Trevor Stanley Smith

Rapid, Nongenomic Responses to Ecdysteroids and Catecholamines Mediated by a Novel Drosophila G-Protein-Coupled Receptor

Nongenomic response pathways mediate many of the rapid actions of steroid hormones, but the mechanisms underlying such responses remain controversial. In some cases, cell-surface expression of classical nuclear steroid receptors has been suggested to mediate these effects, but, in a few instances, specific G-protein-coupled receptors (GPCRs) have been reported to be responsible. Here, we describe the activation of a novel, neuronally expressed Drosophila GPCR by the insect ecdysteroids ecdysone (E) and 20-hydroxyecdysone (20E). This is the first report of an identified insect GPCR interacting with steroids. The Drosophila melanogaster dopamine/ecdysteroid receptor (DmDopEcR) shows sequence homology with vertebrate β-adrenergic receptors and is activated by dopamine (DA) to increase cAMP levels and to activate the phosphoinositide 3-kinase pathway. Conversely, E and 20E show high affinity for the receptor in binding studies and can inhibit the effects of DA, as well as coupling the receptor to a rapid activation of the mitogen-activated protein kinase pathway. The receptor may thus represent the Drosophila homolog of the vertebrate “γ-adrenergic receptors,” which are responsible for the modulation of various activities in brain, blood vessels, and pancreas. Thus, DmDopEcR can function as a cell-surface GPCR that may be responsible for some of the rapid, nongenomic actions of ecdysteroids, during both development and signaling in the mature adult nervous system.

Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry.

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P3 have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography–mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P3 and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP2 are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P3 in unstimulated mouse and human cells (≥105) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

Haemonchus contortus Glycoproteins Contain N-Linked Oligosaccharides with Novel Highly Fucosylated Core Structures

Structural studies on the N-linked oligosaccharides of Haemonchus contortus, an economically important nematode that parasitizes domestic ruminants, have revealed core fucosylation of a type not previously observed in any eukaryotic glycoprotein. Mass spectrometric analyses were performed on detergent extracts of homogenized adult H. contortus and on purified H11, a glycoprotein isolated from intestinal brush borders which has been previously shown to be an effective vaccine antigen. The major N-linked glycans identified in the present study have up to three fucose residues attached to their chitobiose cores. The fucoses are found at the 3- and/or 6-positions of the proximal GlcNAc and at the 3-position of the distal GlcNAc. The latter substitution is unique in N-glycans. Most anti-H11 monoclonal antibodies are known to recognize carbohydrate epitopes, and it is possible that the newly discovered multifucosylated core structures are highly immunogenic in this glycoprotein.

Purification and evaluation of the integral membrane protein H11 as a protective antigen against Haemonchus contorus

A detergent extract of adult Haemonchus contortus enriched in the integral membrane protein H11, previously shown to give protective immunity against the parasite, was fractionated by lectin and ion-exchange chromatography. The fractions were evaluated for their ability to immunize Clun Forest and Dorset Horn sheep against experimental haemonchosis. Most of the protective activity was associated with H11. Used in an approximately 95% pure form it gave a mean reduction in parasite egg output of 94.6% and reduced male and female worm numbers by 86.5 and 93.5%, respectively. Level of protection correlated with serum antibody titre to H11.

WldS protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice

The slow Wallerian degeneration (WldS) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70–amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide–synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of WldS-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the WldS VCP-binding domain with an alternative ataxin-3–derived VCP-binding sequence restores its protective function. Enzyme-dead WldS is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. WldS requires both of its components to protect axons from degeneration.

DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp52/iso-Asp66 species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.

VACCINATION AGAINST HAEMONCHUS CONTORTUS WITH DENATURED FORMS OF THE PROTECTIVE ANTIGEN H11

As part of a systematic examination of the protective epitopes on H11, groups of sheep were vaccinated with preparations of purified H11 used untreated (group A), or progressively denatured (linearized) by incubation with sodium dodecyl sulphate (SDS) (group B) or by boiling with SDS in the presence of dithiothreitol (group C). All the sheep developed antibodies which bound to the untreated H11. When challenged with 10 000 infective larvae of Haemonchus contortus the mean levels of protection relative to the mean values for adjuvant controls were 99.8%, 85% and 79% for faecal egg counts and 95%, 79% and 54% for worm burden at post‐mortem for groups A, B and C respectively. The H11‐specific antibodies inhibited the microsomal aminopeptidase activity of H11 in vitro up to 80%. The levels of inhibition by sera from individual animals correlated with levels of protection with r2, of 0.69–0.87.

The potential value of integral membrane proteins in the vaccination of lambs against Haemonchus conortus

An extract of adult Haemonchus contortus enriched in the parasites intestinal microvillar membrane protein H11 and other integral membrane proteins but free of the protein contortin was evaluated as a potential vaccine in two breeds of sheep. The worm burdens of Clun Forest sheep injected with the extract and challenged with 25,000 infective larvae were reduced 89% by weight compared to the average for the controls. The worm burdens of Dorset sheep (challenged with 10,000 infective larvae) were reduced 72%. In both breeds the reduction in the number of female worms, 92 and 71.8%, respectively, was greater than the reduction in the males (86.5 and 46%). Parasite egg output, determined only for the Dorsets, was reduced 92% protection correlated with serum antibody titre. Most of the antibodies were directed against H11.

Cloning and characterization of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus

In order to characterise the integral membrane glycoprotein H11 from the intestinal microvilli of the nematode Haemonchus contortus, cDNA libraries prepared using mRNA from adult worms from the UK and Australia were immunoscreened with anti-H11 sera. Antibodies affinity purified on the protein expressed by insert DNA (295 bp) of a positive clone from a UK library bound specifically to H11. A longer clone (948 bp) was obtained from the Australian library by hybridisation. Using a primer based on sequence common to these, a polymerase chain reaction product of 3.3 kb was generated from cDNA from UK H. contortus. The sequences from the UK and Australian nematodes were essentially identical over the 929 bp region in which both were represented. All three cloned DNAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian microsomal aminopeptidases, indicated that H11 has a short N-terminal cytoplasmic tail, a single transmembrane region and a long extracellular region with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases. Microsomal aminopeptidase activity co-purifies with H11. It is inhibited by bestatin, phenanthroline and amastatin. The recombinant protein has been expressed in active form in insect cells.

Vaccination of young Dorset lambs against haemonchosis.

Summary Six Dorset Horn lambs were each vaccinated at age 7 weeks and 9 weeks with 50 μg of glycosylated integral membrane proteins, particularly enriched in the protein H11 from the intestinal brush border of adult Haemonchus. At 11 weeks of age the lambs were infected with 10 000 infective third stage Haemonchus larvae. Compared with the average for the control group the vaccinated group of lambs had a 78% reduction in parasite egg output over the patent period, with four of the six better than 93% protected. At autopsy 35 days post‐infection the mean total worm burden of the vaccinated lambs was 83% reduced compared with the controls. The serum antibody titres to H11 correlated with the degree of protection.