Biochemistry

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A. Biosynthesis of Hormones

1. Biosynthesis of Insulin a. Identification of Proinsulin

For years one of the intriguing problems relating to insulin biosynthesis was that of describing the independent synthesis of the separate A and B chains followed by the correct formation of the three disulfide linkages. It was not at all clear whether these formed spontaneously on the basis of peptide chain folding, conformation, and entropic considerations or whether there were specialized "biological mechanisms" that mediated this precise process. The discovery of proinsulin by Steiner and colleagues in 1967 provided a resolution to this knotty problem.

Proinsulin is a precursor form of insulin that exists as a single-chain polypeptide of ~9000 Da; this chain contains, within its sequence, the 21 amino acid residues of the A chain and the 30 residues of the B chain of

3 Leptin is derived from the Greek root leptos, meaning thin.

insulin. In addition, it contains a connecting sequence, known as the C peptide, which falls between the N-terminus of the A chain and the C-terminus of the B chain (see Figure 7-14). It is now clear that proinsulin represents an intermediate species that appears on the biosynthetic pathway, leading from the immediate polysomal translation product to the final granule stored form of insulin. In the case of proinsulin, the excised C peptide is stored in the secretory granule along with mature insulin.

Mammalian proinsulins contain from 76 (hagfish) to 89 (angler fish) amino acid residues; these variations in amino acid units reflect only differences in the length of the connecting C peptide between the A and B chains. Theoretically it is not necessary to have a connecting sequence of 25-38 amino acid residues; from model-building exercises related to the tertiary structure of insulin (Figure 7-9), it appears that only 5-8 residues would be minimally required. The additional residues of the C peptide do not completely "mask" the biological activity of the molecule, since proinsulin contains 3-5% of the biological activity of native insulin.

Both the prospective N- and C-termini of the C peptide of all known mammalian proinsulins end in a pair of basic amino acid residues (either lysine or arginine). These represent the recognition signals for proteolytic cleavage that are necessary for the conversion of proinsulin to insulin (see Figure 7-15). As a consequence of extensive molecular biological studies, two new endo-peptidases PC-2 and PC-3, which are specifically involved in proinsulin processing, were identified. PC-2 and PC-3 are members of a family of subtilisin-related proprotein convertases. PC-2 and PC-3 are also known to be involved in the processing of the proopiomelanocortin precursor (see Figure 1-9B).

The amino acid composition of the C peptide is known for fifteen mammalian and one avian species. It is apparent that there is a 15 X higher rate of mutation in the C peptide domain than in the A or B chains of the related insulins; this is consistent with the hypothesis that the C peptide does not have any extrapancreatic hormonal function. The C peptide is secreted in equi-

figure 7-14 Amino acid sequence of human proinsulin. Dashed circles indicate sites of peptidase cleavage so as to generate the mature insulin and free C peptide. [Modified with permission from Oyer, P. E., Cho, S., Peterson, J. D., and Steiner, D. F. (1971). Studies on human proinsulin: Isolation and amino acid sequence 4 of the human pancreatic C-peptide. /. Biol. Chem. 246, 1375-1386.]

figure 7-14 Amino acid sequence of human proinsulin. Dashed circles indicate sites of peptidase cleavage so as to generate the mature insulin and free C peptide. [Modified with permission from Oyer, P. E., Cho, S., Peterson, J. D., and Steiner, D. F. (1971). Studies on human proinsulin: Isolation and amino acid sequence 4 of the human pancreatic C-peptide. /. Biol. Chem. 246, 1375-1386.]

molar amounts with insulin and is known to circulate in the blood; radioimmunoassays are now available to measure this peptide.

b. Biosynthetic Pathway for Insulin

The structure of the gene is now known for seven species (see Figure 7-16). The insulin genes belong to an insulin superfamily of hormones and growth factors that are concerned with metabolism and growth; the other family members include the insulin-related growth factors IGF-I and IGF-II, as well as the ovarian hormone relaxin (IGF; see Chapter 19).

In contrast to IGF-I and IGF-II, which are biosynthe-sized by most tissues, insulin is only produced in the /3-cells of the pancreatic islet. A summary of the regulation of expression of the insulin gene is given in Figure 7-17. Glucose has been shown to modulate both the transcription and translation processes at several distinct steps. This level of control by glucose is consistent with the observation that an elevation in glucose levels can result in a 20-fold increase in insulin biosynthesis.

As is the case with other secreted proteins (e.g., parathyroid hormone, honeybee mellitin, myeloma L chains, and serum albumin), in insulin-proinsulin the immediate translation product released from the polyribosomes has an N-terminal extension or "leader sequence" of ~23 amino acid residues (see Figure 7-16). This product released from the polysomes is designated preproinsulin [by analogy, see preproparathy-roid hormone (Figure 1-11)].

The conversion of proinsulin to insulin occurs in the rough endoplasmic reticulum (RER). The details of the synthesis and secretion of insulin are described in Figure 7-18. As the insulin is released from the proinsulin, it tends to crystallize in the secretory granules as a consequence of the relatively high levels of zinc ions that are present. Morphological studies have shown that the repeating units characteristic of mature pancreatic granules are quite similar to those found in the in vitro crystallized hexameric zinc insulin.

2. Biosynthesis of Glucagon

The biosynthesis and secretion of the 29-amino-acid glucagon by the pancreas a-cell are under the regulatory control of nutrients. In addition, an intestinal form of glucagon, termed glicentin, is produced that consists of 69 amino acid residues; this includes an 8-amino acid C-terminal extension added to glucagon and a 32-residue N-terminal extension. In contrast to glucagon, the secretion of glicentin from the intestinal L cells is not regulated by nutrients. Glicentin is released from the small intestine during the absorption of glucose, triglycerides, and amino acids; its physiological function remains to be clarified.

Both glucagon and glicentin are encoded by the same gene. As detailed in Figure 7-18, differential processing of the initial gene transcript by the pancreatic a-cell or the intestinal L cells results in the biosynthesis of mature glucagon or the peptides glicentin (GLP-I), IVP-II, and GLP-II, respectively (see Figure 7-19). Intestinal L cells presumably lack the endopeptidases required to complete the processing of proglucagon to glucagon.

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