κO-SrVIA Conopeptide, a Novel Inhibitor Peptide for Two Members of the Human EAG Potassium Channel Family.

The first conotoxin affecting the voltage-gated potassium channels of the EAG family was identified and characterized from the venom of the vermivorous species Conus spurius from the Gulf of Mexico. This conopeptide, initially named Cs68 and later designated κO-SrVIA, is extremely hydrophobic and comprises 31 amino acid residues, including six Cysteines in the framework VI/VII, and a free C-terminus. It inhibits the currents mediated by two human EAG subtypes, Kv10.1 (IC50 = 1.88 ± 1.08 µM) and Kv11.1 (IC50 = 2.44 ± 1.06 µM), and also the human subtype Kv1.6 (IC50 = 3.6 ± 1.04 µM). Despite its clear effects on potassium channels, it shares a high sequence identity with δ-like-AtVIA and δ-TsVIA. Also, κO-SrVIA is the third conopeptide from the venom of C. spurius with effects on potassium channels, and the seventh conotoxin that blocks Kv1.6 channels.

A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

The T-1 conotoxin µ-SrVA from the worm hunting marine snail Conus spurius preferentially blocks the human NaV1.5 channel.

Conotoxin sr5a had previously been identified in the vermivorous cone snail Conus spurius. This conotoxin is a highly hydrophobic peptide, with the sequence IINWCCLIFYQCC, which has a cysteine pattern "CC-CC" belonging to the T-1 superfamily. It is well known that this superfamily binds to molecular targets such as calcium channels, G protein-coupled receptors (GPCR), and neuronal nicotinic acetylcholine receptors (nAChR) and exerts an effect mainly in the central nervous system. However, its effects on other molecular targets are not yet defined, suggesting the potential of newly relevant molecular interactions. To find and demonstrate a potential molecular target for conotoxin sr5a electrophysiological assays were performed on three subtypes of voltage-activated sodium channels (NaV1.5, NaV1.6, and NaV1.7) expressed in HEK-293 cells with three different concentrations of sr5a(200, 400, and 600 nM). 200 nM sr5a blocked currents mediated by NaV1.5 by 33%, NaV1.6 by 14%, and NaV1.7 by 7%. The current-voltage (I-V) relationships revealed that conotoxin sr5a exhibits a preferential activity on the NaV1.5 subtype; the activation of NaV1.5 conductance was not modified by the blocking effect of sr5a, but sr5a affected the voltage-dependence of inactivation of channels. Since peptide sr5a showed a specific activity for a sodium channel subtype, we can assign a pharmacological family and rename it as conotoxin µ-SrVA.

A Novel Dimeric Conotoxin, FrXXA, from the Vermivorous Cone Snail Conus fergusoni, of the Eastern Pacific, Inhibits Nicotinic Acetylcholine Receptors.

We isolated a new dimeric conotoxin with inhibitory activity against neuronal nicotinic acetylcholine receptors. Edman degradation and transcriptomic studies indicate a homodimeric conotoxin composed by two chains of 47 amino acid in length. It has the cysteine framework XX and 10 disulfide bonds. According to conotoxin nomenclature, it has been named as αD-FrXXA. The αD-FrXXA conotoxin inhibited the ACh-induced response on nAChR with a IC50 of 125 nM on hα7, 282 nM on hα3ß2, 607 nM on α4ß2, 351 nM on mouse adult muscle, and 447 nM on mouse fetal muscle. This is first toxin characterized from C. fergusoni and, at the same time, the second αD-conotoxin characterized from a species of the Eastern Pacific.

A short framework-III (mini-M-2) conotoxin from the venom of a vermivorous species, Conus archon, inhibits human neuronal nicotinic acetylcholine receptors.

The venoms of Conus snails contain neuroactive peptides named conotoxins (CTXs). Some CTXs are nicotinic acetylcholine receptor (nAChRs) antagonists. nAChRs modulate the release of neurotransmitters and are implicated in several pathophysiologies. One venom peptide from Conus archon, a vermivorous species from the Mexican Pacific, was purified by RP-HPLC and its activity on human α7, α3ß2, and α7ß2 nAChRs was assessed by the two-electrode voltage clamp technique. At 36.3 µM the purified peptide (F27-1, renamed tentatively ArchIIIA) slowly reversibly inhibited the ACh-induced response of the hα7 subtype by 44.52 ± 5.83%, while it had low or no significant effect on the response of the hα3ß2 and hα7ß2 subtypes; the EC50 of the inhibiting effect was 45.7 µM on the hα7 subtype. This peptide has 15 amino acid residues and a monoisotopic mass of 1654.6 Da (CCSALCSRYHCLPCC), with three disulfide bridges and a free C-terminus. This sequence with a CC-C-C-CC arrangement (framework III) belongs to the M superfamily of conotoxins, corresponding to the mini-M´s (M-1-M-3) conotoxins; due to its size and inter-Cys spacings it is an M-2 conotoxin. This toxin is a novel mini-M conotoxin affecting ligand-gated ion channels, like the maxi-M CTX ψ-conotoxins and α-MIIIJ conotoxin (nAChRs blockers). This peptide seems to be homologous to the reg3b conotoxin (from Conus regius) with an identity of 93.3%, differing only in the third residue in the sequence, serine for threonine, both uncharged polar residues. We obtained, in silico, a probable 3D structure, which is consistent with its effect on neuronal subtypes.

Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

A turripeptide from Polystira nobilis venom inhibits human α3ß2 and α7 nicotinic acetylcholine receptors.

Almost all marine snails within superfamily Conoidea produce venoms containing numerous neuroactive peptides. Most toxins characterized from members of this superfamily are produced by species belonging to family Conidae. These toxins (conotoxins) affect diverse membrane proteins, such as voltage- and ligand-gated ion channels, including nicotinic acetylcholine receptors (nAChRs). Family Turridae has been considerably less studied than their Conidae counterpart and, therefore, turrid toxins (turritoxins) have just been barely described. Consequently, in this work the most prominent chromatographic (RP-HPLC) fractions from the East Pacific species Polystira nobilis venom duct extract were isolated. The biological activity of six selected fractions was assayed on human (h) α7 AChRs expressed in Xenopus laevis oocytes. One of these fractions, F21, inhibited the acetylcholine-elicited response by 62 ± 12%. Therefore, this fraction was further purified and the F21-2 peptide was obtained. This peptide (at 5.6 µM) strongly and irreversibly inhibited the acetylcholine-induced response on hα7 and hα3ß2 nAChRs, by 55 ± 4 and 91 ± 1%, respectively. Electrospray mass spectrometry indicates that the average molecular mass of this toxin is 12 358.80 Da. The affinity for hα3ß2 nAChRs is high (IC50 of 566.2 nM). A partial sequence without cysteines was obtained by automated Edman degradation: WFRSFKSYYGHHGSVYRPNEPNFRSFAS…; blastp search revealed that this sequence has low similarity to some non-Cys-containing turripeptides. This is the first report of a turritoxin from a species of the American Pacific and the second description of a turripeptide inhibiting nAChRs.

αD-Conotoxins in Species of the Eastern Pacific: The Case of Conus princeps from Mexico.

Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 µM, and it does not affect the hα3ß2 subtype at 6.5 µM.

Conorfamide-Sr3, a structurally novel specific inhibitor of the Shaker K+ channel.

Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.

A Comprehensive Proteomic Study of the Skin Secretions of the Frog Lithobates spectabilis.

Disulfide C-terminal loop fragments derived from AMPs and the presence of peptidases have been previously reported in the skin secretions of different amphibians. However, there are only a few studies on the identification of enzymes in frog skin secretion based on the primary structure of these proteins. Similarly, little data exist regarding the identification of disulfide C-terminal loops at large scale. Therefore, a comprehensive study on this issue certainly could bring in much more information for understanding this molecular process and its biochemical consequences. Thus, the aim of this work was to characterize the presence of disulfide C-terminal loop fragments of AMPs and identify the proteins and probable enzymes present in the completely unknown secretion contents of the frog Lithobates spectabilis. For this purpose, high-resolution mass spectrometry was applied to analyze the skin secretions processed by two different protocols: (1) using a cocktail of enzymatic inhibitors and 2) without any protease inhibitors, maintaining the solution for 2 hours at 10°C. Results from procedure-1, revealed 122 molecular masses, whereas procedure-2 permitted 253 different molecular masses to be identified. Fifty-nine peptides including 22 disulfide C-terminal loop-containing peptides were obtained following procedure-2. Polyacrylamide gel electrophoresis separation, tryptic digestion and LCMS/ MS were used for "de novo" sequencing of 111 different peptides and the unequivocal identification of fifteen proteins including at least three different peptidases. Additionally, it was possible to fully sequence eight peptides, including a ranatuerin-related peptide identified here as Spectabilin, that was subsequently chemically synthesized and showed high antibacterial, antiparasitic and cytotoxic activities.

A New Member of Gamma-Conotoxin Family Isolated from Conus princeps Displays a Novel Molecular Target.

A novel conotoxin, named as PiVIIA, was isolated from the venom of Conus princeps, a marine predatory cone snail collected in the Pacific Southern Coast of Mexico. Chymotryptic digest of the S-alkylated peptide in combination with liquid chromatography coupled to tandem mass spectrometry, were used to define the sequencing of this peptide. Eleven N-terminal amino acids were verified by automated Edman degradation. PiVIIA is a 25-mer peptide (CDAOTHYCTNYWγCCSGYCγHSHCW) with six cysteine residues forming three disulphide bonds, a hydroxyproline (O) and two gamma carboxyglutamic acid (γ) residues. Based on the arrangement of six Cys residues (C-C-CC-C-C), this conotoxin might belong to the O2-superfamily. Moreover, PiVIIA has a conserved motif (-γCCS-) that characterizes γ-conotoxins from molluscivorous Conus. Peptide PiVIIA has 45% sequence identity with γ-PnVIIA-the prototype of this family. Biological activity of PiVIIA was assessed by voltage-clamp recording in rat dorsal root ganglion neurons. Perfusion of PiVIIA in the µM range produces a significant increase in the Ca(2+) currents, without significantly modifying the Na⁺, K⁺ or proton-gated acid sensing ionic currents. These results indicate that PiVIIA is a new conotoxin whose activity deserves further studies to define its potential use as a positive modulator of neuronal activity.

A novel arrangement of Cys residues in a paralytic peptide of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting snail from the Gulf of Mexico.

The present study details the purification, the amino acid sequence determination, and a preliminary characterization of the biological effects in mice of a new conotoxin from the venom of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting cone snail collected in the western Gulf of Mexico (Mexico). The 23-amino acid peptide, called as25a, is characterized by the sequence pattern CX1CX2CX8CX1CCX5, which is, for conotoxins, a new arrangement of six cysteines (framework XXV) that form three disulfide bridges. The primary structure (CKCPSCNFNDVTENCKCCIFRQP*; *, amidated C-terminus; calculated monoisotopic mass, 2644.09Da) was established by automated Edman degradation after reduction and alkylation, and MALDI-TOF and ESI mass spectrometry (monoisotopic mass, 2644.12/2644.08Da). Upon intracranial injection in mice, the purified peptide provokes paralysis of the hind limbs and death with a dose of 240 pmol (~0.635 µg, ~24.9 ng/g). In addition, a post-translational variant of this peptide (as25b) was identified and determined to contain two hydroxyproline residues. These peptides may represent a novel conotoxin gene superfamily.

Arrhythmogenic effect of a crude extract from sea anemone Condylactis gigantea: possible involvement of rErg1 channels.

Sea anemones possess a number of peptide toxins that target ion channels which provide powerful tools to study the molecular basis of diverse signaling pathways. It is also acknowledged that currents through Erg1 K(+) channels in cardiac myocytes are important for electrical stability of the heart and alterations in its activity has been linked to the onset of a potentially life-threatening heart condition named long QT syndrome type 2. Here, we report that a crude extract from sea anemone Condylactis gigantea significantly increases the QT interval and has arrhythmogenic effects in the rat heart. Furthermore, a bioassay-guided purification procedure allowed the isolation of a chromatographic fraction containing a major component with a molecular mass of 4478 Da from the crude extract, which causes a significant inhibition of whole-cell patch-clamp currents through recombinant Erg1 channels, responsible of the rapid delayed rectifying current crucial for electrical activity in the heart. Further studies could provide relevant information on the molecular mechanism of C. gigantea peptide toxins which represent promising tools in studying the physiology of diverse ion channels.

Precursor De13.1 from Conus delessertii defines the novel G gene superfamily.

Peptide de13a was previously purified from the venom of the worm-hunting cone snail Conus delessertii from the Yucatán Channel, México. This peptide has eight cysteine (Cys) residues in the unique arrangement C-C-C-CC-C-C-C, which defines the cysteine framework XIII ("-" represents one or more non-Cys residues). Remarkably, δ-hydroxy-lysine residues have been found only in conotoxin de13a, which also contains an unusually high proportion of hydroxylated amino acid residues. Here, we report the cDNA cloning of the complete precursor De13.1 of a related peptide, de13b, which has the same Cys framework and inter-Cys spacings as peptide de13a, and shares high protein/nucleic acid sequence identity (87%/90%) with de13a, suggesting that both peptides belong to the same conotoxin gene superfamily. Analysis of the signal peptide of precursor De13.1 reveals that this precursor belongs to a novel conotoxin gene superfamily that we chose to name gene superfamily G. Thus far superfamily G only includes two peptides, each of which contains the same, distinctive Cys framework and a high proportion of amino acid residues with hydroxylated side chains.

Adaptive radiation of venomous marine snail lineages and the accelerated evolution of venom peptide genes.

An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, each containing approximately 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (∼500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage-specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.

Biochemical characterization of a bacteriocin-like inhibitory substance produced by Enterococcus faecium MXVK29, isolated from Mexican traditional sausage.

BACKGROUND: Enterococci are lactic acid bacteria that can produce bacteriocins, which may offer an additional hurdle to control the growth of food-borne pathogens; moreover, these bacteriocins may have great potential as natural biopreservatives. The aim of this work was to characterize a bacteriocin-like inhibitory substance (BLIS) with antilisterial activity produced by an enterococcal strain. RESULTS: The bacteriogenic strain was isolated from Mexican fermented sausages and identified as Enterococcus faecium with 99% sequence similarity. Maximal activity was detected at 16 h, where bacterial growth was in middle of the stationary phase. The producer strain was not inhibited by its own antimicrobial peptide. BLIS showed a strong anti-Listeria activity and was inactivated by proteinase K. Heating (121 °C for 15 min) induced some inactivation, but thermotolerance was higher at acid pH values. The yield obtained with a pH-mediated purification process was 32.7%, showing a band with an estimated molecular weight of 3.5 kDa. Automated N-terminal Edman degradation showed the following sequence: YYGNGVTCGSHHCSVD. CONCLUSION: Biochemical characteristics of BLIS produced by E. faecium MXVK29 suggested that it belongs to Class IIa of the Klaenhammer classification and could be considered as a natural food preservative, although further studies need to be performed.

Peptide sr11a from Conus spurius is a novel peptide blocker for Kv1 potassium channels.

More than a hundred conotoxins are known today and from them, only seven conopeptides have been identified to target voltage-gated potassium channels (Kv). Conotoxin sr11a belongs to the I(2)-superfamily which is characterized by four disulfide bridges and provokes muscle stiffness when injected intracranially in mice. The aim of this work was to test the biological activity of sr11a on recombinant voltage-gated Kv1 potassium channels expressed in Xenopus laevis oocytes. Peptide sr11a was purified by high-performance liquid chromatography from the venom of the vermivorous Conus spurius. We found that peptide sr11a inhibits the delayed rectifiers Kv1.2 and Kv1.6 but had not effect on the slowly inactivating Kv1.3 channel. The functional dyad composed of a basic Lys and a hydrophobic amino acid residue is a crucial structural element, regarding the binding properties and blocking activities of more than a hundred K(+) channel toxins. Peptide sr11a does not contain Lys residues and then, it lacks the functional dyad. Molecular modeling of peptide sr11a reveals the presence of exposed basic residues of Arg and suggests that Arg17 and Arg29 are important on its biological activity.

Prolactin released in vitro from the pituitary of lactating, pregnant, and steroid-treated female or male rats stimulates prolactin secretion from pituitary lactotropes of male rats.

We have previously shown that soluble factor(s) in conditioned media (CM) from the central and peripheral regions of the anterior pituitary (AP) gland of lactating rats promoted the in vitro dose-related release of prolactin (PRL) from pituitary glands of male rats. In the present experiments we sought to determine whether CM from rats in different physiological states provoked similar effects (like those of lactating rats), and the nature of the factors, whether 23K PRL or other variants of the hormone, were responsible for these effects. Stimulatory effects were induced by CM from pregnant females and steroid-treated castrated males or females, but not from untreated castrated rats, intact males, or by a PRL standard. More potent effects occurred with CM from APs of early- than from mid- or late-lactating rats, and from rats unsuckled for 8 or 16 h than from those unsuckled for 32 h. With respect to the nature of factor(s) responsible for these effects, immunoprecipitation of PRL from the CM of lactating females and of steroid-treated, castrated males eliminated, whereas dephosphorylation or deglycosylation of CM of lactating rats greatly increased its effects upon PRL release. Also, electrophoretic analysis and Western blotting of the CM proteins under native and denaturing conditions revealed a variety of PRL variants, ranging from 14 to <90 kDa, in CM from lactating rats, and the main effects on PRL release were provoked by the 23- to 46-kDa PRL variants. These results indicate that specific effects upon male rat lactotropes may be exerted by PRL variants released from APs of lactating and non-lactating rats.

Cathepsin D is the primary protease for the generation of adenohypophyseal vasoinhibins: cleavage occurs within the prolactin secretory granules.

Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.

Molecular cloning and expression of a putative crustacean hyperglycemic hormone of Litopenaeus vannamei in Pichia pastoris

Crustacean hyperglycemic hormone (CHH) is the most abundant and best studied member of the CHH/MIH/GIH neuropeptide hormone family. CHH plays a major role in controlling glucose levels in the hemolymph, and it also has significance in regulating molting, reproduction, and osmoregulation. In contrast, molt-inhibiting hormone (MIH) is responsible for maintaining animals in an intermolt stage. In this study, Liv-MIH-1 cDNA, which encodes a mature neuropeptide from the eyestalk of white shrimp, Litopenaeus vannamei, was expressed in methylotrophic yeast (Pichia pastoris KM71) under the control of an alcohol oxidase promoter. Recombinant Liv-MIH-1 was secreted into the culture medium using the á-factor prepro-sequence without Glu-Ala repeats. The expected protein, which had an apparent molecular mass of 12.1 kDa, was detected by Tricine-SDS-PAGE analysis and confirmed by Western blot. Pure recombinant Liv-MIH-1 was obtained by affinity chromatography, and N-terminal sequence analysis confirmed expression of the protein. Biological assays for CHH and MIH activity were also performed. Purified recombinant Liv-MIH-1 showed the ability to elevate the glucose level of hemolymph of L. vannamei, but molting was unaffected. Since these results are in agreement with the high structural and phylogenetic similarity that has been observed between Liv-MIH-1 and other CHH neuropeptides we propose to rename the protein Liv-CHH-SG1.