Identification and Characterization of Acyl-Protein Thioesterase 1/Lysophospholipase I As a Ghrelin Deacylation/Lysophospholipid Hydrolyzing Enzyme in Fetal Bovine Serum and Conditioned Medium

Abstract

Ghrelin contains an octanoic acid at the third residue serine, and the presence of octanoic acid on ghrelin is critical to its physiological functions. The precise mechanism for the deacylation of ghrelin in circulation remains to be clarified, although the level of deacylated ghrelin (des-acyl ghrelin) is higher than that of acylated ghrelin in serum. In this study, rapid identification of ghrelin deacylation activity was achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and a ghrelin deacylation enzyme was purified 1515-fold from fetal bovine serum. Chromatographic separation showed a 24-kDa band on SDS-PAGE corresponding to ghrelin deacylation activity, and the protein band was identified as acyl-protein thioesterase 1 (APT1)/lysophospholipase I. A ghrelin deacylation enzyme in medium from HepG2 cells was also purified and identified as APT1. Although it lacks a secretion signal sequence, APT1 may be released by cells expressing APT1, mainly from liver in vivo. APT1 was originally purified as a cytosolic lysophospholipid hydrolyzing enzyme (lysophospholipase I), and recombinant APT1 exhibited deacylation activity as well as lysophospholipase activity in vitro. APT1 is released at high levels from RAW264.7 macrophage-like cells into the culture medium after stimulation with lipopolysaccharide (LPS), and LPS suppresses APT1 mRNA and protein expressions in these cells. More potent ghrelin deacylase activities were detected in sera from LPS-treated rats than in control sera. These results suggested that the serum activity of APT1 may play an important role in determination of the concentration of des-acyl ghrelin in circulation, especially under septic inflammation.

Identification of activated protein C as a ghrelin endopeptidase in bovine plasma

“…Although the acyl moiety at the third residue of ghrelin is unstable, the peptide bonds in the main chain of ghrelin remain stable after incubation with sera derived from cattle (Satou et al 2010) or rats (De Vriese et al 2004). As mentioned previously, the C-terminus of the ghrelin peptide is dispensable for the activation of GHSR1a in vitro (Bednarek et al 2000, Van Craenenbroeck et al 2004.…”

“…administered acyl ghrelin from the circulation has been observed (Gauna et al 2004, Mayorov et al 2008. We previously reported that acyl protein thoesterase 1 (APT1) purified from bovine serum is a ghrelin deacylation enzyme that hydrolyzes the lipid moiety from ghrelin (Satou et al 2010). Sera derived from septic rats exhibit higher ghrelin deacylation activity as compared with those of naive animals (Satou et al 2010).…”

“…We previously reported that acyl protein thoesterase 1 (APT1) purified from bovine serum is a ghrelin deacylation enzyme that hydrolyzes the lipid moiety from ghrelin (Satou et al 2010). Sera derived from septic rats exhibit higher ghrelin deacylation activity as compared with those of naive animals (Satou et al 2010). These results may explain why the concentrations of desacyl ghrelin in circulation are higher than those of acylated ghrelin (Gauna et al 2004, Marzullo et al 2004.…”

A Lipid-Anchored NAC Transcription Factor Is Translocated into the Nucleus and Activates Glyoxalase I Expression during Drought Stress

“…In animals, acyl-protein thioesterase 1 (APT1) was initially identified as a lysophospholipase that catalyzes fatty acid release (Sugimoto et al, 1998;Satou et al, 2010). Subsequently, APT1 was the first authentic participant in the regulated depalmitoylation of intracellular signaling proteins to be identified Gilman, 1998, 2002), and increasing evidence suggests that APT1 and APT2 are the major APTs that mediate the depalmitoylation of diverse cellular substrates (Lin and Conibear, 2015).…”

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer

“…Although both ghrelin and desacyl ghrelin circulate in the blood, the levels of desacyl ghrelin are significantly higher (Date et al 2000, Patterson et al 2005, Holmes et al 2009). This may be the result of the rapid conversion of ghrelin to desacyl ghrelin by esterases in the circulation (Beaumont et al 2003, De Vriese et al 2004, Satou et al 2010, Eubanks et al 2011. Alternatively, desacyl ghrelin may be produced from proghrelin in the absence of GOAT expression or when the fatty acid substrates for GOAT are unavailable (Takahashi et al 2009).…”