NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.     

Identification of relaxin-3/INSL7 as an endogenous ligand for the orphan G-protein-coupled receptor GPCR135

GPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35S-labeled guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) incorporation in cells over-expressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTPgammaS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry.     

Novel Fatty Acid Modifications of Transportan 10

Cell-penetrating peptides (CPPs) are able to efficiently internalize into cells and can therefore be used as vectors for non-viral cellular delivery of different cargoes. Previous studies have shown that hydrophobic modifications of different CPPs can increase their transfection efficiency dramatically. In this study we have modified the cell penetrating-peptide transportan 10 (TP10) with a variety of hydrophobic molecules to determine the role of hydrophobicity in the uptake of these molecules. The results can be used to synthesize more efficient delivery vectors. To evaluate how these constructs are able to transport cargoes into cells we used 2′-OMe splice correcting oligonucleotides. Non-covalent peptide-cargo complexes were formed and their transfection efficiency was measured using a luciferase readout system. The hydrophobicity of the novel modifications was correlated with their biological efficacy. We determined the most efficient range of hydrophobicity for TP10 analogs for delivering oligonucleotides into cells. In order to assess how the transfection efficacy of these particles is dependent on their size the hydrodynamic diameter of the formed nanoparticles was measured using dynamic light scattering. These findings will be used to develop highly efficient non-viral gene therapy vectors.     

DCDC2 Mutations Cause a Renal-Hepatic Ciliopathy by Disrupting Wnt Signaling

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits β-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.     

INSL5 is a high affinity specific agonist for GPCR142 (GPR100)

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of (125)I-relaxin-3 to GPCR142 with a high affinity (K(i) = 1.5 nM). In a saturation binding assay, (125)I-INSL5 binds GPCR142 with a K(d) value of 2.5 nM. In functional guanosine (gamma-thio)-triphosphate binding and cAMP accumulation assays, INSL5 potently activates GPCR142 with EC(50) values of 1.3 and 1.2 nM, respectively. In addition, INSL5 stimulates Ca(2+) mobilization in HEK293 cells expressing GPCR142 and G alpha(16). Overall, INSL5 behaves as an agonist for GPCR142 similar to relaxin-3. However, unlike relaxin-3, which is also a potent agonist for GPCR135 and LGR7, INSL5 does not activate either GPCR135 or LGR7. INSL5 inhibits (125)I-relaxin-3 binding to GPCR135 with a low potency (K(i) = 500 nM). A functional assay shows that INSL5 (1 microm) is a weak antagonist for GPCR135. In addition, INSL5 (up to 1 microm) shows no affinity or activity at LGR7 or LGR8 either in a binding assay or a bio-functional assay. Previously, we have demonstrated that GPCR142 mRNA is expressed in peripheral tissues, particularly in the colon. Here we show that INSL5 mRNA is expressed in many peripheral tissues, similar to GPCR142. The high affinity interaction between INSL5 and GPCR142 coupled with their co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for GPCR142.     

A novel form of neurotensin post-translationally modified by arginylation

A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the gamma-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide "arginylated neurotensin" (R-NT, N-(neurotensin-C5-4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1.     

Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.

Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.     

Demonstration of [125I]VIP binding sites and effects of VIP on cAMP-formation in rat insulinoma (RINm5F and RIN14B) cells.

Vasoactive intestinal polypeptide (VIP)-immunoreactive nerves have been demonstrated in close association with the islets of Langerhans, and VIP has been shown to stimulate insulin and somatostatin secretion. Using [125I]VIP and membranes prepared from rat insulinoma (RIN) cells, i.e., the subclones m5F (m5F; mainly insulin-secreting) and 14B (14B; mainly somatostatin-secreting), it was found that VIP (10(-10)-10(-7) M) competitively inhibited the binding of [125I]VIP. A single class of high affinity binding sites with Kd values of 0.40 +/- 0.06 nM and 0.36 +/- 0.08 nM for m5F and 14B, respectively, with a corresponding number of binding sites (Bmax) of 163 +/- 20 and 254 +/- 51 fmol/mg protein was observed. The rank order of potency in inhibiting [125I]VIP binding was in both cell lines: VIP greater than helodermin greater than pituitary adenylate cyclase activating polypeptide 1-27 (PACAP27) greater than peptide histidine isoleucine (PHI) greater than secretin. VIP caused a dose-dependent increase in cAMP-formation in both m5F and 14B cell membranes with EC50 values of 3.0 and 3.5 nM, respectively, but VIP (1.10(-9)-3.10(-6) M) had no effect on insulin secretion (over 2 h) from the m5F cells. Thus, the data suggest that the VIP-receptors in these neoplastic rat cell lines, despite an apparent coupling to adenylate cyclase activity, seem to be functionally uncoupled to an effect on insulin secretion following an acute exposure to VIP.     

Isolation and characterization of galanin from sheep brain.

Galanin is a well-known, naturally occurring peptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinally derived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarity to pig galanin, differing by one amino acid substitution, that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26.