Cloning, Expression And Functional Analysis Of Legumain In Amphioxus Branchiostoma Belcheri

Legumains(C13) (EC3.4.22.34) , or asparaginyl endopeptidases, are a recently identified family of cyteine-class endopeptides. They have been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates, the amphioxus Branchiostoma belcheir. Amphioxus or lancelet, a cephalochordate, has long been regarded as the closest relative of vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of amphioxus legumain gene (BbLGMN).The full-length cDNA of BbLGMN was 1 637 bp long, containing an open reading frame (ORF) of 1 308 bp which encoded a protein of 435 amino acids with a predicted molecular mass of approximately 48.9 kDa . The deduced protein had a signal peptide (15 amino acids) and had a single peptidase_C13 superfamily domain with the conserved KGD (Lys123-Gly124-Asp125) motif and the catalytic dyad His153 and Cys195, which are both characteristic of legumains. It also possessed all the highly conserved cysteines at the residual positions 226, 386, 398, 418 and 435. Alignment analysis showed that B. belcheri legumain shared 36.6%, 45.9%, 46.1%, 49.8%, 49.4%, 47.1% and 48.5% identity to the legumains of S. mansoni, H. longicornis, C. intestinalis, D. rerio, X. laevis, B. taurus and H. sapiens, respectively. The phylogenetic tree constructed using the sequences of representative legumains including that of B. belcheri demonstrated that BbLGMN formed an independent group together with C. intestinalis legumain, which is positioned at the base of vertebrate cluster, suggesting that BbLGMN gene may be the archetype of vertebrate legumain genes.Both rBbLGMN expressed in yeast and endogenous BbLGMN are able to be converted into active protein of ~37 kDa via a C-terminal autocleavage at acid pH values to efficiently degrade the legumain-specific substrate Z-Ala-Ala-Asn-MCA. The endogenous BbLGMN efficiently hydrolyses the legumain-specific substrate at optimum pH5.5. Both autocatalytic activity and the enzymatic activity are inhibited potently by idoacetamide and N-ethylmaleimide, partially by egg white cystatin, but not by E-64, PMSF and pepstain A.In addition, legumain is expressed in vivo in a tissue-specific manner, with main expression in the hepatic caecum and hind-gut of B. belcheri. The hepatic caecum has been thought to be the precursor of liver/pancreas in vertebrates The tissue-specific distribution of BbLGMN therefore suggests that this enzyme may play a role in the degradation of macromolecules in food.Considering that mammalian legumain is involved in processing of the microbial tetanus toxin C fragment for MHCâ…¡antigen presentation in the lysosomal system, we initially approached immune function of BbLGMN. Expression of BbLGMN is up-regulated in the digestive system of B. belcheri challenged with LPS, indicating BbLGMN may be involved in immune defense in B. belcheri, But rBbLGMN expressed in P. pastoris dose not inhibit the growth of E. coli and S. aureus.In summary, this study reports the first characterization of legumain in lower chordates, and demonstration in the hepatic caecum and hind-gut of B. belcheri suggests a function for this enzyme associated with the degradation of food.