Expression And Function Of Peptidoglycan Recognition Protein Gene From Amphioxus Branchiostoma Japonicus

Peptidoglycan recognition proteins （PGRPs） are a crucial family ofinnate immunity molecules owing to their prominent ability in detectinginvading bacteria and promoting immunity. They have been identified inmollusks, insetcts, echinoderms, and vertebrates including humans.PGRPs are structurally conserved through evolution, but their functionsin innate immunity are different in invertebrates and vertebrates.To date，little is known about PGRPs in amphioxus, an intermediatary speciesfrom invertebrates to vertebrates. Herein a short PGRP gene （pgrp-s）from amphioxus Branchiostoma japonicum，its expression and functionswere examined.The amphioxus PGRP-S cDNA obtained was895bp long, whichcontains an open reading frame （ORF） of753bp, a5’-untranslated region（UTR） of18bp and a3’-UTR of124bp. The ORF codes for a deducedprotein of250amino acids with a predicted molecular mass ofapproximately26.5kDa. Analyses by SignalP3.0and TMHMM2.0revealed that the deduced protein has a putative N-terminal signal peptideof18amino acids, but lacks a transmembrane helix, suggesting it is asecreted form of PGRP-S. Multiple sequence alignment of amphioxusPGRP-S against available short PGRPs showed that like all known short PGRPs, amphioxus PGRP-S includes a PGRP domain at its C-terminus,with the active site H¹¹⁶-Y¹⁵¹-H²²⁵-T²³¹-C²³³required for T7lysozymeamidase activity and Zn²⁺binding. However, it possesses a chitin-binding（ChtBD1） domain at its N-terminus, which is not identified in all knownPGRPs, suggesting that amphioxus PGRP-S is a new member of shortPGRPs. Comparison of the genomic DNA with cDNA sequence showedthat amphioxus PGRP-S gene spans2640bp, and consists of six exonsinterspaced by five introns. For the first two exons in amphioxus PRGP-Sgene, no counterparts with high sequence homology were identified ininvertebrate and vertebrate PGRP-S genes. This is in line with thatamphioxus PGRP-S is a new member of short PGRPs. The expression ofamphioxus pgrp-s is tissue-specific, and it is predominantly expressed inthe hepatic caecum, hind-gut, and muscle. The predominant expression ofpgrp-s in the hepatic caecum and hind-gut provides an additionalevidence supporting that the digestive system is central in the immuneresponse in amphioxus. To determine the functions of amphioxusPRGP-S and the structure-activity relationship between PGRP domainand ChtBD1domain, the recombinant amphioxus PRGP-S mature protein（without signal peptide） rPGRP-S and truncated protein （without ChtBD1domain） rP86/250were expressed. Amphioxus rPGRP-S and rP86/250are both able to bind to Lys-type PGN, Dap-type PGN and chitin.Consistently, rPGRP-S and rP86/250both bind to S. aureus, E. coli, and P. pastoris as well. These indicate that like insect and mammalian PGRPs,amphioxus PGRP-S is a PRR with a broad-spectrum activity, capable ofrecognizing the bacteria S. aureus and E. coli, and the fungus P. pastoris.Additionally, rPGRP-S and rP86/250can degrade both Lys-type andDap-type PGNs, with the former being a preferrential substrate. Thesedata indicated that rPGRP-S and rP86/250are typical amidases, anddeletion of ChtBD1domain has little effect on the amidase activity.Amphioxus rPGRP-S and rP86/250were directly microbicidal, capableof killing both bacteria such as S. aureus and E. coli and fungus like P.pastoris, suggesting that microbicidal activity may be a common featureshared by all chordate PGRPs.The PGRP domain can be divided into three sub-PGRP domains （I,II, and III） based on the conserved homology in both insects andmammals with intervening less conserved sequences. PGRP domains I, II,and III are highly conserved in mammals and are also well conservedbetween insects and mammals. The functional significance of theseconserved regions, however, is not clear. To determine the functions ofthese domains, the recombinant rP120/250（without PGRP domains III）and rP174/250（without PGRP domains III and II） were expressed.rP120/250and rP174/250also showed complete binding to insolublePGNs and chitin，and they are both able to bind to S. aureus, E. coli, andP. pastoris. But its binding activity against S. aureus and P. pastoris was significantly reduced. Both rP120/250and rP174/250had lost enzymaticactivity. This suggested that the complete PGRP domain is required foramidase activity. Deletion of PGRP domain III and II from amphioxusPGRP-S did not affect its antimicrobial activities against E. coli, but itresulted in an abolition decrease in the antimicrobial activity against S.aureus and P. pastoris. These data indicated that an antibacterialmechanism may not be solely responsible for the amphioxus PGRP-Smicrobicidal activity against S. aureus, E. coli, and P. pastoris.