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INTRODUCTION |
The 500 species of predatory cone snails have evolved extremely
complex venoms. Each individual species typically has at least 100 different pharmacologically active components of varying molecular weights in its venom; most are small peptides (<30 amino acids) specifically targeted to receptors or ion channels. These small, biologically active peptides found in Conus venoms can be
divided into two general classes, those with sequences that contain few or no cysteine residues and those that are cysteine-rich (conotoxins). Among those in the first group, we have identified conantokins with no
disulfide bridges (1, 2), whereas conopressins (3), contryphans (4, 5),
and the bromoheptapeptide (6) all contain one disulfide bridge. The
Conus peptides rich in disulfide bridges (7, 8) are
synthesized from only a few conotoxin superfamilies with their
diversity generated by hypermutation.
Compared with other gene translation products, Conus
peptides are unusually enriched in a variety of posttranslational
modifications. Some of these are widely distributed among
Conus peptides (such as hydroxylation of proline to
4-trans-hydroxyproline or the amidation of the C terminus by
conversion of the C-terminal glycine residue). Others appear more
highly specialized (
-carboxylation of glutamate to
-carboxyglutamate, bromination of tryptophan to 6-bromotryptophan, sulfation of tyrosine, or the epimerization of L-tryptophan
to D-tryptophan).
In this communication, we describe the purification and biochemical
characterization of a novel Conus peptide that has proven to
be the first known example of the neurotensin family of peptides from a
nonvertebrate source. This 16-amino acid peptide, contulakin-G, contains a posttranslational-modified amino acid not previously found
in a Conus peptide, an O-glycosylated threonine
residue. Although evidence for O-glycosylation (at a Ser
residue) was recently obtained for another Conus peptide,
A-conotoxín SIVA (9), contulakin-G is the first
O-glycosylated Conus peptide for which the
complete structure of both polypeptide and glycan have been determined.
In addition, the successful chemical synthesis of an intact,
biologically active O-glycosylated gene product, as reported
here, has not to our knowledge previously been achieved.
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EXPERIMENTAL PROCEDURES |
Crude Venom--
Conus geographus specimens were
collected from Marinduque island in the Philippines. The crude venom
was obtained by dissection of the venom duct gland and then
freeze-dried and stored at
70 °C.
Peptide Purification--
Freeze-dried C. geographus
venom (1 g) was extracted with 1.1% acetic acid and chromatographed on
a Sephadex G-25 column eluted with 1.1% acetic acid as described
previously (10). A peptide that makes mice sluggish and unresponsive
was purified by a series of
RP-HPLC1 purifications on
preparative and semipreparative and analytical reverse phase
C18 columns as indicated in Fig. 1. A gradient of acetonitrile in 0.1% trifluoroacetic acid was used to elute the peptide from the columns. The major species shown in panel C
was repurified before further characterization.
Bioactivity--
Typically, mice-injected icv with the partially
purified native peptide initially had trouble righting after 5 min,
became sluggish after 12 min, and then rested on their stomachs after 30 min. These signs were used as an assay to identify the biologically active peptide during purification.
Enzyme Hydrolysis--
Approximately 180 pmol of the peptide (6 µl) was incubated with 7 milliunits of
-galactosidase (bovine
testes) (2 µl) in 50 µl of 50 mM citrate/phosphate
buffer (pH 4.5) for 53 h at 32 °C. Approximately 60 pmol of the
peptide (2 µl) was incubated with 2 milliunits of
O-glycosidase (Diplococcus pneumoniae) (2 µl)
in 50 µl of 20 mM cacodylic acid (pH 6.0) for 19 h
at 32 °C.
Chemical Sequence and Amino Acid Analysis--
Automated
chemical sequence analysis was performed on a 477A protein sequencer
(Applied Biosystems, Foster City, CA). Amino acid analysis was carried
out using precolumn derivatization. Approximately 500 pmol of the
contulakin-G was sealed under vacuum with concentrated HCl, hydrolyzed
at 110 °C for 24 h, lyophilized, and then derivatized with
o-phthalaldehyde. The derivatized amino acids were then
analyzed with RP-HPLC.
Mass Spectrometry--
Matrix-assisted laser desorption (MALD)
(11) mass spectra were measured using a Bruker REFLEX (Bruker
Daltonics, Billerica, MA) time-of-flight (12) mass spectrometer fitted
with a gridless reflectron, an N2 laser, and a 100 MHz
digitizer. An accelerating voltage of +31 kV and a reflector voltage
between 1.16 and 30 kV were employed for the post-source decay (13)
measurements. The sample (in 0.1% aqueous trifluoroacetic acid) was
applied with
-cyano-4-hydroxycinnamic acid. Liquid secondary
ionization (LSI) (14) mass spectra were measured using a Jeol HX110
(Jeol, Tokyo, Japan) double-focusing mass spectrometer operated at 10 kV accelerating voltage, 1000 or 3000 resolution. The sample (in 0.1%
aqueous trifluoroacetic acid and 25% acetonitrile) was mixed in a
thioglycerol and dithiothreitol matrix. Nano-electrospray (nano-ESI)
mass spectra were measured using an Esquire ion trap mass spectrometer
(Bruker Daltonics, Billerica, MA). The RP-HPLC-purified sample,
collected in 0.1% aqueous trifluoroacetic acid and acetonitrile, was
diluted in methanol 1% acetic acid, transferred to a nanospray capillary, and analyzed. The mass accuracy was typically better than
1000 ppm for the time-of-flight instrument, 200 ppm for the ion trap
instrument, and 20-100 ppm for the double-focusing mass spectrometer,
depending on the resolving power settings of the magnetic sector
instrument employed.
Synthesis of Contulakin-G--
The solid-phase glycopeptide
synthesis was carried out manually using Fmoc chemistry, with
t-butyl ether side chain protection for tyrosine and serine,
N-tert-butyloxycarbonyl side chain protection for
lysine, and t-butyl ester side chain protection for glutamic acid (protected amino acids were obtained from Bachem, Torrance, CA).
Starting with a Wang resin, the amino acids were coupled with
benzotriazoyloxyl-tris(dimethylamino)phosphonium
hexafluorophosphate/diisopropylethylamine/N-methylpyrrolidone/dichloromethane (15, 16), and the N-deprotections were done with
N-methylpyrrolidone/piperidine (15, 16). The Wang resin was
prepared at The Salk Institute with a substitution of 0.2 nmol/g. After
coupling of the first six amino acids, the resin was coupled with
peracetylated
Fmoc-O(
-D-Galp-(1
3)-
-D-GalpNAc-(1
0)-threonine, synthesized as described elsewhere
(17),2 followed by single
coupling of the remaining nine amino acids in the sequence. The
nonglycosylated peptide was similarly synthesized using Fmoc-threonine
(Bachem, Torrance, CA). The resin was subjected to cleavage conditions
(95% trifluoroacetic acid, 5% anisole (15)), and in the case of the
glycopeptide, the resulting peracetylated glycopeptide was isolated
with RP-HPLC, with the major component, m/z
2322.3 (MALD analysis), corresponding to the desired product (2322.0 Da). After lyophilization, the peracetylated glycopeptide was treated
with 20 µl of sodium methoxide (Sigma) (50 mM) in dry
methanol for 1 min (to remove O-acetyl groups on the sugar (18)) and lyophilized at
20°C. The deacetylated sample was loaded
onto a Waters Prep LC/System 500A equipped with gradient controller,
Waters model 450 variable wavelength detector, and Waters 1000 PrepPack
cartridge chamber column (65.5 × 320 mm) packed with Vydac
C18 15-20-µm particles. Flow conditions: wavelength 230 nm; absorbance units at full scale, 2.0; flow, 100 ml/min; gradient,
20-60% B for 60 min (where the A buffer was 0.1% trifluoroacetic acid in water, and the B buffer was 0.1% trifluoroacetic acid in 60%
aqueous acetonitrile). The fractions (200 ml) were collected manually.
The major component, m/z 2069.9 (LSI analysis),
corresponded to the desired product (2069.98 Da). After preparative
RP-HPLC purification, sufficient purified contulakin-G was obtained for analytical characterization and biological studies. A more extensive characterization of the synthetic contulakin-G including 1H
NMR data will be presented elsewhere.
Co-elution--
The native and synthetic contulakin-G were
analyzed separately (Fig. 6, panels A and B,
respectively) and co-eluted with RP-HPLC (panel C), using a
2.1 × 150-mm Vydac C18 column and a 0.5%/min gradient from 0% to 40% B (where the A buffer was 0.55%
trifluoroacetic acid in water, and the B buffer was 0.05%
trifluoroacetic acid in 90% aqueous acetonitrile).
Binding Studies--
The nonglycosylated Thr10
contulakin-G and synthetic contulakin-G were assayed with the human
neurotensin type 1 receptor using a Biomek 1000 robotic workstation for
all pipetting steps in the radioligand binding assays as described
previously (19). Competition binding assays with
[3H]neurotensin1-13 (1 nM) and
varying concentrations of unlabeled neurotensin1-13,
nonglycosylated Thr10, contulakin-G, or synthetic
contulakin-G were carried out with membrane preparations from HEK-293
cell line. Nonspecific binding was determined with 1 µM
unlabeled neurotensin1-13 in assay tubes with a total
volume of 1 ml. Incubation was at 20 °C for 30 min. The assay was
routinely terminated by the addition of cold 0.9% NaCl (5 × 1.5 ml), followed by rapid filtration through a GF/B filter strip that had
been pretreated with 0.2% polyethylenimine. Details of binding assays
have been described before (20). The data were analyzed using the
LIGAND program (21).
The nonglycosylated Thr10 contulakin-G and synthetic
contulakin-G were separately assayed with the rat neurotensin type 1 and type 2 receptors (rNTR1 and rNTR2) and mouse neurotensin type 3 receptor (mNTR3). 125I-Tyr3
neurotensin1-13 was prepared and purified as described previously (22). Stable transfected CHO cells expressing either the
rNTR1 (23) or the rNTR2 (cloned in the laboratory of J. Mazella by
screening a rat brain cDNA library (Stratagene)) were grown in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
and 0.25 mg/ml G418 (Sigma). Cell membrane homogenates were prepared as
initially described (24). Protein concentration was determined by the
Bio-Rad procedure with ovalbumin as the standard.
Binding Experiments on Cell Membranes--
Membranes (25 µg
for NTR2 and 10 µg for NTR1) were incubated with 0.4 nM
125I-Tyr3 neurotensin1-13 (2000 Ci/mmol) and increasing concentrations of neurotensin1-13,
nonglycosylated Thr10 contulakin-G, or synthetic
contulakin-G for 20 min at 25 °C in 250 µl of 50 mM
Tris-HCl (pH 7.5) containing 0.1% bovine serum albumin and 0.8 mM 1-10-phenanthroline. Binding experiments were terminated by the addition of 2 ml of ice-cold buffer followed by
filtration through cellulose acetate filters (Sartorius) and washing
twice. Radioactivity retained on filters was counted with a
-counter.
Binding Experiments on Solubilized
Extracts--
CHAPS-solubilized extracts (100 µg) were incubated
with 0.2 nM 125I-Tyr3
neurotensin1-13 for 1 h at 0 °C in 250 µl of
Tris-glycerol buffer containing 0.1% CHAPS. Bound ligand was separated
from free ligand by filtration on GF/B filters pretreated with 0.3% polyethylenimine. Filters were rapidly washed twice with 3 ml of
ice-cold buffer and counted for radioactivity.
For binding experiments on mNTR3, membrane homogenates from mouse brain
were resuspended in 25 mM Tris-HCl buffer (pH 7.5) containing 10% (w/v) glycerol, 0.1 mM phenylmethylsulfonyl
fluoride, 1 µM pepstatin, 1 mM iodoacetamide,
and 5 mM EDTA (Tris-glycerol buffer). Solubilization was
carried out by incubating homogenates at a concentration of 10 mg/ml in
the Tris-glycerol buffer with 0.625% CHAPS containing 0.125%
cholesteryl hemisuccinate (25). Solubilized extracts were recovered by
centrifugation at 100,000 × g during 30 min at 4 °C
and used either immediately or stored at
20 °C.
Phosphoinositides Determination--
Cells expressing the rNTR1
or NTR2 were grown in 12-well plates for 15-18 h in the presence of 1 µCi of myo-[3H]inositol (ICN) in a
serum-free Ham's F-10 medium. Cells were washed with Earle buffer (pH
7.5; 25 mM Hepes, 25 mM Tris, 140 mM NaCl, 5 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgCl2, 5 mM glucose) containing 0.1% bovine serum albumin and
incubated for 15 min at 37 °C in 900 µl of 30 mM LiCl
in Earle buffer. Neurotensin1-13 was then added at the
indicated concentrations for 15 min. The reaction was stopped by 750 µl of ice-cold 10 mM HCOOH (pH 5.5). After 30 min at
4 °C, the supernatant was collected and neutralized by 2.5 ml of 5 mM NH4OH. Total
[3H]phosphoinositides (PIs) were separated from free
[3H]inositol on Dowex AG-X8 (Bio-Rad) (26) chromatography
by eluting successively with 5 ml of water and 4 ml of 40 mM and 1 M ammonium formate (pH 5.5). The
radioactivity contained in the 1 M fraction was counted
after the addition of 5 ml of Ecolume (ICN).
Identification of a cDNA Clone Encoding
Contulakin-G--
Contulakín-G encoding clones were selected
from a size-fractionated cDNA library constructed using mRNA
obtained from a C. geographus venom duct as described
previously (27). The library was screened using a specific probe
corresponding to amino acids 10-15 of the peptide (5'-ATR ATN GGY TTY
TTN GT-3'), where R = A or G, N = A, C, G, or T, and Y = C or T. The oligonucleotide was end-labeled and hybridized, and a
secondary screening by polymerase chain reaction was performed on 10 clones that hybridized to this probe as described previously (4).
Clones identified in the secondary screen were prepared for DNA
sequencing as described previously (28). The nucleic acid sequence was
determined according to the standard protocol for Sequenase version 2.0 DNA sequencing kit as described previously (4).
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RESULTS |
Purification of Contulakin-G--
A fraction of C. geographus venom was detected, which made mice exceedingly
sluggish (see Fig. 1). Normally, when
mice that are sitting down are poked with a rod, they immediately get
up and run a considerable distance. Upon icv injection of the fraction from C. geographus indicated in Fig. 1, panel A,
the mice had to be poked with much more force before they got up at
all, and after getting up, they would walk one or two steps and
immediately sit down again. This sluggish behavior was followed through
several steps of purification (panel B and C),
and the apparently homogeneous peptide (panel D) was further
analyzed. We have designated this peptide contulakin-G (the Filipino
word tulakin' means "has to be pushed or prodded," from
the root word tulak, to push). The "G" indicates that
the peptide is from C. geographus; we have used an analogous
nomenclature for other cysteine-sparse Conus peptides
(i.e. conantokin-G, conopressin-G, contryphan-R).

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Fig. 1.
Purification of contulakin-G. One gram
of crude lyophilized venom from C. geographus was extracted
and applied on a Sephadex G-25 column as described previously (10).
Three successive fractions containing paralytic and sleeper activities
(Ve/Vo = 1.37 to 1.41) were pooled, applied on a preparative
reversed phase Vydac C18 column, and eluted with a gradient
of acetonitrile in 0.1% trifluoroacetic acid (panel A). The
component indicated by an arrow in panel A caused
wobbling and death when administered icv in mice. This was applied on a
semipreparative C18 column and eluted with 12-42%
acetonitrile gradient in 0.1% trifluoroacetic acid (panel
B). The component marked by an arrow in panel
B made mice unresponsive when administered icv. This component was
further purified with an isocratic elution at 20.4% acetonitrile in
0.1% trifluoroacetic acid (panel C). A mouse injected icv
with an aliquot of the component had trouble righting itself in 5 min
and became very sluggish within 12 min. In approximately 25-30 min,
the mouse was stretched out and lay on its stomach.
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Biochemical Characterization of the Purified
Contulakin-G--
Attempted amino acid sequence analysis of the
purified peptide revealed that the peptide was blocked at the N
terminus. Because most N-terminal-blocked Conus peptides
have a pyroglutamate residue at position 1, the peptide was treated
with pyroglutamate aminopeptidase. This resulted in a shift in
retention time, suggesting removal of a pyroglutamate residue. After
enzyme treatment, the sequence Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Xaa-Lys-Lys-Pro-Tyr-Ile-Leu was obtained by standard Edman methods confirming removal of the
pyroglutamate residue, where Xaa indicates no amino acid was assigned
in the 9th cycle (at position 10), although a very low signal for
threonine was obtained. Amino acid analyses were consistent with the
presence of one threonine residue in the peptide.
To confirm the nature of the amino acid residue in position 10, a
cDNA clone encoding the peptide was isolated. The nucleotide sequence and presumed amino acid sequence revealed by the clone are
shown in Fig. 2. The amino acid sequence
of contulakin-G obtained by direct Edman sequencing is found encoded
toward the C-terminal end of the only significant open reading frame in
the clone (at residues 51-66); the predicted amino acid sequence
reveals that position 10 of the mature peptide (residue 60 of the
precursor) is encoded by a codon for threonine. Thus, the Edman
sequencing, together with cloning results, suggested that a modified
threonine residue was present in position 10.
Mass spectrometric analyses (MALD, LSI, and nano-ESI) of the purified
contulakin-G fraction revealed a variety of intact species as
summarized in Table I. Some variation in
the intensity of the different species was observed with different
ionization techniques, which was ascribed to differences in the bias
(29) with each ionization technique. In the following analysis, we have
concentrated on the major glycoform with intact mass M1 = 2069 observed with all of the ionization techniques investigated. The
difference between the observed mass (2069 Da) and the mass calculated
for the sequence assuming Thr at residue 10 (1703.83 Da) was 365 Da. Because one possible modification of threonine is
O-glycosylation, we proposed, based on this mass difference,
that the unidentified residue was
hexose-N-acetylhexosamine-threonine (Hex-HexNAc-Thr), which
would result in the addition of 365.13 Da. The observed masses (Table
I) are consistent with the calculated monoisotopic mass of the
[M1 + H]+ or [M1 + 2H]2+ of the proposed disaccharide-linked peptide (2069.98 or 1035.5 Da, respectively). Intense fragment ions were observed in the nano-ESI MS/MS mass spectrum of the doubly charged [M1 + 2H]2+ intact molecule ion of contulakin-G (Fig.
3), corresponding to the loss of the
complete Hex-HexNAc glycan (denoted p(
3)10
(30) or loss of the terminal hexose residue
(p(
8)10).
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Table I
Species observed with MALD, LSI, and nano-ESI analysis of purified
contulakin-G
ToF, time of flight; IT, ion trap.
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Fig. 3.
Nano-ESI MS/MS spectrum
(m/z 1035 precursor) of native
contulakin-G (286-1886 Da) (the MS/MS experiment is denoted using a
suggested shorthand (37) where the closed circle
represents m/z 1035 [M + 2H]2+
precursor, and the arrows are directed toward the
open circles which represent the fragments generated
from the precursor). Above the spectrum, the structure of the
glycoamino acid is represented where the arrows indicate 2 sites that lead to major fragment ions observed in the MS/MS spectrum
(30).
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Evidence That Thr-10 is O-glycosylated--
The results of the
enzymatic treatment of the peptide are shown in Figs.
4 and 5.
Native contulakin-G was treated with
-galactosidase isolated from
bovine testes. This enzyme preferentially hydrolyzes terminal
1
3galactopyranosyl residues from the nonreducing end of
glycoconjugates. After
-galactosidase treatment of the native sample, a new component was observed on RP-HPLC (see Fig. 4,
inset). This component was collected and analyzed with
MALD-MS in which a species was observed at m/z
1907 (Fig. 4). The difference in mass and the specificity of the enzyme
are consistent with a terminal galactose residue being released. Based
on the
-galactosidase hydrolysis results, we reasoned that the
glycan moiety might be susceptible to O-glycosidase
treatment, which liberates the disaccharide
-D-Galp-(1
3)-
-D-GalpNAc
bound to serine or threonine as a core unit of glycopeptides.
O-Glycosidase treatment of the native contulakin-G did in
fact result in a new species after the enzyme hydrolysis mixture was
analyzed on RP-HPLC (see Fig. 5, inset). The new component was collected and analyzed with MALD-MS, where an
m/z 1704 species was observed, consistent with
loss of Hex-HexNAc (Fig. 5) (i.e. the mass was consistent
with that predicted for the peptide with an unmodified threonine
residue at position 10). The enzyme hydrolysis results are consistent
with the presence of a
-D-Galp-(1
3)-
-D-GalpNAc-(1
) glycan. Based on the O-glycosidase and the
-galactosidase
hydrolysis results, we propose that the structure of the most abundant
glyco-peptide is
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Fig. 4.
MALD mass spectra of native contulakin-G
before (A) and after (B)
-galactosidase treatment. The inset
shows the RP-HPLC chromatograms of native contulakin-G alone
(C), -galactosidase alone (D), and native
contulakin-G (E) after -galactosidase treatment, where
the arrow indicates the component that was analyzed in
panel B (in panels C thru E, the
dashed lines represent the solvent gradient).
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Fig. 5.
MALD mass spectra of native contulakin-G
before (A) and after
(B) O-glycosidase
treatment. The inset shows the RP-HPLC chromatograms of
native contulakin-G alone (C), O-glycosidase alone (D), and
native contulakin-G after O-glycosidase treatment
(E), where the arrow indicates the component that
was analyzed in panel B (in panels C thru
E, the dashed lines represent the solvent
gradient).
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Synthesis of the Nonglycosylated and Glycosylated
Contulakin-G--
The 16-amino acid nonglycosylated peptide was
chemically synthesized. The synthetic material was found to have
the same retention time as the enzymatically deglycosylated
contulakin-G on RP-HPLC. The 16-amino acid-glycosylated contulakin-G
containing Gal(
1
3)GalNAc(
1
) attached to Thr10
was also synthesized. This synthetic glycosylated contulakin-G co-eluted with the native contulakin-G on RP-HPLC (see Fig.
6, panels A, B, and
C). The post-source decay fragmentation spectra observed for both native and synthetic contulakin-G showed very similar
fragmentation patterns (panels D and E).

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Fig. 6.
RP-HPLC chromatograms of native contulakin-G
(A), synthetic contulakin-G (B), and
co-injection of native and synthetic contulakin-G (C,
in panels A thru C, dashed
lines represent the solvent gradient) and the MALD-MS
post-source decay spectrum (m/z 2068 precursor) of native (D) and synthetic contulakin-G
(E) (100-2070 Da) (the post-source decay experiment
is denoted using a suggested shorthand (37), where the closed
circle represents m/z 2068 [M + H]+ precursor, and the arrows are
directed toward the open circles, which represent the
fragments generated from the precursor).
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Biological Potency of Synthetic Glycosylated and
Nonglycosylated Contulakin-G--
The loss of motor control for which
the native contulakin-G was originally isolated, together with gut
contraction, absence of preening/grooming, and reduced sensitivity to
tail depression were signs observed when
neurotensin1-13, nonglycosylated Thr10
contulakin-G, or synthetic contulakin-G were administered icv. To
investigate these observations in more detail, we undertook a dose
response comparison as detailed in Table
II. Although the nonglycosylated
Thr10 contulakin-G analog was active at doses of 1 nmol and
higher, it was inactive at 300-pmol doses. In contrast, contulakin-G
was found to elicit loss of motor control at doses of 30 pmol or
approximately 5 pmol/g.
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Table II
Effect of icv administration of neurotensin1-13, Thr10
contulakin-G and contulakin-G in 14-18-day-old mice
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The six C-terminal amino acids of contulakin-G show significant
similarity to the sequences of neurotensin1-13,
neuromedin, xenin, and the C terminus of xenopsin (see Table
III). Because of the similar symptoms
observed when either contulakin-G or neurotensin1-13 were
administered icv and the significant homology between contulakin-G and
neurotensin1-13, we tested the affinity of contulakin-G for a number of the cloned neurotensin receptors. As shown in Table
IV, the nonglycosylated
Thr10 contulakin-G analog was found to bind the human
neurotensin type I receptor (hNTR1) with 10-fold lower affinity than
neurotensin1-13 and even lower affinities for the other
NTRs. Contulakín-G exhibited significantly lower affinity than
the nonglycosylated Thr10 contulakin-G analog for all of
the NTRs tested.
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Table III
Sequence comparison of contulakin-G and some members of the neurotensin
family of peptides
The asterisk (*) indicates an O-linked-glycosylated
threonine/serine residue; the percentage identity (Id) and similarity
(Si) of the 6 C-terminal amino acids are compared to
contulakin-G11-16. <E is pyroglutamate.
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Table IV
Comparison of binding affinity of neurotensin1-13,
Thr10 contulakin-G, and contulakin-G for the cloned human and
rat NTR1, rNTR2, and mNTR3
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Both contulakin-G and the nonglycosylated Thr10
contulakin-G analog acted as agonists when tested on CHO cells
expressing the rNTR1 as shown in Fig. 7.
No response was observed with CHO cells expressing the rNTR2. In Fig.
7, the nonglycosylated Thr10 contulakin-G analog
resulted in slightly lower potency (0.6 nM) but with
similar efficacy as compared with neurotensin1-13. The
synthetic glycosylated contulakin-G potency was significantly lower
(20-30 nM), and the agonistic efficacy was approximately half that observed for neurotensin1-13.

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Fig. 7.
A plot of the IP accumulation
versus concentration of neurotensin1-13,
nonglycosylated Thr10 contulakin-G, or synthetic
contulakin-G applied to CHO cells expressing rNTR1.
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DISCUSSION |
Contulakín-G and Post-translational Modification--
The
Conus peptide characterized in this report, contulakin-G,
has a novel biochemical feature: a post-translationally
O-glycosylated threonine not previously found in
Conus peptides. We demonstrated, using mass spectrometry and
specific enzymatic hydrolyses, that Thr10 was modified with
the disaccharide
-D-Galp-(1
3)-
-D-GalpNAc-(1
). We synthesized the corresponding glycosylated and nonglycosylated forms
of contulakin-G and were able to confirm the molecular structure of
this major glycosylated form of the native molecule based on RP-HPLC
co-elution and MS fragmentation criteria. The other more minor molecule
species observed with mass spectrometry (see Table I) are not yet fully
determined; their masses are consistent with glycan structural
variations at peripheral sites on the characterized oligosaccharide
core unit (31).
An analysis of a cDNA clone encoding contulakin-G (Fig. 2) reveals
that the prepropeptide organization of the contulakin-G precursor is
similar to that of other Conus peptide precursors (8). A
typical signal sequence is found, and immediately N-terminal to the
contulakin-G sequence are two basic amino acids that presumably signal
a proteolytic cleavage to generate the N terminus of the mature peptide
(the glutamine residue would cyclize to pyroglutamate either
spontaneously or because of the action of glutaminyl cyclase (32)).
Although in most respects the contulakin-G precursor has the same
organization as all other Conus venom peptide precursors and
would be predicted to be processed in the same way, the 10 C-terminal
amino acids predicted by the clone are not present in contulakin-G
purified from venom. One possibility is that the clone represents a
different variant; for example, one that was alternatively spliced.
Alternatively, further proteolytic processing at the C terminus may be
required to generate mature contulakin-G.
Over the last 20 years, an increasing number of biologically important
glycopeptides and glycoproteins have been identified. Vespulakinin 1, first identified by Pisano and co-workers (33), is to our knowledge the
only other O-glycosylated peptide toxin that has been
isolated from venom other than Conus. Vespulakinin 1 was
extracted from the venom sacs of the yellow jacket wasp, Vespula
maculifrons. The peptide (TAT*T*RRRGRPPGFSPFR-OH, where the
asterisk indicates an O-linked glycosylated threonine
residue) contains two sequential sites of O-linked
glycosylation. The C terminus of vespulakinin is identical to the
sequence of bradykinin (RPPGFSPFR-OH), and the peptide was found to
elicit a number of signs also elicited by bradykinin. Vespulakinin is
therefore another example of an O-linked glycosylated
peptide toxin in which the C terminus appears to target a mammalian
neurotransmitter receptor. Thus, both contulakin-G and vespulakinin I
contain glycosylated N-terminal extensions to sequences with very high
homology to mammalian neuropeptides. We note that
A-conotoxin SIVA,
a K+ channel inhibitor, is unusual among disulfide-rich
Conus peptides in having a long N-terminal tail that has an
O-glycosylated residue (9).
For most Conus peptides, a specific conformation appears to
be stabilized either by multiple disulfide linkages or by the appropriate spacing of
-carboxyglutamate residues to promote formation of
-helices (34). Conus peptides without
multiple disulfides comprise a most eclectic set of families, including the conopressins, conantokins, contryphans, and now contulakin-G. The
conopressins are probably endogenous molluscan peptides, clearly homologous to the vasopressin/oxytocin family of peptides; these are
more widely distributed in molluscan tissues than in Conus venom ducts. However, the other nondisulfide-rich peptides
(conantokins, contryphans, and contulakin-G) may be specialized venom
peptides exhibiting unusual post-translational modifications. In
addition to the O-glycosylated threonine moiety of
contulakin-G described here,
-carboxylation of glutamate residues
and the post-translational epimerization and bromination of tryptophan
residues were discovered in conantokins and contryphans. Table
V summarizes all of the post-translational modifications found in Conus peptides to
date.
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Table V
Post-translational modifications found in Conus peptides
Gla, -carboxy-glutamate; Z, pGlu, pyroglutamic acid; Y ,
tyrosine sulfate; w, D-tryptophan; W ,
6-L-bromotryptophan; 0, Hyp,
4-trans-hydroxyproline; S§,
Hex3HexNAc2-Ser; T§, Gal-GalNAc-Thr; * indicates the
amidated C-terminus; PAM, protein amidating monooxygenase; PP, GalNAc
transferase, polypeptide N-acetylgalactosaminyl transferase;
Hyp, hydroxyproline; glyco-Ser, glycosylated Ser.
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Contulakín-G, a Member of the Neurotensin
Family--
Several lines of evidence are consistent with contulakin-G
being the first member of the neurotensin family of peptides to be
isolated from an invertebrate source. First, the C-terminal region of
contulakin-G exhibits a striking degree of similarity to other members
of the neurotensin family (all from vertebrates), as shown in Table IV.
Furthermore, we have shown that contulakin-G competes for binding to
three known neurotensin receptor subtypes; evidence that contulakin-G
acts as an agonist on a cloned neurotensin receptor is also presented
above. Most convincingly, however, when contulakin-G is injected into
mice, the same behavioral signs are elicited as with administration of
neurotensin. Thus, structural data, binding data, and in
vivo behavioral symptomatology are all consistent with the
assignment of contalukín-G to the neurotensin family of peptides.
Establishing that contulakin-G is an invertebrate member of the
neurotensin family of peptides raises the question of whether this
peptide has endogenous functions in Conus and other
molluscs. Although contulakin-G may well have evolved exclusively as
part of the prey capture strategy of C. geographus, we note
that the only other neuropeptide homologs isolated from
Conus venom, the conopressins (3), were later shown to be
endogenous neuropeptides of the vasopressin family in
Lymnaea, a gastropod mollusc only distantly related to
Conus. Thus, the discovery of a contulakin-G makes a
potential endogenous role for the neurotensin family of peptides in
invertebrates worthy of further investigation.
One possible role for contulakin-G in the capture of fish prey by
C. geographus is to suppress sensory circuitry in the prey. We have indicated elsewhere (8) that in contrast to fish-hunting Conus that use a "hook-and-line" strategy, C. geographus uses a "net" strategy. We suggest that instead of
the exitotoxic shock (35) elicited by the hook-and-line fish hunters,
net hunters appear to elicit a sedated state in their fish prey (8);
contulakin-G may be part of such a physiological program initiated upon
venom injection.
O-Glycosylation Confers Increased Potency to Contulakin-G in
Vivo--
Clearly, both contulakin-G and the nonglycosylated
Thr10 contulakin-G analog are rNTR1 agonists at
physiologically relevant concentrations (20-30 and 0.6 nM,
respectively). The observed agonistic effects of both contulakin-G and
the nonglycoslyated analog as well as the absence of any agonistic
effect of these ligands on CHO cells expressing rNTR2 using the IP
accumulation assay does not correlate with the in vitro
binding data; both peptides are agonists at concentrations
significantly below their IC50 binding affinity (524 and 79 nM, respectively). Most unexpected, therefore, given its
apparently lower binding affinity, is the increased potency of
glycosylated contulakin-G compared with the nonglycosylated analog
after icv administration.
Thus, the role of the glycan is somewhat paradoxical. In
vitro, the glycan neither increases the binding affinity, the
agonistic potency, nor agonistic efficacy. In contrast, in
vivo, the glycan significantly increases the potency of the
peptide. One simple explanation is that the increased potency of
contulakin-G compared with the nonglycosylated analog is because of
increased stability. An alternative mechanism for the increased potency
is transport to the site of action facilitated by the glycan.
Additionally, the glycosylated peptide may act with high affinity on an
as yet undefined neurotensin receptor subtype (36) or may be a
selective high affinity ligand for a particular state of a neurotensin
receptor subtype. Yet another possibility is that the relevant targeted neurotensin receptors may be closely co-localized with carbohydrate binding sites and that the glycan may serve as an "address label," a mechanism postulated for certain opiate peptides.
We have begun to evaluate these different possibilities. Preliminary
data supporting the increased stability hypothesis has been obtained;
proteolytic degradation of contulakin-G is inhibited by the presence of
the glycan moiety.3 The
increased stability may well result in an enhanced supply of the
glycopeptide at the receptor. However, the increased in vivo
potency of contulakin-G conferred by O-glycosylation clearly requires a more balanced evaluation of the possibilities outlined above.