ghrelin octanoylation mediated by an orphan lipid transferase · ghrelin octanoylation mediated by...

Ghrelin octanoylation mediated by an orphanlipid transferaseJesus A. Gutierrez*†, Patricia J. Solenberg*, Douglas R. Perkins*, Jill A. Willency*, Michael D. Knierman*, Zhaoyan Jin*,Derrick R. Witcher‡, Shuang Luo§, Jude E. Onyia‡, and John E. Hale*

*Integrative Biology, Eli Lilly and Company, 2001 West Main Street, Greenfield, IN 46140; and ‡Biotechnology Discovery Research and §Cancer Inflammationand Cell Survival, Eli Lilly and Company, Indianapolis, IN 46285

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved February 28, 2008 (received for reviewJanuary 23, 2008)

The peptide hormone ghrelin is the only known protein modifiedwith an O-linked octanoyl side group, which occurs on its thirdserine residue. This modification is crucial for ghrelin’s physiolog-ical effects including regulation of feeding, adiposity, and insulinsecretion. Despite the crucial role for octanoylation in the physi-ology of ghrelin, the lipid transferase that mediates this novelmodification has remained unknown. Here we report the identi-fication and characterization of human GOAT, the ghrelin O-acyltransferase. GOAT is a conserved orphan membrane-bound O-acyltransferase (MBOAT) that specifically octanoylates serine-3 of theghrelin peptide. Transcripts for both GOAT and ghrelin occurpredominantly in stomach and pancreas. GOAT is conserved acrossvertebrates, and genetic disruption of the GOAT gene in mice leadsto complete absence of acylated ghrelin in circulation. The occur-rence of ghrelin and GOAT in stomach and pancreas tissuesdemonstrates the relevance of GOAT in the acylation of ghrelin andfurther implicates acylated ghrelin in pancreatic function.

acylation � membrane-bound O-acyl transferase

Ghrelin is a 28-aa peptide hormone produced principally bystomach tissue with an unusual acyl modification on its critical

serine-3 residue. Ghrelin is the endogenous ligand for the growthhormone secretagogue receptor 1a (GHSR1a), and its acyl modi-fication is critical for the activation of the GHSR1a (1). In additionto stimulating growth hormone release from pituitary, ghrelin alsopromotes food intake, carbohydrate utilization, and adiposity (2–5).Accordingly, ghrelin levels are modulated by changes in nutritionalstatus such as feeding and fasting or exposure to high-fat diets (5,6). Importantly, ghrelin is the only peptide hormone of peripheraltissue origin that increases food intake (2).

More recent studies have identified roles for acylated ghrelin inregulating insulin secretion and blood glucose. Acylated ghrelinoccurs in pancreas tissues, and GHSR1a receptor blockade withspecific antagonists or treatments with antiserum against acylatedghrelin enhance glucose-induced increases in insulin releasewhereas acylated ghrelin decreases insulin release (5, 7–10). Theseobservations have further implicated acylated ghrelin in the regu-lation of metabolism.

In stomach tissue and in circulation, acylated forms of ghrelin aremodified via an ester linkage with n-octanoic acid and to a lesserextent with decanoyl and decenoyl fatty acids (1, 3). Importantly,the acyl modification in ghrelin is essential for function, withoctanoyl and decanoyl fatty acids being optimal (11). Ghrelin ishighly conserved in vertebrates, and the third serine residue, whichis uniquely modified by the ester-linked acyl group, occurs in allmammal, avian, and fish species (3).

The enzyme(s) responsible for acylation of ghrelin has remainedunknown. Work by Takada et al. (12) described that porcupine, anenzyme with structural similarities to membrane-bound O-acyltransferases (MBOAT), is required for serine-209 acylation withpalmitoleic acid and for transport of Wnt3a from the endoplasmicreticulum for secretion. Ghrelin and Wnt3a are the only proteinsknown to possess acylated serine residues. Intriguingly, the enzyme

porcupine has been localized at the endoplasmic reticulum, thesame cellular compartment through which ghrelin is expected topass during its processing (13). These observations raised thepossibility that perhaps an acyl transferase belonging to theMBOAT family of enzymes may also mediate the acyl modificationin ghrelin.

In 2001, Kanamoto et al. (14) exploited immunohistochemistryand radioimmunoassays with reagents capable of detecting des-octanoyl ghrelin (des-acyl ghrelin) or acyl ghrelin to show that thehuman medullary thyroid carcinoma cell line (TT cell line) pro-duces ghrelin peptides, with des-acyl ghrelin being the most prom-inent form. Importantly, however, low levels of acyl ghrelin immu-noreactivity were also reported, suggesting that these cells possessthe necessary enzymatic machinery for the acylation of ghrelin.

To further understand the mechanism by which ghrelin is acy-lated, we used the TT cell line along with siRNA gene-silencingstrategies with MS-based assays to search for genes capable ofmodulating the octanoylation of ghrelin. Here we report theidentification and characterization of a member of the MBOATfamily of acyl transferases capable of specifically octanoylatingghrelin on its critical serine-3 residue.

ResultsStimulation of Ghrelin Octanoylation in TT Cells. We used an immu-noprecipitation MALDI-TOF assay to detect both acyl and des-acyl forms of ghrelin peptides from cell culture media of humanmedullary thyroid carcinoma (TT) cells (14). We detected onlydes-acyl ghrelin peptides 1–28 (m/z 3,244) and 1–27 (m/z 3,088) (Fig.1A), suggesting that the cells generate low levels of acylated ghrelin.We reasoned that limiting fatty acid levels in our culture system maypreclude ghrelin acylation. Accordingly, we supplemented cellculture media with various levels of octanoic acid and then readilydemonstrated the octanoylation of ghrelin 1–28 and 1–27 (Fig. 1B).Additional peptide fragmentation and tandem MS (MS/MS) anal-yses confirmed that the octanoylation occurs exclusively at serine-3(data not shown). These TT cell culture conditions provided auseful system for identifying the ghrelin acyl transferase.

Author contributions: J.A.G. and P.J.S. contributed equally to this work; J.A.G., P.J.S., D.R.P.,J.E.O., and J.E.H. designed research; J.A.G., P.J.S., D.R.P., J.A.W., M.D.K., Z.J., and S.L.performed research; D.R.W. contributed new reagents/analytic tools; J.A.G., P.J.S., D.R.P.,J.A.W., M.D.K., Z.J., and J.E.H. analyzed data; and J.A.G. and J.E.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The GOAT sequence reported in this paper has been deposited in theGenBank database [EU518498 (human), EU518496 (rat), EU518495 (mouse), and EU518497(zebrafish)].

See Commentary on page 6213.

†To whom correspondence should be addressed. E-mail: gutierrez�jesus�[email protected].

This article contains supporting information online at www.pnas.org/cgi/content/full/0800708105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

6320–6325 � PNAS � April 29, 2008 � vol. 105 � no. 17 www.pnas.org�cgi�doi�10.1073�pnas.0800708105

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Searching for the Acyl Transferase. Candidate sequences were se-lected for gene-silencing experiments according to the followingcriteria: (i) similarity to known acyltransferase sequences; (ii)presence of a human homologue; and (iii) the function of the genewas unknown. Twelve candidate genes were identified, most ofwhich were orphan MBOATs. Silencing RNAs were generated andused for determination of ghrelin acylation in TT cells. Fig. 2 showsthe effects of candidate gene silencing and the accumulation ofoctanoylated ghrelin (m/z 3,370). Remarkably, TT cell treatmentwith reagents designed to knock down one of the candidate genes(candidate 7; also described as FKSG89, MBOAT4, or OACT4),but none of the other sequences, including several MBOAT can-didates, greatly diminished octanoyl ghrelin synthesis (Fig. 2A).Dose–response studies with siRNA7–3, targeting candidate 7,showed that diminished ghrelin octanoylation levels correlated withknockdown of targeted transcripts (Fig. 2 B and C). Furthermore,exposure of TT cells to five distinct siRNAs targeting the FKSG89transcript decreased ghrelin octanoylation by �50–90% (Fig. 2D).These data defined an essential role for the transcripts targeted bythese siRNA reagents in ghrelin acylation.

Candidate 7 Is a Member of the MBOAT Family of Proteins. Candidate7 encodes an uncharacterized protein with structural motifs presentin the MBOAT family of acyltransferases [supporting information(SI) Fig. S1]. The predicted proteins described in the publicdatabases with homology to FKSG89 exhibit large diversity specificfor their respective N termini. To ensure authenticity of the humangene for candidate 7, RT-PCR and 5� RACE reactions wereperformed with human TT cell mRNA using primers specific forthis gene. The 5� RACE results from TT cells or human stomachmRNA showed only the presence of larger amplification productsand not those expected for the FKSG89 gene. Nucleotide analysesof human chromosome 8 (region 8p12) supported the presence oftwo additional exons upstream of the single-exon FKSG89 gene,which confirmed the full-length transcript that we observed exper-imentally. We named the predicted protein encoded by the longertranscript of candidate 7 the ghrelin O-acyl transferase, GOAT(Fig. S2).

GOAT Acylates Ghrelin at the Critical Serine-3. To ascertain whetherGOAT can octanoylate ghrelin protein, we performed transient

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Fig. 1. Generation of octanoylated ghrelin peptides by octanoic acid treatment in TT cells. (A) Ghrelin immunoprecipitation MALDI-TOF MS (IPMS) analysesof TT cell culture media under control conditions. Des-acyl ghrelin 1–28 (m/z 3,244) and 1–27 (m/z 3,088) were observed within 6 days. (B) Exposure of TT cellsto octanoic acid (125 �g/ml) induced production of octanoylated ghrelin 1–28 (m/z 3,370) and 1–27 (m/z 3,214) peptides by the cells. Treatment-dependentgeneration of octanoylated ghrelin peptides is denoted by downward arrows. Ghrelin peptide standards were added at the start of the culture period (m/z 3,187,3,314, and 3,393 for rat des-acyl ghrelin, rat octanoylated ghrelin, and human SIL octanoylated ghrelin peptides).

Gutierrez et al. PNAS � April 29, 2008 � vol. 105 � no. 17 � 6321

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transfections in human embryonic kidney (HEK-293) cells. Mediafrom HEK-293 cells 72 h after transfection with ghrelin alonecontained only the des-acyl forms of ghrelin 1–28 and 1–27 (Fig.3A). Strikingly, cotransfection with GOAT yielded an intense peakat m/z 3,370 and a minor peak at m/z 3,214, corresponding tooctanoylated ghrelin 1–28 and 1–27 (Fig. 3B). Further MS frag-

mentation confirmed that the octanoylation occurs only at serine-3(Fig. 3C). Interestingly, we also detected minor levels of serine-3acetylated and serine-3 butyrylated (Fig. 3B, m/z 3,286 and 3,314,

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Fig. 2. GOAT is essential for ghrelin octanoylation in TT cells. (A) TT cells wereexposed to targeting siRNAs (2 �g) specific for candidate 7 (Cand-7),MBOAT-1, MBOAT-2, MBOAT-3, MBOAT-5, human BB1, SOAT-1, or nontar-geting control siRNAs and assayed for ghrelin octanoylation by using theghrelin IPMS assay. Ghrelin octanoylation levels were normalized to cellstreated with nontargeting siRNA control. (B) Dose-dependent decrease inoctanoylated ghrelin levels in TT cells treated with candidate 7 gene siRNA 7-3.(C) Dose-dependent effects of candidate 7 gene siRNA 7-3 on normalizedGOAT transcripts (GOAT/18s rRNA). (D) Exposure of TT cells to targetingsiRNAs (2 �g) to five distinct regions of the candidate 7 transcript decrease thelevels of octanoylated ghrelin (1–28) relative to siRNA control treatment.

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3088 Human Des Acyl Ghrelin 1-273188 Rat Des Acyl Ghrelin 1-28 (STD)3214 Human C8 Acyl Ghrelin 1-273228 Dog C8 Acyl Des Q Ghrelin 1-28 (STD)3245 Human Des Acyl Ghrelin 1-28 3370 Human C8 Acyl Ghrelin 1-28

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Ser(CO-(CH2)6-CH3)

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Fig. 3. GOAT octanoylates ghrelin peptide in HEK-293 cells. (A) HEK-293 cellstransiently transfected with human preproghrelin cDNA secreted des-acylghrelin peptides 1–28 (m/z 3,244) and 1–27 (m/z 3,088). (B) Transient cotrans-fection of HEK-293 cells with human preproghrelin and GOAT cDNAs pro-duced principally octanoylated ghrelin peptides 1–28 (m/z 3,370) and 1–27(m/z 3,214). Ghrelin peptides standards were rat des-acyl ghrelin (m/z 3,188)and dog octanoyl des Q ghrelin (m/z 3,228). (C) MS fragmentation analysesshowing the GOAT-mediated octanoyl modification of serine-3 in ghrelin.Immunoprecipitated ghrelin peptides from cotransfected cells were subjectedto MS/MS analyses. (Upper) Fragmentation pattern for �5 ions (m/z 649.56)for des-acyl ghrelin denoting the presence of daughter y� ions up to theunmodified serine-3 residue. (Lower) Fragmentation pattern for �5 ions (m/z674.78) for octanoylated ghrelin. Daughter y� ion fragmentation pattern foroctanoylated ghrelin shows a shift for the modified serine-3 residue corre-sponding to the covalent octanoylation of this residue. Arrows denote massshift at serine-3 for des-acyl and octanoylated ghrelin. The amino acid se-quence for human octanoylated ghrelin is shown within the figure.

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respectively) ghrelin peptides, suggesting that GOAT can also useadditional fatty acid substrates.

To determine whether other fatty acids, besides octanoic acid,can be used as substrates by HEK-293 cells in the ghrelin acylationsystem, we supplemented the media with fatty acids ranging fromacetate (C2) to hexadecanoic acid (C16). These studies showed thatGOAT can acyl modify the serine-3 residue of ghrelin with fattyacids up to tetradecanoic acid, suggesting that GOAT has greaterfatty acid selectivity beyond octanoic and decanoic acid (Fig. S3).

The specificity of GOAT as the acyl transferase for ghrelin anda member of the MBOAT family of proteins was further tested byusing the HEK-293 cell system. Of the MBOATs tested only GOATis capable of acyl modifying ghrelin (Fig. S4). We also wanted todetermine whether the MBOAT-conserved histidine residue(GOAT H338) is critical for ghrelin acylation. Alanine replacementof histidine-338 in GOAT completely abolished its ability to oc-tanoylate ghrelin, further supporting the observation that GOAT isa member of the MBOAT family of proteins (Fig. S5).

GOAT Is Conserved Across Vertebrate Animals. GOAT is predicted tobe conserved across vertebrates. To determine whether this con-servation was indeed functional, we obtained the GOAT cDNAsfor a diverse group of vertebrates, including rat, mouse, andzebrafish, and coexpressed them with ghrelin in HEK-293 cells.These studies showed that the mouse, rat, and zebrafish forms ofGOAT can faithfully octanoylate human ghrelin (Fig. S6). Aminoacid sequence comparisons for human, mouse, rat, and zebrafishGOAT showed percentage amino acid similarities of �90% forhuman, mouse, and rat GOAT proteins and �60% for mammalianand zebrafish GOAT proteins (data not shown).

GOAT Is the Lipid Acyl Transferase for Ghrelin. GOAT gene knockoutmice were generated and used to characterize circulating acyl anddes-acyl ghrelin peptides with the ghrelin IPMS assay. Results fromthese initial studies show the complete absence of octanoylatedghrelin in the blood of GOAT-null mice in contrast to wild-typelittermate animals (Fig. 4). These results show that GOAT is theacyl transferase required for the acylation of ghrelin.

Stomach and Pancreas Tissues Express GOAT Transcripts. We evalu-ated the distribution of GOAT by transcript profiling in 48 humantissues. We found that GOAT is a message of relatively lowabundance because its measured cycle threshold value was 32.7 forstomach tissue. This is in contrast to ghrelin’s transcript cyclethreshold measurements of 20.6 in the same tissue (data notshown). However, there are elevated levels of GOAT transcripts instomach and pancreas and very low levels in most other tissues (Fig.5A). By comparison, ghrelin showed abundant transcript expressionin stomach tissue, modest expression in pancreas, and relatively lowlevels in most other tissues (Fig. 5B). The concordant expression ofboth ghrelin and GOAT in stomach agrees with observations thatoctanoylated ghrelin is principally produced in gastric tissue. Inaddition, the elevated levels for both GOAT and ghrelin in pancreasare consistent with recent work showing that octanoylated ghrelinregulates insulin secretion (8).

DiscussionThe structure of ghrelin is unprecedented, with a unique octanoylmodification on its serine-3 residue. This modification is absolutelyessential for its growth hormone, orexigenic, metabolic, and insulinsecretion effects. It is always present on the third hydroxyl-containing residue. In vivo, ghrelin is mainly modified with octanoyland to lesser degree decanoyl fatty acids. The mechanism respon-sible for this modification has remained elusive (3–5, 15). Becauseof the essential nature of ghrelin acylation, the enzyme or enzymecomplex responsible for this modification has been sought as a keyregulatory step. Our discovery of human GOAT describes themetabolic activation of the peptide hormone ghrelin.

Establishment of a ghrelin octanoylation cell culture system waskey to our identification of GOAT. Kanamoto et al. (14) reportedthat TT cells secrete ghrelin into cell culture media. Our supple-mentation studies with octanoic acid and our stabilization of ghrelinwith an antibody specific to the acylated form were crucial. Nev-ertheless, no more than 10% of ghrelin secreted by our TT cells wasacylated, suggesting that acylating machinery is of relatively lowabundance in these cells (Fig. 1B).

Gene silencing of GOAT from these cells decreased acyl ghrelinproduction, indicating that GOAT is essential. This inhibition ofghrelin acylation was achieved with several siRNAs to distinctregions of GOAT, confirming its role in ghrelin acylation. TheGOAT gene-silencing effect was dose-dependent and correlatedwith ghrelin octanoylation levels in TT cells (Fig. 2 B and C).Silencing of other MBOAT members in TT cells had no effect onthe octanoylation of ghrelin, implicating GOAT’s function specif-ically in the acylation of ghrelin. Transient coexpression of GOATand ghrelin in HEK-293 cells recapitulated the secretion of oc-tanoylated ghrelin. HEK-293 cells do not express either GOAT orghrelin (data not shown). MS/MS fragmentation analyses of GOATacylated ghrelin shows that the acylation is on serine-3 identical tostomach-produced acyl ghrelin.

GOAT shares structural similarities with members of theMBOAT family of acyl transferases (16–18). When we coexpressedghrelin with other members of the MBOAT family, includingMBOAT1, MBOAT2, MBOAT3, MBOAT5, human-BB1, porcu-pine, and FKSG89, we were able to achieve ghrelin octanoylationonly with GOAT. Moreover, mutating the conserved catalytichistidine residue in GOAT to alanine (H338A) abolished itsactivity, demonstrating that GOAT is an MBOAT.

GOAT can also acylate ghrelin with other fatty acids, besidesoctanoate, ranging from acetate to tetradecanoic acid. Peak inten-sities for acyl-modified forms of ghrelin corresponding to C7 to C12

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Fig. 4. GOAT gene-null mice lack octanoylated ghrelin in circulation. Bloodprofiles for acylated and des-acyl ghrelin in either wild-type (Upper) or GOATgene-disrupted (Lower) mice were determined by using the ghrelin IPMSassay. Arrow denotes location of octanoylated ghrelin. Ghrelin peptide stan-dards were mouse SIL acyl (m/z 3,338) and des-acyl ghrelin (m/z 3,211) pep-tides, respectively.


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appear most intense; however, it is important to point out that thesedata are affected by at least three factors: (i) the uptake of fattyacids by the HEK-293 cells; (ii) the selectivity of the protectingantibody in our assay (to prevent ghrelin desacylation); and (iii) thefatty acid selectivity by GOAT. Nevertheless, these data are rele-vant because tissue-derived and circulating ghrelin forms are mainlymodified with octanoate and decanoate fatty acid residues. Ourobservations suggest that there is some specific and perhaps uniquefatty acid metabolism occurring in the ghrelin-producing gastricX/A cells ensuring that primarily octanoate and decanoate fattyacid substrates are available for GOAT to acylate ghrelin.

GOAT is conserved across vertebrates. Our GOAT and ghrelincoexpression studies show that functional GOAT is also present inrats, mice, and zebrafish. These observations are consistent with theidentification of octanoylated forms of ghrelin peptides acrossvertebrates, including lower vertebrates such as zebrafish (3, 5).Zebrafish GOAT, which shares �60% amino acid similarity tohuman GOAT, is capable of acyl modifying human ghrelin, sug-gesting that structural domains important for acylation are con-served across the distinct GOAT species. Structure to activityrelationship studies for GOAT’s function will be needed to definethese domains. The ability of zebrafish GOAT to octanoylatehuman ghrelin also highlights the structural conservation of ghrelinas a substrate for octanoylation by GOAT. Human and zebrafishforms of ghrelin differ in amino acid length and composition.However, they share a 7-aa conserved region in their N terminicomposed of GSSFLSP and GTSFLSP for human and zebrafishghrelin, respectively. GOAT may recognize structural aspects ofthis motif to specifically acyl modify ghrelin.

GOAT is essential for the acylation of ghrelin in mice. In vivo, lossof function of GOAT in mice yields the absence of octanoylatedghrelin, conclusively demonstrating the critical role of GOAT in itsacylation (Fig. 5). Future studies with these GOAT-null mice are

needed to determine the physiological consequences of the specificdeficiency of acylated ghrelin.

Stomach and pancreas tissues express transcripts for ghrelin andGOAT. This concordant transcript expression for GOAT andghrelin in human stomach and pancreas tissues is consistent withGOAT being the acyltransferase for ghrelin. It is well establishedthat stomach is the principal tissue for acylated ghrelin productionand that changes in ghrelin production in this tissue greatly impactfluctuations caused by metabolic adaptation in organisms (19, 20).Acylated ghrelin also regulates insulin secretion; however, thesource of acylated ghrelin in pancreas remains controversial. ThatGOAT is coexpressed with ghrelin in pancreas may indicate thatlocally produced acylated ghrelin mediates the observed effects oninsulin secretion.

Disrupting ghrelin signaling by targeting the ghrelin or ghrelinreceptor genes blunts weight gain from a high-fat diet. Specifically,GHSR-null mice eat less food, metabolize more fat, become lessadipose, and remain more insulin-sensitive (21). Similarly, ghrelin-null mice resist weight induced by early exposure to diets containinghigh fat. These mice display decreased adiposity and an increase inenergy expenditure (20). Of added interest is the phenotype of theghrelin and ghrelin receptor double knockout mice, which showdecreased body weight, increased energy expenditure, and in-creased motor activity on a standard chow diet (22). Becauseoctanoylated ghrelin promotes food intake and adiposity and alsosuppresses insulin secretion and impairs glucose tolerance, GOATmay provide a critical molecular target in developing novel thera-peutics for obesity and type 2 diabetes.

Materials and MethodsReagents. Rat and dog ghrelin peptides (1–28) were obtained from PhoenixPharmaceuticals.Stable isotope-labeled(SIL)humanghrelinpeptides (1–28)werefrom Midwest Biotech. Two monoclonal antibodies against ghrelin were used forthese studies and prepared at Lilly Research Laboratories. Their specificities weretoward the octanoylated N terminus (antibody C2-5A1) and the carboxyl termi-nus (antibody D4-7.1). Fatty acids and N-octyl glucopyranoside detergent (cata-log no. O3757) were from Sigma. TT Cells were from American Type CultureCollection (catalog no. CRL-1803). Cell culture reagents, magnetic beads (cat-alog no. 142.04), and GripTite 293 MSR Cells (catalog no. R795-07) were fromInvitrogen.

Transcript Silencing in TT Cells. TT cells were cultured in Ham’s F12K media (ATCC30-2004) as described by the supplier. Introduction of specific siRNA complexesinto cells was done with the Amaxa Nucleofector II Device using 5 � 106 cellsfollowing the manufacturer’s recommended protocol. The following fivedouble-stranded RNA-silencing sequences were used to target GOAT transcript:siRNA7-1, 5�-UGU UGC AGA CAU UUG CCU UCU-3� (siRNA 7-1); siRNA7-3, 5�-AAUGCC UAA ACG UGG CAG UGA-3� (siRNA 7-3); stealth-1, 5�-CAG AUU CUU GGACUA GAA UGC CUA A-3� (siRNA 7-5); stealth-2, 5�-CGG GAC UGA CUG AUU GCCAGC AAU U-3� (siRNA 7-6); and stealth-3, 5�-AGC UGA CUA CCU GAU UCA CUCCUU U-3� (siRNA 7-7). As a control, cells were treated with the nontargetingcontrol (NTC-2) siRNA (catalog no. D-001210-02-05 from Dharmacon). Double-stranded silencing RNAs stealth 1–3 were from Invitrogen, and siRNA7-1 andsiRNA7-3 (catalog no. 1027020) were custom siRNAs from Qiagen. Cells in TT cellmedia were allowed to adhere overnight to T-25 tissue culture flasks. Cell mediawere replaced with TT cell culture media supplemented with 125 �g/ml octanoicacid, 0.4 ng/ml SIL human octanoylated ghrelin, and 10 �g/ml C2-5A1 antibody toprevent ghrelin desacylation. Cells were allowed to incubate for 6 days. After thisperiod, cell media were acidified to 50 mN HCl and stored at �80°C until ready forghrelin IPMS analyses. Cell pellets were scraped and stored at �80°C until readyfor total RNA isolation.

Determination of GOAT Transcript Levels in TT Cells. GOAT mRNA levels weredetermined with total RNA by using the RNeasy kit from Qiagen. One microgramof total RNA was converted to cDNA by using a High Capacity cDNA ReverseTranscription kit (Applied Biosystems). Quantitative RT-PCR was performed byemploying a standard curve method using a 7900HT instrument (Applied Biosys-tems).Twenty-microliterPCRswerepreparedcontaining1�Universalmastermix(catalog number 4305719, Applied Biosystems), 0.8 �M custom forward primer(5�-GGCTCTCTGTGCTCCTTCCA-3�), 0.8 �M custom reverse primer (5�-AGAGT-GTCTGGGATGCAAAGC-3�), 0.2 �M probe containing a 5� 6-FAM label with 3�black hole quencher 1 (BHQ1) (5� FAM-CTGGACCCTTGAACACGAGCCTGAAA-

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Fig. 5. GOAT and ghrelin transcript profiles in human tissues. Origene’sTissueScan Real-Time 48 human tissue panel was used for these studies.Ghrelin and GOAT relative transcript levels were normalized to �-actin tran-script amounts and then calibrated to stomach expression in each profile,which was given an arbitrary value of 1. Relative transcript levels for 22 majortissues are shown. (A) GOAT is expressed mainly in stomach and pancreashuman tissues. (B) Ghrelin transcripts were abundantly detected in stomachand modestly in pancreas human tissues.

6324 � www.pnas.org�cgi�doi�10.1073�pnas.0800708105 Gutierrez et al.

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BHQ1–3�), and 4 �l of template cDNA diluted 1:50 in 10 mM Tris (pH 7.5). Primersand probes were from Biosource International. Ribosomal RNA (rRNA) levelswere with the assay from Applied Biosystems (catalog no. 4310893E). PCR con-ditions for GOAT and 18s rRNA were as follows: 50°C for 2 min, 95°C for 10 min,followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The data from GOATwere normalized to the 18s rRNA and calibrated relative to nontargeting control.

Ghrelin and GOAT Gene Transient Transfection in Griptite 293 MSR Cells. Thefull-length ghrelin cDNA was obtained from Origene (catalog no. TC123546). The5� GOAT sequence was obtained with the SMART RACE kit (5� RACE; catalog no.634914, Clontech) with TT cell RNA. GOAT transcript specific primers (5�-CCTCCTCTCCAGGGCTCTGACCAAGCTC-3� and 5�-GGCAGTGCCTTACACACAT-GCTCAGAC-3�), SMART II A oligo, and the universal primer mix provided with theSmart RACE kit were used as described by the manufacturer. Nucleotide primers5�-CACCATGGAGTGGCTTTGGCTG-3� and 5�-TCAGTTACATTTGTGCTTTCTCT-TCGCC-3�, designed to encompass the entire coding region for the GOAT cDNA,were used with TT cell cDNA generated with the SMART RACE kit. The amplifiedGOAT gene was cloned into pcDNA3.2/V5/GW/D-TOPO and sequenced on bothstrands(Agencourt).Cell transfectionstudieswereperformedinGriptite293MSRcellsbyusingMirus transfectionreagents.Cellswereallowedtoadhereovernightto T-25 cell culture flasks in DMEM containing 10% FBS, 0.1 mM MEM nonessen-tial amino acids, and 600 �g/ml geneticin (Griptite 293 MSR growth medium).After overnight incubation, the medium was replaced with the Griptite 293 MSRgrowth medium supplemented with 125 �g/ml octanoic acid, 0.4 ng/ml SILhuman octanoylated ghrelin, and 10 �g/ml C2-5A1 antibody. Cells were allowedto incubate for 3 days. Cell media were acidified to 50 mM HCl and stored at�80°C until ready for ghrelin IPMS analyses.

Ghrelin Immunoprecipitation Reactions. Monoclonal antibodies (D4-7.1) werecovalently coupled to Invitrogen/Dynal magnetic beads following the manufac-turer’s procedure. Acidified media were extracted on tC18 Sep Pak cartridges(catalog no. WAT036805, Millipore). Peptides were eluted with 60% acetonitrilein 0.1% trifluoroacetic acid and lyophilized. Pellets were suspended in 275 �l ofbuffer (140 mM Tris�HCl/50 mM Hepes/150 mM NaCl/0.1% N-octyl glucopyrano-side, pH 7.5), exposed to �1 �g of anti-ghrelin antibody (D4-7.1) bound tomagnetic beads and incubated either overnight at 4°C or for 2 h at roomtemperature. Antibody–antigen complexes were washed (500 �l) twice in 50 mMTris�HCl, 50 mM Hepes, and 150 mM NaCl (pH 7.5) and twice in distilled water.Immunocomplexes were acidified with 10 �l of a solution of 0.1% trifluoroaceticacid, removed from magnetic beads, and processed by using C18 ZipTips (catalogno. ZTC18S, Millipore). Ghrelin peptides were eluted by using 3.0 �l of 50%

acetonitrile/0.1% TFA saturated with �-cyano-4-hydroxy-cinammic acid matrix. A1-�l volumefromeacheluatewasspottedontargetplatesasdescribedpreviouslyand analyzed by using MALDI-TOF MS (23). We refer to the ghrelin immunopre-cipitation reactions combined with the MALDI-TOF MS analysis as the ghrelinIPMS assay.

MALDI-TOF MS. An Applied Biosciences 4700 MALDI-TOF mass spectrometer(Applied Biosystems) was used for MS analysis under optimized conditions forghrelin peptide detection.

Transcript Profiling in Human Tissues. Transcript profiling was performed byusing TissueScan Real-Time human cDNA panels (Origene Technologies) accord-ing to the manufacturer’s protocol. The cDNA-specific gene expression assayswere obtained from Applied Biosystems: ghrelin, HS00175082�m1; �-actin,4310881E. GOAT custom primers and a probe specific for the exon 1 and exon 2splice junction were designed with Primer Express (Applied Biosystems) andsupplied by Applied Biosystems [forward primer, 5�-CCCTTTGCACTTCTCTTCAAT-TATC; reverse primer, 5�-CGAGCACGGCGTAGGAA; probe, 5�-FAM-TCGTGCCAG-GTACCT-MGBNFQ-3� containing 5�-6-FAM label and 3�-minor groove bindingnonfluorescent quencher (MGBNFQ)].

Blood Ghrelin Profiling in GOAT-Null Mice. GOAT-null mice were generated byTaconic/Artemis using C57BL/6N cells from C57/TacN mice. Blood from homozy-gous GOAT-null or wild-type littermate animals was collected and immediatelymixed 1:1 in a solution of 25 mM EDTA, 1 mg/ml protease inhibitor mixture(catalog no. 11 873 580 001, Roche), 1 M NaCl, and 200 mM HCl to prevent ghrelindesacylation. Ghrelin peptide standards for acyl and des-acyl ghrelin were addedat 500 and 250 pg/ml, respectively. The acidified whole blood was ethanol-precipitated (3:1 ethanol:blood ratio), and the resulting supernatants were fur-ther ether-precipitated. Pellets were briefly dried, suspended in Tris-Hepesbuffer, and processed as described above for ghrelin immunoprecipitation andMS analyses.

Note Added in Proof. While this manuscript was in revision, Yang et al. describeda similar observation for GOAT as the acyl transferase for ghrelin (24).

ACKNOWLEDGMENTS. We thank Dr. David S. Bredt for his constructive com-ments in the preparation of this manuscript and Drs. Tamer Coskun, LawrenceGelbert, Niles Fox, and Mark Heiman for discussions and support during theimplementation of these studies. We also acknowledge Jeff Arnold, Yue-WeiQian, Cara Ruble, Brent Sexton, and He Wang for their bioinformatic and tech-nical assistance in this effort.

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