Chapter Nine - From Ghrelin to Ghrelin's O-Acyl Transferase
Introduction
Ghrelin is an extraordinary peptide hormone. By acting on its receptor, namely, the growth hormone secretagogue receptor 1a (GHSR1a), it stimulates growth hormone release from the pituitary gland, food intake, carbohydrate utilization, and adiposity and regulates insulin secretion and blood glucose (Kojima and Kangawa, 2005, Kojima et al., 1999, Tschop et al., 2000, Van der Lely et al., 2004). Ghrelin is the only peptide hormone of peripheral tissue origin that increases food intake (Cummings and Overduin, 2007). To achieve these physiologically critical activities, it requires an unusual acyl modification by a labile ester linkage on its critical serine-3 residue. This modification, unique to ghrelin, involves the mid-chain fatty acids octanoate and decanoate (Kojima et al., 1999).
Several investigators have described highly sensitive radiologic or immunologic methods for acylated and des-acylated ghrelin in tissues and circulation (Akamizu et al., 2005, Groschl et al., 2002, Hosoda et al., 2000, Hosoda et al., 2004, Liu et al., 2008, Rauh et al., 2007). However, much debate has occurred in the ghrelin field as to the consistency of these observations because of the known instability of the acylated forms of ghrelin (Prudom et al., 2010). An understanding of this instability is increasingly important, as hypotheses have emerged involving the ratio of acyl and total ghrelin (acyl + des-acyl) as being physiologically relevant (van der Lely, 2009). Many investigators simply rely on measurements of total ghrelin, as these are reproducible across many studies. The core of this problem lies within the instability of the acyl ester linkage on the modified serine residue. This ester bond is particularly labile to tissue and circulating esterase activities that rapidly convert ghrelin to its inactive form, namely, des-acyl ghrelin (De Vriese et al., 2004). Much of this conversion takes place post tissue or sample collection and becomes a significant artifact of sample processing. As we embarked on the establishment of mass spectrometry methodologies to measure acyl and des-acyl ghrelin, we rapidly determined the need to establish sample collection and processing protocols to minimize this artifact. Observations made with these protocols enabled us to (1) measure reliably the specific acylated and des-acyl forms of ghrelin; (2) determine that ghrelin is modified, in vivo, by other short-chain fatty acids; (3) hypothesize as to genes that may be mediating the acylation of ghrelin; (4) establish a cell culture system to search for ghrelin's acyltransferase and describe its mechanism for acylation; and (5) demonstrate, in vivo, that GOAT is ghrelin's acyltransferase. In this chapter, we describe the specific methodologies, and their significance, that allowed us to make these relevant observations.
Section snippets
Development of acyl and des-acyl ghrelin assays
In order to simplify the measurement of acyl and des-acyl ghrelin from biological samples, we chose to use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Our reasoning was that, with a single immunoprecipitation (IP), both species could be isolated, and with MALDI-ToF MS, we could detect both species in a single measurement. In addition, by including appropriate internal standards, the observations could be made quantitative, control for
Stabilizing Ghrelin in Biological Matrices
One of the major problems with accurate measurements of ghrelin is the instability of the fatty acid ester bond on the modified serine residue. Biological fluids contain esterase activities that can rapidly convert acylated ghrelin into des-acyl ghrelin. This is a particular problem with blood samples, especially rodent blood. Acidification can quickly inactivate esterase activity, but this can also initiate protein aggregation. We evaluated a number of conditions for stabilization of ghrelin
TT Cell Culture System for Ghrelin Production
Establishment of a cell culture system capable of reproducibly secreting acyl-modified forms of ghrelin was essential to initially implicate GOAT in the acylation of ghrelin. Kanamoto and collaborators demonstrated that the human medullary thyroid carcinoma cell line (TT cell line) produced des-acylated and acylated ghrelin, suggesting that these cells possessed ghrelin's acylation components (Kanamoto et al., 2001). We obtained TT cells from ATCC (Cat. No. CRL-1803) and cultured them in Ham's
Functional Screening for Ghrelin's O-Acyl Transferase
To select candidate sequences for transcript silencing in the TT cell ghrelin acylation functional assay system, we used the following criteria: (1) similarity to previously defined acyltransferases, (2) presence of a human homolog, and (3) genes of unknown function. With these criteria, we mainly identified members of the recently described membrane-bound O-acyl transferase (MBOAT) family of proteins (Hofmann, 2000). This led us to hypothesize that an orphan MBOAT protein mediated the
GOAT is Ghrelin's Acyl Transferase
Our results from the TT cell culture system silencing GOAT and the HEK-293 cells recapitulating the production of acylated ghrelin clearly implicated GOAT in this modification and demonstrated the sufficiency of ghrelin and GOAT in the acyl modification of ghrelin. These data, however, did not demonstrate the essentiality in vivo of GOAT for ghrelin's octanoylation and its physiological functions. One approach to determine whether GOAT is the only gene capable of carrying out this important
Summary
The metabolic hormone ghrelin is an extraordinary 28-amino acid peptide with a unique acyl ester modification on its serine 3 residue. This posttranslational modification is absolutely essential for its growth hormone, orexigenic, metabolic, and insulin secretion effects, yet the modification is highly susceptible to circulating esterases, which can convert the active form of ghrelin to the des-acylated form. This conversion of acyl to des-acyl ghrelin can occur within minutes after sample
Acknowledgments
We wish to thank Drs. Mark L. Heiman and Jude E. Onyia for support, stimulating discussions, and input on the topic of ghrelin and the discovery of GOAT. We also thank Dr. Derrick R. Witcher for providing the antighrelin antibodies used in these studies.
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Pure human butyrylcholinesterase hydrolyzes octanoyl ghrelin to desacyl ghrelin
2015, General and Comparative EndocrinologyCitation Excerpt :Manifestation of all these properties requires that ghrelin be acylated on serine 3. Either octanoate or decanoate satisfy this requirement (Gutierrez et al., 2012). Ghrelin is inactivated by hydrolysis of the acyl moiety from serine 3 to yield desacyl ghrelin.
Clinical protein mass spectrometry
2015, MethodsCitation Excerpt :After addition of an isotopic labeled internal reference standard in blood samples, larger proteins are precipitated and all ghrelin isoforms are immunoprecipitated prior to MALDI-TOF analysis. Peptide concentrations in the pg/ml range can be measured with this method [71]. Hepcidin is a 25 amino acid cysteine rich peptide that regulates iron metabolism.
Pyroglutamyl apelin-13 identified as the major apelin isoform in human plasma
2013, Analytical BiochemistryCitation Excerpt :Most proteases, except aspartic proteases, have an optimal pH of neutral or basic, so lowering the pH of the plasma effectively inhibits their activities. A similar approach has been used to successfully stabilize acylated ghrelin in plasma [24]. In the current study, we found that lowering the pH of plasma to pH 4.5 significantly improves the detection and recovery of several apelin peptides.