Refolding of Amphioxus Insulin-like Peptide:  Implications of a Bifurcating Evolution of the Different Folding Behavior of Insulin and Insulin-like Growth Factor 1

Abstract

Insulin and insulin-like growth factor 1 (IGF-1) share high sequence homology, but their folding behaviors are significantly different: insulin folds into one unique thermodynamically controlled structure, while IGF-1 folds into two thermodynamically controlled disulfide isomers. However, the origin of their different folding behaviors is still elusive. The amphioxus insulin-like peptide (ILP) is thought to be the common ancestor of insulin and IGF-1. A recombinant single-chain ILP has been expressed previously, and now its folding behavior is investigated. The folding behavior of ILP shows the characteristics of both insulin and IGF-1. On one hand, two thermodynamically controlled disulfide isomers of ILP have been identified; on the other hand, the content of isomer 1 (its disulfides are deduced identical to those of swap IGF-1) is much less than that of isomer 2 (its disulfides are deduced identical to those of native IGF-1); that is, more than 96% of ILP folds into the native structure. The present results suggest that the different folding behaviors of insulin and IGF-1 are acquired through a bifurcating evolution: the tendency of forming the thermodynamically controlled non-native disulfide isomer is diminished during evolution from ILP to insulin, while this tendency is amplified during evolution from ILP to IGF-1. Moreover, the N-terminal Gln residue of ILP can spontaneously form a pyroglutamate residue, and its cyclization has a significant effect on the folding behavior of ILP: the percentage of isomer 1 is approximately 2-fold that of isomer 1 of the noncyclized ILP; that is, isomer 1 becomes more favored when the N-terminal residue of ILP is cyclized. So, we deduce that the N-terminal residues have a significant effect on the folding properties of insulin, IGF-1, and ILP.In the 1960s Anfinsen and co-workers first demonstrated the three-dimensional structure of a globular protein is uniquely determined by its amino acid sequence (1). Since then, significant advances have been made in the understanding of protein folding through experimental and theoretical approaches. For small proteins with two-state folding, topology is a major determinant of the folding rate and greatly influences the structure of the transition-state ensemble (2-4). Studies on the disulfide-coupled folding of some small globular proteins, such as BPTI, 1 RNase A, and EGF, have revealed a sequence of preferred kinetic intermediates which define a folding pathway (5-12). In vivo the protein folding is assisted by the molecular chaperones, especially for large proteins (13-15); some chaperones even can provide the missing steric information for protein folding (16).Insulin is an extensively studied small globular protein with A-and B-chains linked by three disulfides (one intrachain bond, A6-A11; two interchain bonds, A7-B7 and A20-B19). Its three-dimensional structure has been well solved by X-ray crystallography (17,(18)(19)(20)(21) since the 1970s. Although the separate A-and B-chains of insulin can be recombined success...

Sequences of B-Chain/Domain 1−10/1−9 of Insulin and Insulin-like Growth Factor 1 Determine Their Different Folding Behavior

“…So the bifurcating folding ability is an intrinsic property of the IGF-1 molecule. Recently, we revealed that the bifurcating folding behavior of IGF-1 was evolved from aILP (35), which has been thought to be the common ancestor of insulin and IGF-1 (13,14). Our current investigation further suggests that the bifurcating folding behavior of IGF-1 has evolved from mutations in the N-terminal residues 1-9 of B-domain.…”

Identification, Evolution and Expression of an Insulin-Like Peptide in the Cephalochordate Branchiostoma lanceolatum

“…1C suggests that amphioxus ILPs represent in fact the sister group (that is, an unduplicated version) of both the vertebrate insulins and vertebrate IGFs. Interestingly, amphioxus ILPs show structural and folding characteristics of both insulin and IGF [ 1 , 18 ] suggesting that subfunctionalization may have occurred after duplication in vertebrates even if functional data are still missing to substantiate this point.…”

“…Amphioxus ILP sequences display structural characteristics of both insulin and IGF sequences from vertebrates: sequence analyses suggest that amphioxus ILP is proteolytically cleaved like proinsulin, releasing the C-terminal peptide but it also contains extended D and E domains as found in vertebrate IGFs [ 3 , 15 , 18 ]. It has been shown that in adult B .…”

The Role of Insulin-Like Growth Factor Binding Proteins in Development

“…It is tempting to speculate that IGFBPs could have evolved with a non-IGF related function but that the presence of IGFBPs in some way aided the evolutionary separation of insulin and IGF peptides. This notion is supported by the observation that insulin folds into one thermodynamically controlled structure whereas IGF-I folds into two disulphide isomers, with an Amphioxus ILP exhibiting intermediate folding behaviour [10]; importantly, the N-terminal residues of these peptides have a significant role in determining folding properties and therefore ability to associate IGFBPs (which interact with the N-terminal) could have influenced co-evolution of insulin and IGFs.…”