Contulakin-G, an O-Glycosylated Invertebrate Neurotensin*

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide β-d-Galp-(1→3)-α-d-GalpNAc-(1→) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(β→3) GalNAc(α→)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakı́n-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10contulakin-G was approximately an order of magnitude lower than that of neurotensin1–13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.

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 (Ͻ30 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-transhydroxyproline 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 Oglycosylated 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.

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 Ϫ70°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 * This work was supported by National Institute Health Grant GM 48677 and National Science Foundation Major Research Instrumentation Program Grant DDBI-972450, conducted in part by the Foundation for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF121108.
¶ ¶ To whom correspondence should be addressed.
a series of RP-HPLC 1 purifications on preparative and semipreparative and analytical reverse phase C 18 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.
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 N 2 laser, and a 100 MHz digitizer. An accelerating voltage of ϩ31 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-4hydroxycinnamic acid. Liquid secondary ionization (LSI) (14) mass spectra were measured using a Jeol HX110 (Jeol, Tokyo, Japan) doublefocusing 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-(133)-␣-D-GalpNAc-(130)-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 peracety-lated 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 l of sodium methoxide (Sigma) (50 mM) in dry methanol for 1 min (to remove O-acetyl groups on the sugar (18)) and lyophilized at Ϫ20°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 ϫ 320 mm) packed with Vydac C 18 15-20-m 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 1 H 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 ϫ 150-mm Vydac C 18 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).
The nonglycosylated Thr 10 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). 125 I-Tyr 3 neurotensin 1-13 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 g for NTR2 and 10 g for NTR1) were incubated with 0.4 nM 125 I-Tyr 3 neurotensin 1-13 (2000 Ci/mmol) and increasing concentrations of neurotensin 1-13 , nonglycosylated Thr 10 contulakin-G, or synthetic contulakin-G for 20 min at 25°C in 250 l 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 g) were incubated with 0.2 nM 125 I-Tyr 3 neurotensin 1-13 for 1 h at 0°C in 250 l 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 M 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 ϫ g during 30 min at 4°C and used either immediately or stored at Ϫ20°C.
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 (5Ј-ATR ATN GGY TTY TTN GT-3Ј), where R ϭ A or G, N ϭ A, C, G, or T, and Y ϭ 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).

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 tulakinЈ 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).
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 pres-ent 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 M 1 ϭ 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)  Native contulakin-G was treated with ␤-galactosidase isolated from bovine testes. This enzyme preferentially hydrolyzes terminal ␤133galactopyranosyl 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 Oglycosidase treatment, which liberates the disaccharide ␤-D-Galp-(133)-␣-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,

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(␤133)GalNAc(␣13) attached to Thr 10 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).
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 neurotensin 1-13 , nonglycosylated Thr 10 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 Thr 10 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.
The six C-terminal amino acids of contulakin-G show significant similarity to the sequences of neurotensin 1-13 , neuromedin, xenin, and the C terminus of xenopsin (see Table III). Because of the similar symptoms observed when either contulakin-G or neurotensin [1][2][3][4][5][6][7][8][9][10][11][12][13] were administered icv and the significant homology between contulakin-G and neurotensin 1-13 , we tested the affinity of contulakin-G for a number of the cloned neurotensin receptors. As shown in Table IV, the nonglycosylated Thr 10 contulakin-G analog was found to bind the human neurotensin type I receptor (hNTR1) with 10-fold lower affinity than neurotensin 1-13 and even lower affinities for the other NTRs. Contulakín-G exhibited significantly lower affinity than the nonglycosylated Thr 10 contulakin-G analog for all of the NTRs tested.

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 Thr 10 was modified with the disaccharide ␤-D-Galp-(133)-␣-D-GalpNAc- (13). 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 Olinked 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 ϩ 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 contu-  [1][2][3][4][5][6][7][8][9][10][11][12][13] , Thr 10 contulakin-G and contulakin-G in 14 -18- a Average time after which specific behavior was observed (observations were made every 2 min for first 15 min and every 15 min thereafter). Symptoms A and B were observed when a mouse was placed onto a bench top after lifting it by the tail for a second. Symptom A, the mouse moved at most a few steps and rested with the hind legs spread out. Mouse remained stationary unless pushed or lifted. Symptom B, the mouse remained sluggish, but the position of the hind part of the body when at rest resembled that of the normal saline solution controls. Symptom R (recovery), the mouse moved freely when released.
b No symptoms observed.  lakin-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.
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 Thr 10 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 IC 50 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. Contulakin-G ZSEEGGSNAT §KKPYIL PP GalNAc transferase Glyco-Thr