title: Evolution of insulin-like growth factor (IGF) function: production and characterization of recombinant hagfish IGF.
abstract: While there is considerable structural evidence that insulin-like growth factors (IGFs) share a long evolutionary history, little is known about the conservation of IGF function. In order to address this, we have made recombinant hagfish IGF, hence allowing characterization of an IGF from a representative of the primitive vertebrate class, Agnatha. The production of recombinant hagfish IGF has been complicated by a number of factors including the requirement of a longer leader peptide for fusion protein expression, reduced solubility of the protein, as well as problems in the refolding procedure. However, we were able to produce a small quantity of hagfish IGF with an N-terminal glycine addition which is biologically active. Furthermore, N-terminal amino acid sequencing and mass spectrometry confirm that we have produced hagfish IGF. In vitro assessment of recombinant hagfish IGF in cultured cells indicates that hagfish IGF indeed shares functional properties with mammalian IGFs. Thus, hagfish IGF stimulates protein synthesis in rat myoblasts, but 20- and 5-fold more peptide, respectively, is required to achieve the same half-maximal responses as with human IGF-I (hIGF-I) or IGF-II (hIGF-II). Hagfish IGF also competes for binding to the type-1 IGF receptor present both on rat myoblasts and on salmon embryo fibroblasts, though with somewhat lower affinity than either hIGF-I or hIGF-II. However, studies investigating binding to the IGF-II-specific type-2 receptor suggest that hagfish IGF may in fact be more closely related to IGF-I than to IGF-II. These results indicate that motifs important for functions associated with mammalian IGFs appear to have evolved prior to the Agnathans diverging from the main line of vertebrate evolution 550 million years ago. Accordingly, we now have functional as well as structural evidence that the IGFs have a long evolutionary history.
authors.affiliation: Cooperative Research Centre for Tissue Growth and Repair, University of Adelaide, Adelaide, South Australia, 5000, Australia. zeeu@dhn.csiro.au
authors.forename: Z
authors.initials: Z
authors.lastname: Upton
authors.forename: G L
authors.initials: GL
authors.lastname: Francis
authors.forename: S J
authors.initials: SJ
authors.lastname: Chan
authors.forename: D F
authors.initials: DF
authors.lastname: Steiner
authors.forename: J C
authors.initials: JC
authors.lastname: Wallace
authors.forename: F J
authors.initials: FJ
authors.lastname: Ballard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D015870:Gene Expression / Q000235:genetics*; D006196:Hagfishes / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000032:analysis / Q000378:metabolism; D007457:Iodine Radioisotopes; D007930:Leucine / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D016415:Sequence Alignment; D012756:Sheep; D013002:Somatomedins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D014316:Tritium
chemical_list: D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins; D013002:Somatomedins; D014316:Tritium; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D007930:Leucine