Elsevier

Vitamins & Hormones

Volume 77, 2007, Pages 13-30
Vitamins & Hormones

Biochemistry of Ghrelin Precursor Peptides

https://doi.org/10.1016/S0083-6729(06)77002-9Get rights and content

Abstract

Since its discovery in 1999, the stomach‐derived hormone ghrelin has been studied intensively. Proghrelin is 94 amino acids long in mammals and this undergoes proteolytic processing to produce ghrelin [residues 1–28 of proghrelin(1–94)] and the C‐terminal peptide C‐ghrelin, which likely contains the entire 66 amino acids of the prohormone C‐terminus. The accumulating data identifies ghrelin as having important roles in growth hormone (GH) release, appetite, metabolism, energy balance, cardiovascular function, reproduction, and bone growth. The most striking feature of ghrelin is that it can be acylated at its third amino acid residue (usually Ser), usually in the form of n‐octanoyl group (C8:0). Approximately 10–20% of circulating ghrelin is acylated and this feature confers its GH releasing ability, mediated by the GH secretagogue receptor (GHSR). In contrast, the remaining 80–90% of circulating ghrelin is desacylated. Desacyl ghrelin was initially thought to be inactive, but recent in vivo and in vitro evidences have identified biological actions for this peptide, independent of GHSR. Whether C‐ghrelin has bioactivity remains to be determined, but it is known that plasma concentrations of this peptide respond to endocrine and metabolic manipulations in the same fashion as ghrelin itself. A third putative proghrelin peptide, termed “obestatin” has been mooted, but confirmatory biochemical and functional evidences supporting the existence of this peptide have not been forthcoming, suggesting it to be a biochemical miscalculation. This chapter will address biochemical aspects of proghrelin peptides and point to potential avenues for future work.

Introduction

The discovery of ghrelin as an endogenous ligand regulating growth hormone (GH) secretion occurred against the backdrop of an intense research effort focused around the GH secretagogue receptor (GHSR) and has been described in detail elsewhere (Kojima 2005, Korbonits 2004, van der Lely 2004). That ghrelin is primarily synthesized in and secreted from stomach tissue (Kojima et al., 1999) was an unexpected, but upon reflection, not surprising discovery because, in its role as the prime recipient of energy intake, the stomach/gastrointestinal tract is in a unique position to signal to the pituitary/hypothalamus specific information regarding nutrient content and volume. Such a pattern of discovery is not without parallel as the identification of the cardiac natriuretic peptides resulted from careful testing of atrial tissue extracts for their ability to induce the natriuretic and diuretic activity of the kidney (De Bold, 1979).

The single most important feature of the discovery of ghrelin was the painstaking attention to detail observed regarding the hormone's biochemistry. Thus, ghrelin was purified from the rat stomach through four steps of chromatography: gel filtration, two ion‐exchange HPLC steps, and a final reverse‐phase high performance liquid chromatography (RP‐HPLC) procedure in combination with its ability to stimulate GHSR‐mediated Ca2+ influx in a stable cell line (Kojima et al., 1999). However, after the 28‐amino acid sequence was obtained from Edman degradation, verification that synthetic ghrelin eluted consistent with endogenous peptide on RP‐HPLC was not forthcoming and synthetic peptide did not stimulate GHSR‐mediated GH release. Thus, analysis of the endogenous material on mass spectrometry (MS) revealed a previously unappreciated posttranslational modification consisting of an acylate addition to Ser3. When C8:0 modified synthetic ghrelin was rerun on RP‐HPLC, it eluted consistent with endogenous purified peptide and it stimulated GH release. Without such a careful biochemical approach, it is unlikely that ghrelin's physiological roles would have been identified so rapidly, with a remarkable 2200‐plus publications appearing in the 8 years since its discovery.

Section snippets

Organization of the ghrelin gene

The gene encoding ghrelin is present in mammalian and nonmammalian species including birds, fish, and amphibians. The gene for human proghrelin is located on chromosome 3 at position p25–26 (Kishimoto et al., 2003), whereas the gene for GHSR is at position q26–27 on the same chromosome (Smith et al., 2001). In humans, rats, and mice, the ghrelin gene comprises five exons (Kanamoto 2004, Kojima 1999, Tanaka 2001a). The first exon is only 20 bp and appears to be a noncoding region (Fig. 1).

Ghrelin and desacyl ghrelin

The amino acid sequences of mammalian proghrelin(1–94) precursors are well conserved (Fig. 2). In humans and rodents, preproghrelin is a 117‐amino acid peptide. After translocation through the endoplasmic reticulum membrane, the 23‐amino acid signal peptide is cleaved, resulting in proghrelin(1–94). Mature ghrelin is generated from proghrelin(1–94) by cleavage between residues Arg28‐Ala29, indicating Pro27‐Arg28 is used as a proteolytic cleavage recognition site (Kojima et al., 1999). Such a

Distribution of Proghrelin Peptides

In mammals, the site of maximum gene expression of ghrelin is the stomach, followed closely by the gastrointestinal tract (Kojima et al., 1999). In agreement with this, the tissue with the highest enrichment of proghrelin peptides (i.e., ghrelin and C‐ghrelin) is the stomach (Ariyasu et al., 2001; Bang et al., 2006; Hosoda et al., 2000a) with the next highest amounts found in the duodenum, jejunum, ileum, cecum, and colon (Hosoda et al., 2000a). Other sites of notable proghrelin peptide

Proghrelin Peptides in Lower Vertebrates

Ghrelin appears to be well conserved in lower vertebrates as fish (Kaiya et al., 2003a), birds (Kaiya et al., 2002), and amphibians (Galas 2002, Kaiya 2001); all express the gene and produce functional ghrelin peptide that releases GH in all species (Fig. 3). Their homology to human ghrelin is low, ranging from 29% for bullfrog through to 44% for fish and 54% for bird forms of the hormone, respectively (Kojima and Kangawa, 2005). A feature of interest is that fish encode for short versions of

Summary

Ghrelin presents as a unique hormone in all vertebrates, and it is given that status by a posttranslational modification that had not been previously observed. A huge number of studies have been carried out addressing its biological activity in multiple disciplines which has resulted new and important insights in these disciplines. However, several key pieces of information concerning ghrelin are still unknown: (1) What is the enzyme responsible for acylating ghrelin? (2) Why does genetic

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