Ghrelin-like peptide with fatty acid modification and O-glycosylation in the red stingray, Dasyatis akajei.

Abstract

Ghrelin (GRLN) is now known to be an appetite-stimulating and growth hormone (GH)-releasing peptide that is predominantly synthesized and secreted from the stomachs of various vertebrate species from fish to mammals. Here, we report a GRLN-like peptide (GRLN-LP) in a cartilaginous fish, the red stingray, Dasyatis akajei.
The purified peptide contains 16 amino acids (GVSFHPQPRS10TSKPSA), and the serine residue at position 3 is modified by n-octanoic acid. The modification is the characteristic of GRLN. The six N-terminal amino acid residues (GVSFHP) were identical to another elasmobranch shark GRLN-LP that was recently identified although it had low identity with other GRLN peptides. Therefore, we designated this peptide stingray GRLN-LP. Uniquely, stingray GRLN-LP was O-glycosylated with mucin-type glycan chains [N-acetyl hexosamine (HexNAc)3 hexose(Hex)2] at threonine at position 11 (Thr-11) or both serine at position 10 (Ser-10) and Thr-11. Removal of the glycan structure by O-glycanase made the in vitro activity of stingray GRLN-LP decreased when it was evaluated by the increase in intracellular Ca2+ concentrations using a rat GHS-R1a-expressing cell line, suggesting that the glycan structure plays an important role for maintaining the activity of stingray GRLN-LP.
This study reveals the structural diversity of GRLN and GRLN-LP in vertebrates.

Full text

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BioMed Central
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BMC Biochemistry
Open Access
Research article
Ghrelin-like peptide with fatty acid modification and
O-glycosylation in the red stingray, Dasyatis akajei
Hiroyuki Kaiya*3, Shiho Kodama1, Koutaro Ishiguro2, Kouhei Matsuda2,
Minoru Uchiyama2, Mikiya Miyazato3 and Kenji Kangawa3
Address: 1Biochemical research laboratories, ASUBIO PHARMA CO, LTD, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8513,
Japan, 2Department of Biology, Faculty of Science, Toyama University, Toyama 930-8555, Japan and 3Department of Biochemistry, National
Cardiovascular Center Research Institute, Osaka 565-8565, Japan
Email: Hiroyuki Kaiya* - kaiya@ri.ncvc.go.jp; Shiho Kodama - kodama.shiho.hm@asubio.co.jp; Koutaro Ishiguro - kmatsuda@sci.u-
toyama.ac.jp; Kouhei Matsuda - kmatsuda@sci.u-toyama.ac.jp; Minoru Uchiyama - uchiyama@sci.u-toyama.ac.jp;
Mikiya Miyazato - miyazato@ri.ncvc.go.jp; Kenji Kangawa - kangawa@ri.ncvc.go.jp
* Corresponding author
Abstract
Background: Ghrelin (GRLN) is now known to be an appetite-stimulating and growth hormone
(GH)-releasing peptide that is predominantly synthesized and secreted from the stomachs of
various vertebrate species from fish to mammals. Here, we report a GRLN-like peptide (GRLN-
LP) in a cartilaginous fish, the red stingray, Dasyatis akajei.
Results: The purified peptide contains 16 amino acids (GVSFHPQPRS10TSKPSA), and the serine
residue at position 3 is modified by n-octanoic acid. The modification is the characteristic of GRLN.
The six N-terminal amino acid residues (GVSFHP) were identical to another elasmobranch shark
GRLN-LP that was recently identified although it had low identity with other GRLN peptides.
Therefore, we designated this peptide stingray GRLN-LP. Uniquely, stingray GRLN-LP was O-
glycosylated with mucin-type glycan chains [N-acetyl hexosamine (HexNAc)3 hexose(Hex)2] at
threonine at position 11 (Thr-11) or both serine at position 10 (Ser-10) and Thr-11. Removal of
the glycan structure by O-glycanase made the in vitro activity of stingray GRLN-LP decreased when
it was evaluated by the increase in intracellular Ca2+ concentrations using a rat GHS-R1a-expressing
cell line, suggesting that the glycan structure plays an important role for maintaining the activity of
stingray GRLN-LP.
Conclusions: This study reveals the structural diversity of GRLN and GRLN-LP in vertebrates.
Background
Ghrelin (GRLN), which generally consists of 28 amino
acids, was first identified in the stomachs of rats and
humans as an endogenous ligand for the growth hormone
secretagogue-receptor 1a (GHS-R1a)[1]. The serine resi-
due at position 3 of this peptide (Ser-3) contains a unique
octanoyl modification, and the acylation is necessary for
the peptide to bind and activate GHS-R1a [1,2]. In mam-
mals, GRLN is an important hormone involves in various
physiological events such as pituitary, cardiovascular, ster-
oidogenic, and developmental functions and energy
homeostasis [3-6].
Published: 14 December 2009
BMC Biochemistry 2009, 10:30doi:10.1186/1471-2091-10-30
Received: 24 April 2009
Accepted: 14 December 2009
This article is available from: http://www.biomedcentral.com/1471-2091/10/30
© 2009 Kaiya et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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In non-mammals, GRLN has been identified in species
from fish to birds (reviewed by [7-11]). Very recently,
endogenous GRLN form was determined in a bony fish,
goldfish [12]. Non-mammalian mature GRLNs are com-
posed of 17 to 28 amino acids, and are known to involve
in regulating pituitary functions in teleosts [6], amphibi-
ans [13] and birds [14], and feeding in teleosts [15-19]
and birds [7,20-23]. An inhibitory effect of GRLN on
drinking in birds (chicken) was also recently reported
[24].
We identified a GRLN-like peptide (GRLN-LP) in the
stomach of primitive vertebrates, cartilaginous fish, the
hammerhead shark (Sphyrna lewini) and blacktip reef
shark (Carcharhinus melanopterus) [25]. The GRLN-like
peptide from both sharks consisted of 25 amino acids,
and only three amino acids differed between the two spe-
cies. Like other vertebrate GRLN, Ser-3 of GRLN-LP from
sharks had been modified by n-octanoic or n-decanoic
acid. However, the first seven N-terminal residues, which
are generally highly conservative region of GRLN includ-
ing the active core, had low identity between sharks (GVS-
FHPR) and those of other species (GSSFLSP, GTSFLSP and
GSTFLSP). This difference is one of the reasons why this
peptide was designated GRLN-LP though there is Ser-3
acylation. In addition to this, the C-terminal end of the
shark GRLN-LP has not been amidated, which is a specific
feature of teleost GRLN. However, we have proposed that
shark GRLN-LP exhibits ancestral features of GRLN mole-
cule that are present in higher vertebrate.
Here we report the structure of GRLN-LP from the stom-
achs of another elasmobranch, the red stingray, Dasyatis
akajei, and identification of the cDNA that encodes the
precursor protein. Interestingly, we revealed that the sting-
ray GRLN-LP is not only octanoylated at Ser-3 but also
possesses a mucin-type glycan structure at threonine
(Thr)-11 or both Ser-10 and Thr-11.
Methods
Purification of GRLN-LP from stingray stomachs
Stingrays, Dasyatis akajei, were collected at Toyama bay
(Toyama, Japan). Frozen stomachs (approximately 40 g)
were used as the starting material. All animal experiments
were conducted in accordance with Guidelines for the
Care and Use of Animals of the University of Toyama and
of National Cardiovascular Center (ref. no. 8053). GRLN
was purified as previously described [26] with slight mod-
ifications. During the purification process, GRLN activity
was monitored by measuring changes in the intracellular
calcium ion (Ca2+) concentrations in a cell line that stably
expressing rat GHS-R1a (CHO-GHSR62).
Stomach tissues were boiled in five volumes of Milli-Q
level water, minced, acidified with concentrated acetic
acid (AcOH) to 1 M, and homogenized. The supernatant
was obtained by centrifugation. Next, cold acetone was
added to the AcOH-extracts at a final concentration of
66%, after which the mixture was stirred over night and
then centrifuged. The resulting supernatant was evapo-
rated and purified with a Sep-Pak Vac 35 cc C18 cartridge
(Waters, Milford, MA) to enrich the peptide components.
The cartridge was successively eluted with 25% acetonit-
lile containing 0.1% trifluoroacetic acid (TFA) and 60%
acetonitlile containing 0.1% TFA. The lyophilized Sep-Pak
fraction that was eluted with 60% acetonitlile containing
0.1% TFA was dissolved in 1 M AcOH, and then subjected
to cation-exchange chromatography using a SP-Sephadex
C-25 (GE Healthcare UK Ltd., Buckinghamshire, Eng-
land). Successive elution with 1 M AcOH, 2 M pyridine
and 2 M pyridine-AcOH (pH 5.0) yielded three fractions:
SP-I, SP-II and SP-III, respectively.
The basic peptide-enriched SP-III fraction was subjected to
carboxymethyl (CM)-ion exchange HPLC (TSKgel CM-
2SW, 4.6 × 250 mm, Tosoh, Tokyo, Japan) at a flow rate
of 1 ml/min. A two-step gradient was made from solvent
A (10 mM ammonium formate (pH 4.8) in 10% acetonit-
lile) to 25% solvent B (1 M ammonium formate (pH 4.8)
in 10% acetonitlile) for 10 min and then to 55% solvent
B for 90 min. The eluate was collected in 1-ml fractions
from the start of the gradient program. Based on the assay
results, GRLN activities were eluted in clusters. Thus, we
performed secondary CM-HPLC on the active fractions
with a more shallower two-step gradient profile from sol-
vent A to 15% solvent B for 10 min and then to 35% sol-
vent B for 80 min. The eluate was collected in 1-ml
fractions every 1 min, 20 min after injection into the
HPLC system.
Active CM-HPLC fractions were desalted by Sep-Pak treat-
ment, lyophilized, and then subjected to an anti-rat
GRLN1-11 immunoglobulin G (IgG) immuno-affinity
column to purify GRLN-immuno-cross reactive sub-
stances [26]. This immuno-affinity column effectively
absorbed the shark GRLN-LP [25]. The adsorbed sub-
stances were eluted with 60% acetonitlile containing
0.1% TFA and then separated by two different reverse-
phase (RP)-HPLC methods. The samples were first
applied to a preparative RP-HPLC with a Symmetry C18
column (3.9 × 150 mm, Waters) at a flow rate of 1 ml/min
under a linear gradient from 10% to 60% acetonitlile con-
taining 0.1% TFA for 40 min. Active fractions were further
purified on a Symmetry C18 column (2.1 × 150 mm,
Waters) or a diphenyl column (2.1 × 150 mm,
219TP5215, Vydac, Hesperia, CA) at a flow rate of 0.2 ml/
min under a gradient from 10% to 60% acetonitlile con-
taining 0.1% TFA for 40 min. The eluate that corre-
sponded to each absorbance peak was collected. For
peptide sequencing, approximately 5 pmol of the purified

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peptide, which was estimated based on the absorbance
peak height, was subjected to protein sequencing (model
494, Applied Biosystems, Foster City, CA).
Mass spectrometry
The molecular weights of the purified peptides were deter-
mined using matrix-assisted laser desorption ionization-
time of flight-time of flight (MALDI-TOF-TOF) mass spec-
trometry (4700 proteomics analyzer, Applied Biosystems,
Foster City, CA) with α-Cyano-4-hydroxycinnamic acid
(CHCA) (Sigma-Aldrich Chem. Corp., WI) as a matrix.
Prediction of O-glycosylation sites
The detected peptide mass and its profile indicated that
the peptide was possibly glycosylated. The NetOGlyc pro-
gram, which is available at the Center for Biological
Sequences CBS Prediction Server http://www.cbs.dtu.dk,
was used to predict the potential O-glycosylation sites.
Cloning of stingray GRLN-LP cDNA
The nucleotide sequence of stingray GRLN-LP cDNA was
determined using the Rapid Amplification of cDNA Ends
(RACE) PCR Kit (Invitrogen, Carlsbad, CA). Total RNA (2
μg) was extracted from a stingray stomach and then
reverse-transcribed using Omniscript RT (QIAGEN
GmbH, Hilden, Germany). For 3'-RACE PCR, four degen-
erate primers were designed based on the sequence of the
seven N-terminal amino acids in stingray GRLN-LP
(G1VSFHPQ7) that was identified by protein sequencing.
Among these primers, the rayGRL-s2 primer (5'-GGN
GTN AGY TTY CAY CCN CA-3') effectively amplified
expected cDNA fragment. PCR was performed using 50
pmol/reaction of rayGRL-s2, an adaptor primer supplied
in the kit, ExTaq DNA polymerase (TaKaRa, Shiga, Japan)
under the following amplification conditions: 94°C for 1
min, 35 cycles at 94°C for 30 sec, 53°C for 30 sec, and
72°C for 1 min; followed by a final extension for 3 min at
72°C. The amplified products were purified by the Wizard
PCR Preps (Promega, Madison, WI). Second-round
nested PCR was performed on the purified cDNA with
four degenerate sense primers (50 pmol/reaction) that
were based on the amino acid sequence of stingray GRLN-
LP (F4HPQPRS10). Among these primers, two primers,
rayGRL-s6 (5'-TTY CAY CCN CAR CCN CGN AG-3') and
rayGRL-s8 (5'-TTY CAY CCN CAR CCN AGR AG-3') effec-
tively amplified expected cDNA fragment under the fol-
lowing conditions: 94°C for 1 min, 35 cycles at 94°C for
30 sec, 55°C for 30 sec, and 72°C for 1 min; and a final
extension for 3 min at 72°C. The approximately 400-bp
amplified products were subcloned into the pCRII-TOPO
vector (Invitrogen). The nucleotide sequence of the insert
was determined by a DNA sequencer (ABI PRISM 3100
Genetic analyzer, Applied Biosystems), according to the
BigDye® Terminator Cycle Sequencing Kit protocol
(Applied Biosystems) using the M13 forward or reverse
primer.
For 5'-RACE PCR, two gene-specific primers were
designed based on the partial sequence of the stingray
GRLN-LP cDNA that was obtained by 3'-RACE PCR: ray-
GRL-as1, 5'-GGA CGA TGC ATT GAT CTG CGG-3' and ray
GRL-as2, 5'-CCC GTT CAG GTC GGA CGA TGC-3'. Pri-
mary PCR was performed using rayGRL-as1, an anchor
primer supplied in the 5'-RACE Kit, and ExTaq DNA
polymerase under the following reaction conditions:
94°C for 2 min, 35 cycles at 94°C for 30 sec, 56°C for 30
sec, and 72°C for 1 min; and a final extension for 3 min
at 72°C. The second-round nested PCR was performed
with 5 pmol/reaction of the rayGRL-as2, an abridged uni-
versal amplification primer (AUAP) supplied in the 5'-
RACE Kit, and ExTaq DNA polymerase under the same
conditions described above. The rayGRL-as2 primer was
designed inside the rayGRL-as1 primer, but nine nucle-
otides on the 3'-side of the rayGRL-as2 were identical to
the primer sequence of rayGRL-as1. Thus, the target cDNA
could be amplified effectively by the second-round nested
PCR. The amplified products, which were approximately
480 bp, were subcloned into the pCRII-TOPO vector and
sequenced.
Amplification of the full-length stingray GRLN-LP cDNA
To confirm the precise nucleotide sequence of the full-
length stingray GRLN-LP cDNA, we performed PCR using
a proofreading, Pyrobest DNA polymerase (TaKaRa). The
template cDNA for the 3'-RACE PCR was used because the
Tm of the antisense primer for the 3'end of the stingray
GRLN-LP cDNA was too low to amplify the full-length
cDNA. Thus, PCR was conducted using a sense primer
from the 5'end of the stingray GRLN-LP cDNA (5'-ACG
ACC ACA GAT CCA ACT CGA-3') and AUAP under the
following conditions: 98°C for 30 sec, 30 cycles at 98°C
for 15 sec, 60°C for 30 sec, and 72°C for 1 min. For TA
cloning, an additional 10-min cycle at 72°C was per-
formed using ExTaq DNA polymerase (overhang reac-
tion). The amplified product was subcloned into the
pCRII-TOPO vector and sequenced.
The putative stingray GRLN-LP sequence was analyzed by
BLAST against the NCBI database, and amino acid
sequence alignment and identity analysis were performed
by multiple comparison and maximum matching pro-
gram using GENETYX-Mac ver. 15.0.1 (gap penalty, insert:
-1; extend: -1 for multiple comparison, and default condi-
tion for maximum matching). Phylogenetic tree was
made using Mega 4 software http://www.megasoft
ware.net/.
Deglycosylation with O-glycanase
Stingray GRLN-LP was O-glycosylated with mucin-type
sugar chains. To examine the role of this sugar chain mod-
ification in the GRLN-like activity of stingray GRLN-LP, a
high-yield preparation of purified stingray GRLN-LP
(peak 2, Table 1) was treated with the O-glycanase, end-α-

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N-acetylgalactosaminidase (Calbiochem, Darmstadt, Ger-
many). This enzyme catalyzes the hydrolysis of the unsub-
stituted Galβ1,3GalNAc core dissaccharide attached to a
Ser or Thr of glycopeptides to generate free oligosaccha-
rides. Approximately 5 pmol of native stingray GRLN-LP
(peak 2), synthesized stingray des-acyl GRLN-LP, and
octanoylated stingray GRLN-LP were incubated separately
with 1.25 mU O-glycanase in 100 μl 50 mM sodium phos-
phate (NaH2PO3) buffer (pH 5.0) for 16 h at 37°C, fol-
lowed by an incubation for 15 min at 70°C to inactivate
the enzyme. The reaction mixture was subjected to RP-
HPLC on a Symmetry C18 column (2.1 × 150 mm,
Waters) at a flow rate of 0.2 ml/min under a linear gradi-
ent from 10% to 60% acetonitlile containing 0.1% TFA
for 40 min. The absorbance peaks that corresponded to
each catalyzed peptide were collected. Two peaks
appeared from this reaction: one was stingray GRLN-LP
without sugar chains, and the other was the original
native peptide. We did not observe any changes in the elu-
tion pattern of synthetic stingray des-acyl GRLN-LP and
octanoylated GRLN-LP after the catalytic treatment.
Quantitative real-time PCR of stingray GRLN-LP in
stingray tissues
We performed quantitative real-time PCR (qPCR) to
examine the GRLN-LP cDNA expression levels in various
stingray tissues. Total RNA was extracted from 21 tissues
from two separate stingrays to examine the variation in
expression levels. First-strand cDNAs were synthesized
from 1 μg of DNase-I- (Invitrogen) treated total RNA
using the QuantiTect RT Kit (QIAGEN GmbH) and an
oligo-dT12-18 primer. PCR amplification was performed
using the LightCycler system (Roche Applied Science,
Mannheim, Germany) and the QuantiFast SYBR Green
PCR Kit (QIAGEN GmbH). To generate a standard curve,
full-length stingray GRLN-LP cDNA or partial stingray β-
actin cDNA fragment in the pCRII-TOPO vector was line-
arized by Xba-I digestion. The linearized plasmid was seri-
ally diluted from 5 × 106 to 5 × 103 molecules to generate
a standard curve that was used to determine the cDNA
copy number. All qPCR amplifications were performed in
duplicate. All specific quantities were normalized as the
copy number relative to the total RNA [27] and to stingray
β-actin. For the GRLN-LP analysis, sense (5'-TCC CTC
ACC CTC AAG GCA GAG-3') and antisense primers (5'-
TCAT CTC CCA CTG GCA ACT GG-3') were designed to
amplify a 170-bp product. For the β-actin analysis, sense
(5'-GAT CTG TAT GCC AAC AAC GTC-3') and antisense
primers (5'-CAG AGA TGC CAG AAT AGA GCC-3') were
designed to amplify a 194-bp product. The PCR reaction
mixture (20 μl) contained 100 ng of cDNA, 1× QuantiFast
SYBR Green PCR mix, and 5 pmol of each primer was pre-
pared according to the manufacturer's protocol. The PCR
conditions were 95°C for 5 min, 35 cycles at 95°C for 10
sec, 60°C for 30 sec. Amplified products were electro-
phoresed on a 1.5% agarose gel to determine the product
size and quantity.
Results
Purification of stingray GRLN-like peptide
The SP-III fraction from the SP-Sephadex C-25 cation-
exchange chromatography contained GRLN-like activity.
Next, we subjected the basic peptide-enriched SP-III frac-
tion to CM-HPLC (pH 4.8) with a gradient program.
However, the GRLN-like activities are present in positions
where a large number of peptides eluted (fractions 17-29,
data not shown). To reduce the peptide content, active
fractions were subjected again to CM-HPLC with a much
shallower two-step gradient profile. As a result, the GRLN-
like activity was divided into four groups from A to D (Fig.
1A). Each group was purified by anti-rat GRLN1-11 IgG
immuno-affinity column chromatography, followed by
RP-HPLC until a single peak was isolated. Figs. 1B and 1C
show representative preparative RP-HPLC and final RP-
HPLC profiles after immuno-affinity chromatography of
group B (Fig. 1A).
The estimated peptide yield from each peak height is sum-
marized in Table 1. Overall, seven species of peptides were
isolated. The amino acid sequences of peptides in high
yield peaks 2 and 4 were analyzed. The amino acid
sequences of 16 residues were determined: GVXFH-
PQPRXXSKPSA for peak 2 and GVXFHPQPRSXSKPSA for
peak 4 (X, unidentified). The amino acid at position 3 was
not detected, probably due to the characteristic acyl mod-
ification at this position of GRLN. However, the reason
why amino acid at positions 10 and 11 could not be iden-
tified was uncertain.
Cloning of cDNA encoding the stingray peptide precursor
To determine the complete amino acid sequence of the
stingray peptide, we isolated cDNA encoding the peptide
precursor from stingray stomach mRNA based on the
identified amino acid sequence. The identified nucleotide
sequence of the stingray peptide was 527 bp in length,
which consisted of a 90-bp 5' untranslated region (UTR),
a 297-bp coding region, and a 140-bp 3'UTR (accession
number AB480033, Fig. 2A). A polyadenylation signal
Table 1: Summary of stingray GRLN-LP purification
Grou
p
Pea
k
Yields
(pmol)
Mass-1
[M+H]+
Mass-2
[M+H]+
A
B
1
2
3
4
5
6
7
10
24
4
21
4
3
2
2580.15
3351.68
3375.42
2580.26
2604.15
2604.17
2594.15
3513.74
3537.46
2742.33
2766.20
2766.26
2756.21
C
D

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Purification of stingray ghrelin-like peptide (GRLN-LP) from stomach extracts
Figure 1
Purification of stingray ghrelin-like peptide (GRLN-LP) from stomach extracts. Black bars indicate the measured
fluorescence changes in intracellular calcium ion concentrations in CHO cells expressing rat GHS-R1a (CHO-GHSR62). (A)
Carboxymethyl (CM)-cation ion-exchange HPLC (pH 4.8) of the SP-III fraction of stomach extracts. The GRLN-like activity
was divided into four groups (A-D). (B) Preparative reverse-phase (RP)-HPLC (Symmetry C18, 3.9 × 150 mm) of group B after
purification with an anti-rat GRLN1-11 immuno-affinity column. (C) Final purification of the active fraction indicated in (B) by
another RP-HPLC (Vydac diphenyl, 219TP5215, 2.1 × 150 mm).