Ghrelin Octanoylation Is Completely Stabilized in Biological Samples by Alkyl Fluorophosphonates

Abstract

Ghrelin is a peptide hormone involved in multiple physiological processes related to energy homeostasis. This hormone features a unique posttranslational serine octanoylation modification catalyzed by the enzyme ghrelin O-acyltransferase, with serine octanoylation essential for ghrelin to bind and activate its cognate receptor. Ghrelin deacylation rapidly occurs in circulation, with both ghrelin and desacyl ghrelin playing important roles in biological signaling. Understanding the regulation and physiological impact of ghrelin signaling requires the ability to rapidly protect ghrelin from deacylation in biological samples such as blood serum or cell lysates to preserve the relative concentrations of ghrelin and desacyl ghrelin. In in vitro ghrelin O-acyltransferase activity assays using insect microsomal protein fractions and mammalian cell lysate and blood serum, we demonstrate that alkyl fluorophosphonate treatment provides rapid, complete, and long-lasting protection of ghrelin acylation against serine ester hydrolysis without interference in enzyme assay or ELISA analysis. Our results support alkyl fluorophosphonate treatment as a general tool for stabilizing ghrelin and improving measurement of ghrelin and desacyl ghrelin concentrations in biochemical and clinical investigations and suggest current estimates for active ghrelin concentration and the ghrelin to desacyl ghrelin ratio in circulation may underestimate in vivo conditions.

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

“…Membrane fraction was preincubated with 1 μM methyl arachidonyl fluorophosphonate (MAFP) and inhibitor or vehicle as indicated in 50 mM HEPES pH 7.0 for 30 minutes at room temperature. 53 Reactions were initiated with the addition of 500 μM octanoyl CoA and 1.5 μM fluorescently-labeled ghrelin mimetic, GSSFLC Acrylodan , for a total volume of 50 μL, and were incubated for 3 hours at room temperature in the dark. Reactions were stopped with the addition of 50 μL of 20% acetic acid in isopropanol, and solutions were clarified by protein precipitation with 16.7 μL of 20% trichloroacetic acid, followed by centrifugation (1,000x g , 1 minute).…”

Clarifying the Ghrelin System’s Ability to Regulate Feeding Behaviours Despite Enigmatic Spatial Separation of the GHSR and Its Endogenous Ligand

“…Nonetheless, since diurnal CSF ghrelin concentrations or those that accompany food restriction and/or fasting conditions have not been characterized, the capacity of CSF acylated ghrelin to initiate GHSR dependent processes should not be completely discounted. Furthermore, circulating acylated ghrelin is rapidly converted into des-acyl ghrelin by esterases present in the circulation and thus CSF acylated ghrelin levels may be underestimated [59]. Therefore, enhancing the sensitivity of current ghrelin quantification assays and developing more efficient sampling methods that maximize recovery and mitigate degradation of acylated ghrelin may make CSF ghrelin detection possible in rodents and more accurate in other model species.…”

“…Therefore, enhancing the sensitivity of current ghrelin quantification assays and developing more efficient sampling methods that maximize recovery and mitigate degradation of acylated ghrelin may make CSF ghrelin detection possible in rodents and more accurate in other model species. Fortunately, compounds able to reliably prevent hydrolytic degradation of acylated ghrelin into des-acyl ghrelin in biological samples have recently been identified [59]. These ameliorations will go a long way in determining whether endogenous CSF ghrelin levels relevantly influence GHSR dependent behaviours such as feeding.…”

“…However, as ghrelin and GOAT transcript levels within the HYP are increased following food restriction, measuring ghrelin levels in hypothalamic regions during a time of energy insufficiency, may heighten the concentration of ghrelin to a detectable level [56]. Likewise, early addition of alkyl fluorophosphonate compounds, which slow the degradation of acylated ghrelin (through the inhibition of enzymes that cleave the ester bond attaching the octonyl moiety to the rest of the ghrelin peptide), to sample perfusates should also ameliorate detection thresholds [59]. Alternatively, high-performance liquid chromatography-mass spectroscopy techniques could be exploited to assess central acylated ghrelin levels.…”

“…While recent data suggest that the proportion of acyl ghrelin may be higher, des-acyl ghrelin remains the principal form of ghrelin in blood [59]. Des-acyl ghrelin does not seem to increase GHSR signalling at physiological concentrations [11,20,60,61] but see [62].…”