VP28 transcripts by quantitative RT-PCR showed that the concentration of rALFPm3 required for 50% inhibition of viral propagation in vitro (IC₅₀) was less than 2.5 μM.