Expression And Function Of Plasminogen-like And Insulin-like Growth Factor Genes From Amphioxus Branchiostoma Japonicum

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 plasminogen-like gene (BbPlgl) and report the characterization, expression, phylogenetic analysis and functional characterization of it. Moreover, in order to further demonstrate the origin of GH/IGF axis, we report the recombinant expression of the mature peptide of the amphioxus insulin-like growth factor gene (BbIGF) and functional characterization of it.In this study, we isolated a cDNA, designated BbPlgl, which was 1949 bp long with an opening reading frame (ORF) of 1290 bp, a 5'-untranslated region (UTR) of 123 bp and a 3'-UTR of 536 bp. The ORF of BbPlgl encoded a polypeptide of 430 amino acids with a calculated molecular weight of about 48 kDa. SignalP software analysis by the Signal IP 3.0 server revealed that BbPgl had a putative N-terminal signal peptide of 16 amino acids. There was a potential N-linked glycosylation site in BbPlgl located at the residual position 32. The BLASTp searching at NCBI shows that BbPlgl was characterized structurally by the presence of two kringle domains in the N-terminus and a serine protease domain in the C-terminus with a novel domain structure of K-K-SP. Phylogenetic analysis shows that kringles 1 and 2 of BbPlgl are grouped with two more ancient clusters, groupⅠand groupⅡ, respectively, and SP region of BbPlgl is positioned at the base of the subfamily containing Pig, HGF, MSP and APOA, suggesting that BbPlgl may represent the archetype of this subfamily members. Both the kringle domains K1 and K2 in BbPlgl comprised 79 amino acids, sharing an identity of 51.9% to each other. Moreover, the K1 and K2 in BbPlgl were both closely identical to Kl (43.0～58.2% and 41.8～51.9%), K2 (41.8～51.9% and 50.6～60.9%), K3 (49.4～57.0% and 51.9～63.3%), K4 (50.6～55.7% and 46.8～55.7%) and K5 (50.6-57.0% and 44.3～50.6%) of known Pigs. An expression vector including the entire ORF of BbPlgl and a 5'additional tag of pET28a was constructed and transformed into E. coli, which resulted in the original N-terminal Met in the recombinant protein replaced by Met-Gly-Ser-Ser-(His)6-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met. The recombinant protein BbPlgl was purified by affinity chromatography on a Ni-NTA resin column. The purified recombinant BbPlgl with the His6 tag yielded a single band of approximately 48 kDa on SDS-PAGE gel after Coomassie blue staining. Western blotting showed that the mouse anti-human His-antibody (diluted 1:1000) reacted with the inclusion bodies of IPTG-induced E. coli BL21 with expression vector, forming a band of approximately 48 kDa on SDS-PAGE, corresponding to the molecular mass predicted by BbPlgl cDNA. The refolded BbPlgl was readily activated by human uPA and exhibited an enzymatic activity of 8.7 mU/mg protein. Besides, the catalytic activity was enhanced when the amount of the recombinant protein was increased while both human uPA and chromogenic substrate were in fixed quantities, showing that BbPlgl acts in a concentration-dependent manner. Moreover, the refolded BbPlgl was able to auto-activate at neutral and alkaline pH at 4℃without the addition of uPA, displaying an enzymatic activity of 6.5 mU/mg protein toward the chromogenic substrate (p<0.01). However, when human uPA was added, the activation was accelerated (p<0.05), indicating that human uPA can interact with BbPlgl, resulting in its activation. Northern blotting revealed the presence of an approximately 2000 bp transcript in B. belcheri. In situ hybridization histochemistry demonstrated that BbPlgl transcript was most abundant in the hepatic caecum and hind-gut, and at a lower level present in the gill and ovary, while it was absent in the muscle, neural tube, notochord and testis (Fig.8), implicating a tissue-specific expression pattern of BbPlgl in adult B. belcheri. This also suggests that in respect of Pig synthesis, the hepatic caecum in amphioxus is similar to the vertebrate liver, supporting the functional equivalence of amphioxus hepatic caecum to the vertebrate liver.In order to further discuss the functions and mechanisms of the BbIGF and demonstrate GH/IGF axis in amphioxus Branchiostoma belcheri, we constructed an expression vector including the cDNA coding for mature peptide of BbIGF and 5' additional tags of pET28a, then transformed into E. coli cells. The recombinant peptide was induced by IPTG, purified by affinity chromatography on a Ni-NTA resin column and characterized by MALDI TOF/TOF MS analysis. Meanwhile, we isolated the muscle cells from the new-borned Kunming mice and cultured for more than 10 days. The muscle lineage of the cells was demonstrated by the detection of Pax7, myoD and myogenin using RT-PCR. We employed the primary culture of the muscle cells as the in vitro model to reach the following aims:1) to identify if the BbIGF-MP had the abilities of binding to the cells and the partially purified IGF-I receptors as the human IGF-I does,2) to determine if the BbIGF-MP could act as a mitogen, stimulating the prliferation of the cells as the human IGF-I does, and 3) to identify components of the intracellular signaling cascades of BbIGF-MP and IGF-I in the muscle cells and to demonstrate their activation in response to both peptides throughout the in vitro growth and differentiation of muscle cells. The binding assay showed that the FITC-labeled BbIGF-MP could bind to the muscle cells as the human IGF-I does and the enzyme-linked immunosorbent assay (ELISA) confirmed its binding ability to the partially purified IGF-I receptors of mouse muscle and amphioxus as the human IGF-I. MTT Assay showed that BbIGF-MP could stimulate the proliferation of the mouse muscle cells at the concentration of 10 p.g/ml and acts in a concentration-dependent manner, displaying the mitogenic effect as the human IGF-I did. Western blotting revealed that the BbIGF-MP could activate different intracellular signal transduction pathways in mouse skeletal muscle cells depending on the stage of cell culture development as the human IGF-I does. In differentiating and proliferating myoblasts, both the MAPK and Akt pathways are activated by BbIGF-MP and human IGF-I, whereas in fully differentiated myotubes the effects of the peptides were lower than in myoblasts. Our study further confirmed BbIGF is functionally more related to IGF and acts in the similar mechanism to the human IGF-I. Vertebrate IGFs have been shown to be expressed in various tissues but, in all cases, liver is the primary organ producing IGFs. The liver is an important target organ of many hormones in the endocrine system and the hepatic caecum of the cephalochordate amphioxus has long been considered to be the precursor of vertebrate liver. Our results showed the similarity of the functions and mechanisms between BbIGF-MP and human IGF-I, moreover the BbIGF was specifically expressed in the hepatic caecum and hind-gut, further supporting the functional equivalence of amphioxus hepatic caecum to the vertebrate liver, 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.