EVOLUTION OF INSULIN-LIKE GROWTH-FACTOR (IGF) FUNCTION - PRODUCTION AND CHARACTERIZATION OF RECOMBINANT HAGFISH IGF

While there is considerable structural evidence that insulin-like grow th factors (IGFs) share a long evolutionary history, little is known a bout the conservation of IGF function. In order to address this, we ha ve 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 n umber of factors including the requirement of a longer leader peptide for fusion protein expression, reduced solubility of the protein, as w ell as problems in the refolding procedure. However, we were able to p roduce a small quantity of hagfish IGF with an N-terminal glycine addi tion which is biologically active. Furthermore, N-terminal amino acid sequencing and mass spectrometry confirm that we have produced hagfish IGE In vitro assessment of recombinant hagfish IGF in cultured cells indicates that hagfish IGF indeed shares functional properties with ma mmalian IGFs. Thus, hagfish IGF stimulates protein synthesis in rat my oblasts, 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 -I IGF receptor present both on rat myoblasts and on salmon embryo fib roblasts, though with somewhat lower affinity than either hIGF-I or hI GF-II. However, studies investigating binding to the IGF-II-specific t ype-2 receptor suggest that hagfish IGF may in fact be more closely re lated to IGF-I than to IGF-II. These results indicate that motifs impo rtant for functions associated with mammalian IGFs appear to have evol ved prior to the Agnathans diverging from the main line of vertebrate evolution 550 million years ago. Accordingly, we now have functional a s well as structural evidence that the IGFs have a long evolutionary h istory. (C) 1997 Academic Press