Ghrelin Octanoylation Is Completely Stabilized in Biological Samples by Alkyl Fluorophosphonates.
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.
Department of Chemistry (K.R.M.-G., J.E.D., M.A.S., J.L.H.), Syracuse University, Syracuse, New York 13244; and Department of Neuroscience (T.R., A.A.), Carleton University, Ottawa, Ontario, Canada K1S5B6.
Kayleigh R
KR
McGovern-Gooch
Department of Chemistry (K.R.M.-G., J.E.D., M.A.S., J.L.H.), Syracuse University, Syracuse, New York 13244; and Department of Neuroscience (T.R., A.A.), Carleton University, Ottawa, Ontario, Canada K1S5B6.
Trevor
T
Rodrigues
Department of Chemistry (K.R.M.-G., J.E.D., M.A.S., J.L.H.), Syracuse University, Syracuse, New York 13244; and Department of Neuroscience (T.R., A.A.), Carleton University, Ottawa, Ontario, Canada K1S5B6.
Joseph E
JE
Darling
Department of Chemistry (K.R.M.-G., J.E.D., M.A.S., J.L.H.), Syracuse University, Syracuse, New York 13244; and Department of Neuroscience (T.R., A.A.), Carleton University, Ottawa, Ontario, Canada K1S5B6.
Michelle A
MA
Sieburg
Department of Chemistry (K.R.M.-G., J.E.D., M.A.S., J.L.H.), Syracuse University, Syracuse, New York 13244; and Department of Neuroscience (T.R., A.A.), Carleton University, Ottawa, Ontario, Canada K1S5B6.
Alfonso
A
Abizaid
Department of Chemistry (K.R.M.-G., J.E.D., M.A.S., J.L.H.), Syracuse University, Syracuse, New York 13244; and Department of Neuroscience (T.R., A.A.), Carleton University, Ottawa, Ontario, Canada K1S5B6.
James L
JL
Hougland
D000215:Acylation / Q000187:drug effects; D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D005459:Fluorides / Q000494:pharmacology*; D054439:Ghrelin / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D010710:Phosphates / Q000494:pharmacology*; D055550:Protein Stability / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism
D054439:Ghrelin; D010710:Phosphates; C422650:ghrelin, des-n-octanoyl; C012980:fluorophosphate; D012694:Serine; D000217:Acyltransferases; D005459:Fluorides