Assignment #2 - The Structure of Insulin

Insulin Structure

Insulin is a small protein with a molecular weight of about 5800 Daltons. As mentioned in the previous blog posting, insulin consists of two peptide chains (A and B) that contain 51 amino acids in total. However, The insulin mRNA, which is coded for by the INS gene, is translated as a single chain precursor called preproinsulin which has 110 amino acids and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin (Bowan,1999). The proinsulin molecule contains 86 amino acids. Proinsulin differs from insulin in that it contains a connecting peptide, or C-peptide, of 35 amino acids (number may very between species) that connects the carboxy terminus of the B-chain to the amino terminus of the A-chain. The C-peptide has basic residues at each end, Arg-Arg at the amino terminus and Lys-Arg at the carboxy terminus. The Arg-Arg and Lys-Arg residues at either ends are the sites of enzymatic cleavage in the conversion of proinsulin to insulin. This occurs in the Golgi complex (Talwar et al., 2006).

To show how insulin can vary slightly from species to species, I prepared an alignment of preproinsulin mRNA sequences from four different species; 2 large mammals, a small mammal and a non-mammal.

Figure 1: The protein sequence alignment of preproinsulin in four different species. (Alignment produced using ClustalW2 software).

In figure 1, the preproinsulin sequence alignments of four species are compared; a *  means that the amino acids in all sequences are exact matches, : means that the sequences are very similar, .   means the sequences are slightly similar, while no symbol means the sequences are completely different. The table below summarizes the similarities between the alignments.

Table 1:  The similarity scores from comparing the alignments of the preproinsulin sequences produced in figure 1. (Table obtained from ClustalW software)

As shown in table 1, the most similar sequences are the cow and boar. This is plausible because cows and boars are both large mammals with similar lifestyles so most of the sequence is conserved. The largest differences come when the mammals are compared to the non-mammalian jungle foul. However, 65% is still a relatively high conservation. The amino acid sequence of insulin is highly conserved among vertebrates, and insulin from one mammal almost certainly is biologically active in another. Even today, many diabetic patients are treated with insulin extracted from pig pancreas (Bowan, 1999). Both the B and A-chain sequences are highly conserved in mammals and birds, with the exception of those found in the hystricomorph rodents (guinea pig, chinchilla, etc.). Even the fish insulins maintain greater than 50% homology with mammalian insulins (Permutt et al.,1984).

Relationship Between Structure and Activity

 Insulin's two chains are connected by two disulfide bridges. One bridge is found at position 7 on both chains and the other bridge is located at position 20 of chain A and position 19 of chain B. The disulfide bridges are essential for biological activity. In the three-dimensional structure of insulin the A-chain terminal residues are on the surface of the molecule. These residues are invariant and play a large role in the stability, conformation, and activity of the insulin molecule and are therefore always conserved. The C-peptide covers this region in proinsulin which explains the inactivity of proinsulin. The A₂₀ Cys and the A₂₁ Asn are always conserved as well. Any modification to these residues will result in the loss of receptor binding ability and of biological activity (Espinal, 1981). There are also several residues in the B-chain that are equally as important such as B₁ and B₂₉. The removal of eight of the C-terminal residues of the B-chain will also reduce the biological activity of insulin (Talwar et al., 2006). The biological activity of insulin is known to be closely related to these eight C-terminal residues (Shanghai Insulin Research Group, 1973).

Figure 2: The double chain structure of insulin. (Blue = A-chain, Yellow= B-chain, Grey= disulphide bonds)

The above dual chain structure and disulphide bridges are conserved in all species that have been studied which indicates that this structure is a functional requirement for the biological activity of insulin (Espinal, 1981).





*References can be accessed through links on pictures and citations.*