Enzymatic Characterization of a Human Acyltransferase Activity

Abstract

BackgroundNon-histone protein acylation is increasingly recognized as an important posttranslational modification, but little is known as to the biochemical properties of protein serine acylating enzymes.Methodology/Principal FindingsWe here report that we have identified a metal-stimulated serine octanoyltransferase activity in microsomes from human erythroleukemic (HEL) cells. The HEL acylating enzyme was linear with respect to time and protein, exhibited a neutral pH optimum (stimulated by cobalt and zinc), and inhibited by chelating reagents. Hydroxylamine treatment removed most, but not all, of the attached radioactivity. A salt extract of microsomal membranes contained the major portion of enzyme activity, indicating that this acyltransferase is not an integral membrane protein. Sucrose density fractionation showed that the acyltransferase activity is concentrated in the endoplasmic reticulum. In competition experiments, the acyltransferase was well inhibited by activated forms of fatty acids containing at least eight to fourteen carbons, but not by acetyl CoA. The zinc-stimulated HEL acyltransferase did not octanoylate proenkephalin, proopiomelanocortin, His-tagged proghrelin, or proghrelin lacking the amino-terminal His-tag stub of Gly-Ala-Met. The peptides des-acyl ghrelin and ACTH were also not acylated; however, des-acyl ghrelin containing the N-terminal tripeptide Gly-Ala-Met was acylated. Mutagenesis studies indicated a requirement for serine five residues from the amino terminus, reminiscent of myristoyl transferase, but not of ghrelin acylation. However, recombinant myristoyl transferase could not recapitulate the hydroxylamine sensitivity, zinc-stimulation, nor EDTA inhibition obtained with HEL acyltransferase, properties preserved in the HEL cell enzyme purified through four sequential chromatographic steps.Conclusions/SignificanceIn conclusion, our data demonstrate the presence of a zinc-stimulated acyltransferase activity concentrated in the endoplasmic reticulum in HEL cells which is likely to contribute to medium-chain protein lipidation.

Architectural Organization of the Metabolic Regulatory Enzyme Ghrelin O-Acyltransferase

“…Octanoylation is required for activation of the ghrelin receptor, GHSR-1a, and is the only known example of protein octanoylation in higher organisms (4). Because dietary fats and those from local fatty acid metabolism can be directly conjugated to ghrelin (5)(6)(7), it can be viewed as a "fat sensor" from the brain to the body or within the brain itself, modulating energy balance.…”

Integrating GHS into the Ghrelin System

“…Regulation may exist pretranslationally also, since natural antisense ghrelin RNA's can be demonstrated by analysis of human genomic DNA [ 54 , 55 ]. A second acyltransferase, termed microsomal zinc-stimulated serine (Ser 2 ghrelin) octanoyltransferase, distinct from GOAT, was cloned by Ozawa et al in 2009 [ 56 ]. This enzyme is concentrated in the endoplasmic reticulum of human erythroleukemia cells.…”

“…In addition to GOAT, another human ER oxyesterase has been purified and characterized from a stable human erythroleukemia cell line by Ozawa, Speaker, and Lindberg [ 56 ]. The acronym of this additional oxyesterase is ERAT for endoplasmic reticulum O - acyl transferase.…”

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

“…Ghrelin signaling requires octanoylation of Ser3, a unique modification required for activation of the ghrelin receptor, GHSR-1a[3, 5] and catalyzed by the 11-transmembrane integral membrane protein ghrelin- O -acyltransferase (GOAT)[6–9]. Ghrelin may play roles in both energy balance and hunger, depending on the circumstances[10, 11].…”