Demonstration Of Growth Hormone (GH)/insulin-like Growth Factor (IGF) Axis In Amphioxus Branchiostoma Belcheri: Cloning, Expression, Function Of IGF And Its Up-regulation By GH

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 well-known model organism widely used for interspecies comparative genome studies and developmental homology analysis. Sequencing of the whole genome of Florida amphioxus offers a new opportunity to the research of molecular and evolutionary biology of amphioxus. In this paper, we report the cloning, expression, function of amphioxus IGF and its up-regulation by GH, demonstrating GH/ IGF axis in amphioxus Branchiostoma belcheri.The GH/IGF axis is unique to all vertebrate species, but its evolutionary origin is ill-defined. In this study, we have cloned and characterized the cDNA of an IGF polypeptide in amphioxus B. belcheri. The cDNA obtained was 1630 bp long with an open reading frame (ORF) of 924 bp, a 5′-untranslated region (UTR) of 23 bp and a 3′-UTR of 683 bp. The ORF encoded a polypeptide of 307 amino acids with a molecular mass of approximately 35 kDa. The deduced long polypeptide, BbIGF, has an IGF_insulin_like domain at residual positions 23-101. The first one-third (residues 1-101) of the polypeptide was organized much like a typical preproinsulin with a signal peptide (24 residues), B chain, C-peptide and A chain, whereas its remaining sequence (residues 102-307) could be divided into putative D and E domains reminiscent of proIGF. Six conserved cysteine residues required for the formation of inter- and intra-chain disulphide bonds were identified in the B and A domains of BbIGF. Southern blotting demonstrates that BbIGF is a single copy gene, which is in line with that of B. californiensis insulin/IGF gene. Northern blotting revealed the presence of a single band of about 2.1 kb transcript in B. belcheri. In situ hybridization histochemistry showed that BbIGF transcript was specifically expressed in the hepatic caecum and hind-gut. This was further corroborated by the immunohistochemical staining, showing that BbIGF was localized in the cytoplasm of hepatic caecum and hind-gut. This basically agrees with the hypothesis that amphioxus hepatic caecum is homologous to the vertebrate liver in respect to IGF expression pattern. Also, the presence of BbIGF in the humoral fluids, as evidenced by immunoblotting, suggests that the peptide synthesized in the digestive system can be secreted into the blood, circulating via blood stream throughout the body.An expression vector including the cDNA coding for mature BbIGF and 5'additional tags of pET32a was constructed and transformed into E. coli cells. The recombinant peptide was induced by IPTG and purified by affinity chromatography on a Ni-NTA resin column. The mitogenic effect of BbIGF was examined by measuring the growth rate of FG-9307 cells via MTT method. The recombinant BbIGF expressed in vitro was capable of stimulating the flounder gill cell proliferation. Unexpectedly, BbIGF was not able to reduce blood glucose level, suggesting that it had little insulin activity. This suggests that BbIGF is functionally more related to IGF rather than insulin.The hepatic caecums were dissected out of amphioxus B. belcheri, cut into pieces (1 mm3) and cultured in the medium plus 100 ng/ml and 0 ng/ml (control) of the rat recombinant GH. Quantitative real-time PCR demonstrated that exogenous recombinant rat GH was able to induce a significant up-regulation of BbIGF expression in the hepatic caecum. Moreover, GHR was an integral membrane protein in the vertebrate liver, and RIPA was a suitable buffer for the extraction of this molecule. Western blotting revealed the presence of a membrane protein molecule similar to rat growth hormone receptor in the hepatic caecum. These apparently establish a definitive link between GH and IGF in B. belcheri, pushing the evolutionary origin of GH/IGF axis to the primitive chordate. These also pave the way for further characterization of the GH/IGF axis in the protochordate.