Molecular Cloning,Expression And Functional Analysis Of SpALF6 And SpALF7 In The Mud Crab,Scylla Paramamosain

Anti-lipopolysaccharide factors(ALFs)are antimicrobial peptides(AMPs)widely distributed in crustaceans,which have a broad spectrum of the antimicrobial activity that covers a large number of Gram-positive and Gram-negative bacteria,fungi as well as some viruses.The members of the ALF homologues family have sequence difference and functional diversity.So far,we have not known much about the mechanism of how the difference of ALFs cDNA can affect the function of ALFs.To further understand the mechanism,we chose the mud crab,Scylla paramamosain as experimental animals,carried on the related research.In the present study,two new ALF isoforms was isolated from the screening a S.paramamosain hemocyte cDNA library,which are not the members of the family of typical anti-lipopolysaccharide factors.Through the analysis of the sequence and function research,we can preliminarily clarify the relationship between sequence diversity and the immune functions of ALF homologues.In this study,we characterized a novel ALF homolog SpALF6 from mud crab S.paramamosain and its variant SpALF6-V,which was generated by mutations of two amino acids(H46 to R and A110 to P)due to the presence of two single nucleotide polymorphisms(SNPs).SpALF6 was an anionic peptide with isoelectric point(pI)6.79,whereas SpALF6-V was a cationic protein with pI 7.98.These two proteins shared a common lipopolysaccharide(LPS)-binding domain(LBD)with pI 6.05.To investigate the likely functional differences between SpALF6 and SpALF6-V and elucidate the underlying mechanisms,a single amino acid mutant SpALF6-M(from H46 to R,outside but very close to LBD),which had the same pI as SpALF6-V,was harvested by a fusion PCR.Then SpALF6 LBD peptide and its biotin-labeled form were synthesized in this study.SpALF6 was expressed mainly in hemocytes and up-regulated by Vibrio parahaemolyticus or Staphylococcus aureus challenge,indicating that SpALF6 may participate in the antibacterial immune responses.Then,both SpALF6 and SpALF6-M were overexpressed and purified to test antimicrobial activity and binding activity to microbial cells or polysaccharides.SpALF6-M exhibited more potent antimicrobial and cell-binding activity on Gram-positive bacteria and fungi than SpALF6.Furthermore,SpALF6-M possessed stronger lipoteichoic acid(LTA)-binding activity than SpALF6,demonstrating that thisparticular positively charged amino acid outside but close to LBD contributed to the increase in SpALF6-M antibacterial activity.The results of LBD peptide showed that this anionic LBD peptide itself did not exhibit any significant antimicrobial activity against 10 kinds of microorganisms but it possessed strong binding activity to LPS,LTA,and peptidoglycan.These findings suggested that this anionic LBD was still an important active center and required collaboration with some particular positively charged amino acids outside LBD to exhibit antibacterial activity.Thus,SpALF6-M antimicrobial activity was increased by the mutation of H46 to R instead of A110 to P,which did not change the protein charge,suggesting that SpALF6-V may have more potent antimicrobial activity than SpALF6 and play more important roles in antibacterial immunity.In addition we also identified a new ALF isoform named as SpALF7 which is different from the typical ALFs.Comparison of spatial structures between SpALF7 and the typical ALF ALFPm3 demonstrated that SpALF7 and ALFPm3 had similar binding sites,suggesting that SpALF7 may also recognize LPS or other molecular structures,but the spatial structure had a certain difference.SpALF7 only had two?-helices packed against a four-strand ?-sheet but it may form two disulfide bonds,whereas ALFPm3 possessed three ?-helices packed against the four-strand ?-sheet and only have one disulfide bond.So we undertook the reserch of SpALF7 and discovered that it mainly distributed in the hemolymph and can bind with some pathogen-associated molecular patterns of these microorganisms and dispaly more stronger acitivity binding with LPS from E.coli than LTA from S.aureus,PGN from E.coli and S.aureus.This phenomenon indicated that the LBD of SpALF7 was still an vital activity sites.Meanwhile,we discovered that SpALF7 exhibited more potent cell-binding activity Gram-negative bacteria and Pichia pastoris than Gram-positive bacteria.Finally we tested the vitro funciton assay of SpALF7 and detected that SpALF7 can inhibit the most of microorganisms apart from Aeromonas hydrophila.So we speculate that SpALF7 can exert antimicrobial activity via binding with these pathogen-associated molecular patterns of tested microorganisms.Through our study,we can comprehend that the relationship between sequences difference and functional diversity well.Moreover it was useful for us to learn the adaptive evolution and its significance under the selective pressure of environmental pathogens.Our research can also provide a candidate resistant gene for breeding.