Cloning and characterization of a LPS-regulatory gene having an LPS binding domain in kuruma prawn Marsupenaeus japonicus.

ABSTRACT

LPS is known as an effective stimulator of the immune system in various animals, including mammals and horseshoe crabs (HSC). Both of these animal groups have suppressive regulatory proteins for the LPS response, e.g. the bactericidal/permeability increasing protein in mammals and anti-LPS factor (ALF) in HSC. Prawns are a valuable aquaculture species, but the regulatory molecules and/or mechanisms that respond to LPS are largely unknown. To investigate the molecular mechanism of the LPS response in kuruma prawns, we cloned a cDNA having a LPS binding domain. A full-length cDNA gene, denoted as M-ALF (Marsupenaeus japonicus ALF-like peptide) was cloned that consisted of 746bp and encoded 123 amino-acid residues. The 3' non-translated region of this gene had the pentamer of ATTTA repeated four times; this is known as sequences for messenger RNA stabilization. Deduced amino-acid sequences showed a 42% homology with Japanese HSC-ALF. In particular, both have clusters of basic and hydrophobic amino acids, indicating that the region is probably binding to lipid A. The mRNA expression was determined for hemocytes, lymphoid organs, hearts, intestines and gills by RT-PCR. The mRNA expression was augmented 1.5-3h after LPS administration in lymphoid organs, but then decreased to normal level at 6h. Synthetic peptides containing Cys30 to Cys51 had LPS neutralizing activity to the Limulus reaction and NO production in RAW264.7 cells. These data suggest that in kuruma prawns, M-ALF acts as a LPS regulator during the acute phase response after invasion of pathogens.