Cloning, Expression And Antibacterial Activity Of A Tachylectin-related Homolog In Amphioxus Branchiostoma Belcheri

The novel group of lectins identified recently, tachylectin-related proteins, have also been demonstrated to function in innate immunity from sponge to bony fish, yet little is known to date about it in the amphioxus Branchiostoma belcheri. 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 the course of expressed sequence tag (EST) generation from the gut cDNA library of adult amphioxus Branchiostoma belcheri, we isolated a gene fragment exhibiting identity to tachylectin-related genes. The purposes of this study were thus to examine the expression pattern of the tachylectin-related gene in adult amphioxus, to test its expression regulation in response to challenge with lipopolysaccharide (LPS) and to assay its antibacterial activity.We have cloned and characterized the first cDNA of a tachylectin-related molecule in the subphylum Protochordata. The deduced 305 amino acids long protein, BbTL, has an N-terminal signal peptide, suggesting it is a secreted protein. This protein contains 6 tandem repeats all with the consensus sequences, GXXWXpI(V)X GXLK and WGVNSNDpIY(F), that are characteristic of tachylectins and their related proteins. The inter-repeat stretches of BbTL, being all 11 amino acids long, also exhibit striking similarity to that of tachylectin-related proteins like Hydractinia CTRN. Alignment of BbTL with tachylectins, tectonins and other related proteins reveals that the core of BbTL is well conserved. Both sequence comparison and phylogenetic analysis show that BbTL is most similar to CTRN, to horseshoe crab tachylectins, to a sponge lectin and to slime mold tectonins, but is more divergent from carp egg lectin and zebrafish galactose-binding protein. Northern blotting revealed the presence of an approximately 1200 bp trancript corresponding in size to the full-length BbTL cDNA in B. Belchri. In situ hybridization histochemistry demonstrated that BbTL transcript was most abundant in the hepatic caecum and hind-gut,indicating a tissue-specific expression pattern of BbTL in adult amphioxus.Challenge with LPS soon resulted in an up-regulation of BbTL expression in the guts including the hepatic caecum and hind-gut. In contrast, little BbTL expression change was observed in the gut-free body following LPS challenge. The fact that challenge with LPS resulted in an elevation of BbTL expression in the digestive system but not in the gut-free body suggests that BbTL is expressed in a tissue-specific manner. This is further supported by in situ hybridization histochemistry, which definitely shows that BbTL is mainly expressed in the hepatic caecum and hind-gut in B. belcheri. This denotes that amphioxus digestive system rich in mucosa is the major immune tissue responding to LPS challenge.The purified BbTL was used to test its antibacterial activity against E. coli and S. aureus by colony forming unit assay. It was observed that the recombinant BbTL expressed in P. pastoris clearly inhibits the growth of Gram-negative bacterium E. coli, and the inhibition was in a dose-dependent manner.This clearly shows that BbTL is a immune-relevant protein capable of inhibiting the growth of Gram-negative bacteria like E. coli.All these indicate that BbTL is involved in the host immune defense, agreeing with the consensus idea that tachylectins and related proteins are immunity-relevant molecules. Moreover, the quick elevation of BbTL expression following LPS challenge suggests that BbTL may be a candidate of acute phase proteins. It is highly likely that BbTL is synthesized in the digestive system, secreted in the blood, and circulated via the bloodstream, and functions as an immune molecule throughout the amphioxus body.