A. Grey Craig

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 T-superfamily of conotoxins.

We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conusspecies were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (- -CC- -CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conusspecies. These peptides are likely to be functionally diverse; although the peptides are small (11–17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCPKQMRCCTL*; tx5a, γCCγDGW+CCT§AAO; and au5a, FCCPFIRYCCW (where γ = γ-carboxyglutamate, W+ = bromotryptophan, O = hydroxyproline, T§ = glycosylated threonine, and * = COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional γ-carboxylation recognition signal in the −1 to −20 propeptide region, consistent with the presence of γ-carboxyglutamate residues in this peptide.

Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral ...

BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

A Novel Post-translational Modification Involving Bromination of Tryptophan IDENTIFICATION OF THE RESIDUE, L-6-BROMOTRYPTOPHAN, IN PEPTIDES FROM Conus imperialis AND Conus radiatus VENOM

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = γ-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

Three Forms of Gonadotropin-Releasing Hormone, Including a Novel Form, in a Basal Salmonid, Coregonus clupeaformis

Abstract Multiple forms of GnRH within individual brains may have different functions. However, some vertebrates such as salmonids continue to reproduce even though they have lost or do not express 1 of the 3 forms of GnRH found in most other teleosts. We examined a basal salmonid, lake whitefish, to determine the mechanism by which a reduction in the number of GnRH forms occurs. We identified for the first time 3 distinct GnRHs in a salmonid. One form is novel and is designated whitefish GnRH. The primary structure is pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH2. HPLC and RIA were used for purification followed by Edman degradation for sequence determination. Mass spectroscopy was used to confirm the sequence and amidation of the peptide. The other 2 forms, salmon GnRH and chicken GnRH-II, are identical to the 2 forms found in salmon, which evolved later than whitefish. Synthetic whitefish GnRH is biologically active, as it increased mRNA expression of growth hormone and the α-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.

Primary structure of three forms of gonadotropin-releasing hormone (GnRH) from the pacu brain

Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu-His-Trip-Ser-Tyr-Gly-Leu-Ser -Pro-Gly-NH2, 1113.4 Da); chicken GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2, 1236.6 Da); and salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2, 1212.3 Da). In addition the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.

Sequence of two gonadotropin releasing hormones from tunicate suggest an important role of conformation in receptor activation

The primary structure of two forms of gonadotropin releasing hormone (GnRH) from tunicate (Chelyosoma productum) have been determined based on mass spectrometric and chemical sequence analyses. The peptides, tunicate GnRH‐I and ‐II, contain features unprecedented in vertebrate GnRH. Tunicate GnRH‐I contains a putative salt bridge between Asp5 and Lys8. A GnRH analog containing a lactam bridge between Asp5 and Lys8 was found to increase release of estradiol compared with that of the native tunicate GnRH‐I and ‐II. Tunicate GnRH‐II contains a cysteine residue and was isolated as a dimeric peptide. These motifs suggest that the conformation plays an important role in receptor activation.

Potent somatostatin undecapeptide agonists selective for somatostatin receptor 1 (sst1).

A family of analogues of des-AA(1,2,5)-[DTrp(8)/D2Nal(8)]-SRIF that contain a 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 was identified that has high affinity and selectivity for human somatostatin receptor subtype 1 (sst1). The binding affinities of des-AA(1,2,5)-[DTrp(8),IAmp(9)]-SRIF (c[H-Cys-Lys-Phe-Phe-DTrp-IAmp-Thr-Phe-Thr-Ser-Cys-OH], CH-275) (7), des-AA(1,5)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (CH-288) (16), des-AA(1,2,5)-[Tyr(7),DTrp(8),IAmp(9)]-SRIF (23), and des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-SRIF (25) are about (1)/(7), (1)/(4), (1)/(125), and (1)/(4) that of SRIF-28 (1) to sst1, respectively, about (1)/(65), (1)/(130), <(1)/(1000), and <(1)/(150) that of 1 to sst3, respectively, and about or less than (1)/(1000) that of 1 to the other three human SRIF receptor subtypes. A substitution of DTrp(8) by D2Nal(8) in 7 to yield des-AA(1,2,5)-[D2Nal(8),IAmp(9)]-SRIF (13) and in 16 to yield des-AA(1,5)-[Tyr(2),D2Nal(8),IAmp(9)]-SRIF (17) was intended to increase chemical stability, selectivity, and affinity and resulted in two analogues that were less potent or equipotent with similar selectivity, respectively. Carbamoylation of the N-terminus as in des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) increased affinity slightly as well as improved selectivity. Monoiodination of 25 to yield 26 and of 27 to yield 28 resulted in an additional 4-fold increase in affinity at sst1. Desamination of the N-terminus of 17 to yield 18, on the other hand, resulted in significant loss of affinity. Attempts at reducing the size of the ring with maintenance of selectivity failed in that des-AA(1,4,5,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (33) and des-AA(1,4,5,6,12,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (34) progressively lost affinity for all receptors. Both des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) and des-AA(1,2,5)-[DCys(3),DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (29) show agonistic activity in a cAMP assay; therefore, the structural basis for the agonist property of this family of analogues is not contingent upon the chirality of the Cys residue at position 3 as shown to be the case in 18-membered ring SRIF octapeptides. None of the high affinity structures described here showed receptor antagonism. We have prepared the radiolabeled des-AA(1,2,5)-[DTrp(8),IAmp(9),(125)ITyr(11)]-SRIF ((125)I-25) and des-AA(1,2,5)-[DTrp(8),IAmp(9), (125)ITyr(11)]-Cbm-SRIF ((125)I-27), used them as in vitro tracers, and found them to be superior to des-AA(1,5)-[(125)ITyr(2),DTrp(8),IAmp(9)]-SRIF ((125)I-16) for the detection of sst1 tumors in receptor autoradiography studies.

Conophysin-R, a Conus radiatus venom peptide belonging to the neurophysin family.

A novel Conus peptide, conophysin-R, was purified from the venom of Conus radiatus. The distinctive disulfide framework and sequence indicates that it is a member of the neurophysin peptide family. The complete sequence of the peptide is HPTKPCMYCSFGQCVGPHICCGPTGCEMGTAEANMCSEEDEDPIPCQVFGSDCALNNPDNIHGHCVADGICCVDDTCTTHLGCLThis is the first time a neurophysin-like peptide has been found in any venom. In addition, conophysin-R is the first neurophysin family member isolated and biochemically characterized from an invertebrate source.