Expression And Function Of Glutathione S-Transferase And Fibrinogen-Related Protein Genes From Amphioxus Branchiostoma Belcheri Tsingtaunese

Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. It is a well-known model organism widely used for interspecies comparative genome studies and developmental homology analysis. Here we obtain amphioxus glutathione S-transferase (BbGST-1) and fibrinogen-related protein (BbFREP) from gut cDNA library, and report the characterization, expression, phylogenetic analysis and functional characterization of them.The cDNA of BbGST-1 obtained from the gut cDNA library of amphioxus B. belcheri is 1032 bp long, and its longest open reading frame codes for a protein of 227 amino acids with a predicted molecular mass of approximately 26 kDa. The BLASTp searching at NCBI shows that the protein encoded by the cDNA has a GST_N domain at residues 3-79 and a GST_C domain at residues 120-204. To investigate the relationship between BbGST-1 and other GSTs, a phylogenetic tree is constructed using the amino acid sequence of BbGST-1 and that of other representative GSTs from 14 cytosolic classes, It is found that BbGST-1 formed a cluster together with the most GSTs from aquatic organisms including fish and alga, being obviously distinct from other cytosolic GST classes. Further comparison with piscine and algal GSTs reveals that BbGST-1 shared 51.1%, 48.9%, 47.4%, 48.4%, 48.0%, 47.6%, 51.8% and 43.7% identity to red sea bream, fathead minnow, gilthead sea bream, plaice, largemouth bass, flounder, zebrafish putative and green algal GSTs, respectively, while it had less than 27% identity to other GST classes. A search of the B. floridae genome revealed the presence of a Florida amphioxus GST-1 cDNA and its genomic DNA sequence. Sequence comparison demonstrated that BbGST-1 shared 93% similarity to the deduced protein encoded by Florida amphioxus GST-1 gene at the amino acid level, suggesting that GST-1 is highly conserved in intra-species. Analysis of the genomic structure exhibited that Florida amphioxus GST-1 gene consisted of six exons and five introns. It was notable that the zebrafish putative GST, rea sea bream GSTR1 and plaice GSTA1 genes all had six exons and five introns, indicating that amphioxus GST1 gene had an exon-intron organization similar to that of fish including zebrafish, sea bream and plaice. An expression vector including the entire open reading frame of BbGST-1 is constructed and transformed into E. coli. Recombinant protein is expressed and purified, shows a relatively high catalytic activity (3.37±0.1 unit/mg) toward CDNB and a moderate activity toward EA (0.41±0.01 unit/mg), but toward DCNB, CHP, 4NBC and 4HNE were not detectable. In situ hybridization histochemistry demonstrates that BbGST-1 transcript was most abundant in the hepatic caecum, and at a lower level present in the endostyle, epipharyngeal groove, hind-gut, gill, ovary and testis, while it is absent in the muscle, neural tube and notochord. This is further corroborated by the immunohistochemical staining using the mouse antisera against the purified recombinant BbGST-1, which shows that BbGST-1 is mainly localized in the hepatic diverticulum, hind-gut, gill and ovary. All these indicate that the amphioxus glutathione S-transferase belongs to a novel rho-class of glutathione S-transferases with a tissue-specific expression pattern. This also suggests that in respect of GST synthesis, the hepatic caecum in amphioxus is similar to the vertebrate liver, supporting the functional equivalence of amphioxus hepatic caecum to the vertebrate liver.The full length of BbFREP cDNA was 1110 bp with a longest open reading frame of 861 bp, which encoded a protein of 286 amino acids with a calculated molecular weight of about 32 kDa. The deduced protein also had a signal sequence of 29 residues and a potential Asn-linked glycosylation site at residual position 148. BLASTp searching at NCBI revealed that BbFREP had a FBG domain at residues 62-281 in its C-terminal region. Multiple sequence alignments revealed that the FBG domain of BbFREP shared 33.7-43.4% identity to that of known FREPs, and had all the four conserved cysteines. The phylogenetic tree constructed by neighbor-joining method using the conserved FBG domains of representative FREPs including BbFREP showed that BbFREP was clustered with human fibrinogenβandγchains, separating from other FREP members. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). These suggest that BbFREP is an immune defense-relevant molecule. The recombinant BbFREP expressed in pichia pastoris is able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displays strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP is also able to bind to both E. coli and S. aureus. In situ hybridization indicates that BbFREP is mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response.