Insulin porcine, structure

The treatment of type 1 diabetes is the subcutaneous injection of insulin, as insulin cannot be administered orally because it would be broken down in the stomach due to the low pH. Initially, animal insulin was used in the treatment of diabetes, since bovine and porcine insulin are structurally similar to human insulin. Nowadays, most of the insulin used in the treatment of diabetes is human insulin produced via recombinant DNA (see Ch. 27). There are a number of insulin formulations available, e.g. short-, intermediate- or long-acting and biphasic (a mixture short- and intermediate-acting insulin), and these are described in more detail in Chapter 27. There is a range of therapy protocols indicated, based on the individual condition of the patient. 

Mature insulin consists of two polypeptide chains connected by two interchain disulfide linkages. The A-chain contains 21 amino acids, whereas the larger B-chain is composed of 30 residues. Insulins from various species conform to this basic structure, while varying slightly in their amino acid sequence. Porcine insulin (5777 Da) varies from the human form (5807 Da) by a single amino acid, whereas bovine insulin (5733 Da) differs by three residues. 

Fig. 3. Refolding model of insulin protofilaments, from Jimenez et al. (2002). (A) Ribbon diagram of the crystal structure of porcine insulin (PDB ID code 3INS), generated with Pymol (DeLano, 2002). The two chains are shown as dark and light gray with N- and C-termini indicated. The dotted lines represent the three disulfide bonds 1 is the intrachain and 2 and 3 are the interchain bonds. (B) Cartoon representation of the structure of monomeric insulin in the fibril, as proposed by Jimenez et al. (2002). The thick, arrowed lines represent /1-strands, and thinner lines show the remaining sequence. The disulfide bonds are as represented in panel A, and N- and C-termini are indicated. (Components of this panel are not to scale.) (C) Cartoon representation of an insulin protofilament, showing a monomer inside. The monomers are proposed to stack with a slight twist to form two continuous /(-sheets. (Components of this panel, including the protofilament twist, are not to scale.) In the fibril cross sections presented byjimenez et al. (2002), two, four, or six protofilaments are proposed to associate to form the amyloid-like fibrils.

There is a tendency to reserve semisynthetic and totally synthetic methods for the introduction of bonds and residues that cannot be specified by the genetic code. The present chapter will concentrate on these aspects. However, semisynthesis can have a role to play even when building structures that are completely accessible to the genetic code. The first industrial challenge for the emerging technologies of total chemical synthesis, recombinant protein expression, and semisynthesis was the economic production of human insulin in pharmaceutically usable quantity and quality. The semisynthetic human insulin that was made from porcine insulin proved exceptionally convenient to produce, and was the first introduction to human insulin for very many patients. 

Insulin is a polypeptide hormone that consists of two peptide chains bonded by two disulfide bonds. The two chains are designated A and B. The A chain consists of 21 amino acids with a third internal disulfide bond, and the chain contains the remaining 30 amino acids. All vertebrates produce insulin and the structure is similar in these species. For example, the insulin produced in humans and porcine species differs by only one amino acid, and humans and bovine insulin differ by three amino acids. Insulin plays a crucial role in several physiological processes. These include the regulation of sugar in the body, fatty acid synthesis, formation of triglycerides, and amino acid synthesis. 

Structures of human proinsulin and insulin. Insulin is derived from proinsulin by cleavage at the dipeptides Arg-Arg and Lys-Arg to give A and B chains held together by disulfide bonds. In the pig, B30 is Ala. In the cow, A8 is Ala, AlO is Val, and B30 is Ala. Bovine and porcine insulins are used extensively in clinical practice. 

Comparative studies on proteins from different species show that the structures are essentially the same despite different crystallisation conditions. Examples include sperm whale and seal myoglobin, horse and human haemoglobin, horse, tuna, bonito and rice cytochrome c, hen egg white, tortoise egg white and human lysozyme, horse and yeast phosphoglycerate kinase, porcine and hagfish insulin and lobster and Bacillus stearothermophilus glyceraldehyde 3-phosphate dehydrogenase. Coordinates for these proteins are held in the Protein Data Bank [151]. 

Species variations in primary stmctnre are also important in medicine, as illustrated by the comparison of human, beef, and pork insulin. Insulin is one of the hormones that are highly conserved between species, with very few amino acid substitutions and none in the regions that affect activity. Insulin is a polypeptide hormone of 51 amino acids that is composed of two polypeptide chains (Fig. 6.13). It is synthesized as a single polypeptide chain, but is cleaved in three places before secretion to form the C peptide and the active insulin molecule containing the A and B chains. The folding of the A and B chains into the correct three-dimensional structure is promoted by the presence of one intrachain and two interchain disulfide bonds formed by cysteine residues. The invariant residues consist of the cysteine residues engaged in disulfide bonds and the residues that form the surface of the insulin molecule that binds to the insulin receptor. The amino acid substitutions in bovine and porcine insulin (shown in blue in Fig. 6.13.) are not in amino acids that affect its activity. Consequently, bovine and pork insulin were used for many years for the treatment of diabetes mellitus. However, even with only a few different amino acids, some patients developed an immune response to these insulins. 

Fig. 6.13. The primary structure of human insuhn. The substituted amino acids in bovine (beef) and porcine (pork) insulin are shown in blue. Threonine 30 at the carboxy terminal of the B chain is replaced by alanine in both beef and pork insulin. In beef insulin, threonine 8 on the A chain is also replaced with alanine, and isoleucine 10 with vahne. The cysteine residues, which form the disulfide bonds holding the chains together, are invariant. In the bioengineered insulin Humalog (hspro insulin), the position of proline at B28 and lysine at B29 is switched. Insulin is synthesized as a longer precursor molecule, proinsulin, which is one polpeptide chain. Proinsulin is converted to insulin by proteolytic cleavage of certain peptide bonds (squiggly lines in the figure). The cleavage removes a few amino acids and the 31-amino acid C-peptide that connects the A and B chains. The active insulin molecule, thus, has two nonidentical chains.

Fig. 2. Three-dimensional structural representations for zinc metall-oproteins. Comparison of the zinc ion-protein bonding interactions for zinc requiring enzymes (A—C) with the zinc-insulin hexamer (D, E). (A) Human carbonic anhydrase C, redrawn from Ref. (47) with permission. (B) Bovine carboxypeptidase Ay, redrawn from Ref. 30) with permission. (C) Bacillus thermoprotedyticus thermolysin, redrawn from Ref. 45) with permission. (D) and (E) Porcine Zn-insulin hexamer, taken from Ref. 48) with permission. The composite electron density maps in (D) and (E) show that each of the two zinc atoms present in the hexamer is within inner sphere bonding distance of three solvent molecules and three histidyl imidazolyl groups in an octahedral array about the metal ion. The position of one of the three equivalently positioned solvent molecules is indicated in (D). The electron density map in (E) shows the relative orientations of the three histidyl residues (His-BlO). (The atomic positions of one of the three equivalent histidyl groups are shown)...

X-ray, circular dichroism, and centrifuge studies have demonstrated that most mammalian and fish insulins form zinc insulin hexamers similar to those of porcine insulin (Blundell et al, 1972 Blundell and Wood, 1975), with the exception of hagfish insulin, which produces only dimers (Cutfield et al, 1974 Peterson et al, 1974), and guinea pig (Zimmerman et al, 1972), casiragua (Horuk et al, 1979), and porcupine (Horuk et al, 1980) insulins, which exist only as monomers. A complete structural analysis of hagfish insulin dimers shows that, unlike porcine insulin, the two molecules of the dimer in the crystals are exactly equivalent and resemble molecule II of the asymmetric dimer of porcine insulin. This is similar to the structure of porcine insulin in solution as indicated by circular dichroism studies (Wood et al, 1975 Strickland and Mercola, 1976). 

Relaxin is a peptide hormone that is synthesized and stored in the corpus luteum and is responsible for the relaxation of the pubic symphysis in mammals prior to parturition. Porcine relaxin (MW 5600) is composed of an A and a B chain linked by disulfide bonds, and its amino acid sequence is consistent with these links having the same disposition as those in insulin (Schwabe et al, 1976 1977 Kwok et a/., 1977) (see Table I). Using its sequence homology with insulin, Bedarkar et al (1977) postulated a three-dimensional structure for relaxin with the crystal structure of insulin as a basis (Fig. 6). A similar model has been proposed by Isaacs et al (1978). 

The well known specificity of proteinases implies the use of specific amino acids (amides, esters) as acyl donors and—seldom—specific amino acid (derivatives) as acceptors in enzymatic peptide bond formation, since the same structural features of RCONHR that influence the rate of hydrolytic cleavage are also involved in the synthesis. Accordingly trypsin is well suited to the formation of a new -Arg-X or -Lys-X bond. As an example the transformation of the -Lys-AlaOH terminus of the B-chain of porcine insulin into -LysThrOBu of human insuhn may be mentioned. C-terminal Ala was removed by means of car-boxypeptidase A, trypsin-catalyzed condensation of the des-alanine peptide with threonine tert. butylester gave 73% of the ester of human insulin [33] (see also... 

The enzymatic synthesis of peptides (Scheme 6.24) from which proteins can be constructed is not so limited, and chemical synthesis has an even wider application, but these are not yet suitable techniques for manufacture. Moreover, there are no general methods for building the peptides into full protein structures. Nevertheless, enzymes do have a role in the manufacture of peptides themselves. In a mixture of butan-l,4-diol and water, trypsin will catalyse the exchange of the carboxy-terminal alanine of porcine insulin with threonine t-butyl ester (Scheme 6.25). The reaction is essentially a transpeptidation in which the acyl group of lysine is transferred from one amino group on alanine to another on the threonine. This converts porcine insulin into the ester of the human hormone, and a simple deprotection yields one of the commercial products. 

Balschmidt, P., Hansen, F. B., Dodson, E. J., Dodson, G. G., and Korber, F., 1991, Structure of porcine insulin cocrystallized with chipeine ZActa Crysttdlogr. 847 975 986. 

Chronic Diseases. Xenotransplantation has the potential to treat many chronic diseases that cause cell death. For example, in diabetes, pancreatic cells that produce insuhn are destroyed. Encapsulation is being tested as a means of introducing porcine islet cells (pancreatic cell structures from pigs) into human patients with diabetes. The encapsulated porcine cells help these patients produce the insulin that they would otherwise have to inject into themselves. 

Progress in the Insulin field has been rapid in these two years, particularly in the axea, of the blos thesis of the hormone. It is now well established that Insulin, which has two peptide chains (A, 21 amino acids and B, 30 amino acids) cross-linked by two disulfide bridges, is synthesized as a single peptide chain, prolnsulin. in which the A and B chains of inmUn are connected by a "connecting peptide" (C-peptide) chain of 33 (porcine) or 30 (bovine) amino acids. Work on prolnsulin has be reviewed, 1, 10 and the amino acid sequences of bovine and porcine proinsulins have been published. Hie amino acid composition of cod proinsulin has a o appeared Two different proinsulins have been demonstrated in the rat, 8 aod proinsulin has been Isolated from human islet cell tumor tissue cultures. The structures of porcine and bovine prolnsulins are as follows ... 

Porcine islets are at present receiving the greatest attention since pigs produce an insulin which is structurally very similar to human insulin and pigs are, on the other hand, the only large animals slaughtered in sufficient quantities to supply the estimated demand from type I... 

Insulin is produced in the islets of Langerhans in the pancreas. The name comes from the Latin insula meaning island. Its structure varies slightly between species porcine insulin is especially similar to the human version. Insulin is produced and stored in the body as ahexamer [a unit of six insulin molecules, connected by Zn ions (Figure 3.8), while the active form is the monomer. The hexamer is an inactive form with long-term stability, which serves as a way to keep the highly reactive insulin protected, yet readily available. 

An aqueous extract of RJ produced hypoglycemia when injected into larvae of Manduca sexta. The application of specific radioimmunoassay to the partially purified extract showed that RJ contains several insulin-like peptides, the major immunoreactive component of which had an apparent mol. wt similar to that of bovine insulin. These results suggested the existence of a peptide in the honeybee having both biological and structural similarities to vertebrate insulin [90]. Partially purified extracts from honeybees (Apis mellifera), and extracts from their separated heads, cross-reacted in a porcine insulin radioimmunoassay. The active extracts displaced porcine insulin from rat liver insulin receptors and showed insulin-like activity with rat adipocytes that could be abolished with bovine insulin antiserum. The presence of an insulin... 

Porcine insulin differs from human insulin by one amino acid having alanine instead of threonine at B-chain 30 (see Fig. 27.3). The difference in structure from human insulin is not sufficient for antibody production to occur. 

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