Cloning, Expression And Functional Analysis Of A Transferrin-like Homolog In Amphioxus Branchiostoma Belcheri

Transferrin (Tf) is a kind of non-hemeβ-globulin and iron-binding proteins with two iron ions (Fe3+)-binding sites. The structures, functions, sequences and expressions of many kinds of Tfs have been studied in deep. Yet little is known to date about it in the amphioxus Branchiostoma belcheri. Amphioxus or lancelet, a cephalochordate, located at the base 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 transferrin-like genes. In this paper, we report cloning, expression and functional analysis of transferrin-like gene in amphioxus B. belcheri.We have cloned and characterized the cDNA sequence of a transferrin-like molecule in the subphylum Protochordata. The deduced 1256 amino acids long protein, BbTfl, has an N-terminal signal peptide, suggesting it is a secreted protein. This protein contains 2 transferrin conserved domains in its N-terminal and C-terminal region, but there was a large insertion in its C-terminal region. It was different from transferrins from most vertebrates and invertebrates but similar to the MYP of sea urchin which also blongs to transferrin family. The complete N-terminal transferrin domain of BbTfl was compared to the other transferrins revealed that BbTfl shares a lower identity with other Tfs, while 12 cysteine residues in the BbTfl were conserved, indicating that BbTfl had tertiary structures similar to other Tfs. The phylogenetic tree found that BbTfl was clustered together with all the invertebrate Tfs, which was branched from the vertebrate homologs and located at the base of melanotransferrins and other Tfs.Northern blot revealed the presence of an approximate 4000 bp trancript corresponding in size to the full-length BbTfl cDNA we cloned in B. Belchri. In situ hybridization histochemistry demonstrated that BbTfl transcript was most abundant in the hepatic caecum and hind-gut, indicating a tissue-specific expression pattern of BbTfl in adult amphioxus. Challenge with Gram-negative bacteria soon (12 and 24 h) resulted in an up-regulation of BbTfl expression in the guts including the hepatic caecum, suggesting that BbTfl may be a key acute phase protein in amphioxus and involve in the host immune defense.The purified BbTflN was used to test its activities. The recombinant BbTflN has iron-binding activity and bacteriostatic activity. It was observed that BbTflN was capable of significantly inhibiting the growth of E. coli and S. aureus. This clearly shows that BbTfl is an immune-relevant protein capable of inhibiting the growth of Gram-negative bacteria like E. coli and Gram-positive bacteria like S. aureus. Moreover, the bacteriostatic activity of the recombinant BbTflN against E. coli and S. aureus was able to be abolished by iron, thus the iron-binding capacity of the Tf seems to be essential for the inhibition of E. coli and S. aureus. Together these results suggest that transferrin-like iron binding proteins may also be a general part of the insect and cephalochordate immune response by sequestering iron from invading pathogens.