The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis

The G-protein coupled cannabinoid receptors CB(1) and CB(2) are activated by Delta(9)-tetrahydrocannabinol, the psychoactive ingredient of cannabis, and mediate physiological effects of endogenous cannabinoids ('endocannabinoids'). CB(1) genes have been identified in mammals, birds, amphibians and fish, whilst CB(2) genes have been identified in mammals and in the puffer fish Fugu rubripes. Therefore, both CB(1) and CB(2) receptors probably occur throughout the vertebrates. However, cannabinoid receptor genes have yet to be identified in any invertebrate species and the evolutionary origin of cannabinoid receptors is unknown. Here we report the identification of CiCBR, a G-protein coupled receptor in a deuterostomian invertebrate - the urochordate Ciona intestinalis - that is orthologous to vertebrate cannabinoid receptors. The CiCBR cDNA encodes a protein with a predicted length (423 amino-acids) that is the intermediate of human CB(1) (472 amino-acids) and human CB(2) (360-amino-acid) receptors. Interestingly, the protein-coding region of the CiCBR gene is interrupted by seven introns, unlike in vertebrate cannabinoid receptor genes where the protein-coding region is typically intronless. Phylogenetic analysis revealed that CiCBR forms a clade with vertebrate cannabinoid receptors but is positioned outside the CB(1) and CB(2) clades of a phylogenetic tree, indicating that the common ancestor of CiCBR and vertebrate cannabinoid receptors predates a gene (genome) duplication event that gave rise to CB(1)- and CB(2)-type receptors in vertebrates. Importantly, the discovery of CiCBR and the absence of orthologues of CiCBR in protostomian invertebrates such as Drosophila melanogaster and Caenorhabditis elegans indicate that the ancestor of vertebrate CB(1) and CB(2) cannabinoid receptors originated in a deuterostomian invertebrate.     

Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate sensory placodes

Cranial sensory placodes are focused areas of the head ectoderm of vertebrates that contribute to the development of the cranial sense organs and their associated ganglia. Placodes have long been considered a key character of vertebrates, and their evolution is proposed to have been essential for the evolution of an active predatory lifestyle by early vertebrates. Despite their importance for understanding vertebrate origins, the evolutionary origin of placodes has remained obscure. Here, we use a panel of molecular markers from the Six, Eya, Pax, Dach, FoxI, COE and POUIV gene families to examine the tunicate Ciona intestinalis for evidence of structures homologous to vertebrate placodes. Our results identify two domains of Ciona ectoderm that are marked by the genetic cascade that regulates vertebrate placode formation. The first is just anterior to the brain, and we suggest this territory is equivalent to the olfactory/adenohypophyseal placodes of vertebrates. The second is a bilateral domain adjacent to the posterior brain and includes cells fated to form the atrium and atrial siphon of adult Ciona. We show this bares most similarity to placodes fated to form the vertebrate acoustico-lateralis system. We interpret these data as support for the hypothesis that sensory placodes did not arise de novo in vertebrates, but evolved from pre-existing specialised areas of ectoderm that contributed to sensory organs in the common ancestor of vertebrates and tunicates.     

The evolution of the vertebrate metzincins; insights from Ciona intestinalis and Danio rerio

Background

The metzincins are a large gene superfamily of proteases characterized by the presence of a zinc protease domain, and include the ADAM, ADAMTS, BMP1/TLL, meprin and MMP genes. Metzincins are involved in the proteolysis of a wide variety of proteins, including those of the extracellular matrix. The metzincin gene superfamily comprises eighty proteins in the human genome and ninety-three in the mouse. When and how the level of complexity apparent in the vertebrate metzincin gene superfamily arose has not been determined in detail. Here we present a comprehensive analysis of vertebrate metzincins using genes from both Ciona intestinalis and Danio rerio to provide new insights into the complex evolution of this gene superfamily.

Results

We have identified 19 metzincin genes in the ciona genome and 83 in the zebrafish genome. Phylogenetic analyses reveal that the expansion of the metzincin gene superfamily in vertebrates has occurred predominantly by the simple duplication of pre-existing genes rather than by the appearance and subsequent expansion of new metzincin subtypes (the only example of which is the meprin gene family). Despite the number of zebrafish metzincin genes being relatively similar to that of tetrapods (e.g. man and mouse), the pattern of gene retention and loss within these lineages is markedly different. In addition, we have studied the evolution of the related TIMP gene family and identify a single ciona and four zebrafish TIMP genes.

Conclusion

The complexity seen in the vertebrate metzincin gene families was mainly acquired during vertebrate evolution. The metzincin gene repertoire in protostomes and invertebrate deuterostomes has remained relatively stable. The expanded metzincin gene repertoire of extant tetrapods, such as man, has resulted largely from duplication events associated with early vertebrate evolution, prior to the sarcopterygian-actinopterygian split. The teleost repertoire of metzincin genes in part parallels that of tetrapods but has been significantly modified, perhaps as a consequence of a teleost-specific duplication event.     

Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells.

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.     

Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated from invertebrate sources

1. The 4K-prothoracicotropic hormone (PTTH) or bombyxin and the melanization-reddish coloration hormone of the silkworm Bombyx mori resemble insulin and insulin-like growth factors. 2. The family of adipokinetic/red pigment concentrating hormones has some similarity with glucagon. 3. Members of the FMRFamide family are found in vertebrates as well as in invertebrates. 4. In Locusta, a molecule immunologically and biologically related to amphibian melanophore stimulating hormone has been partially characterized. 5. Enkephalins and enkephalin-related peptides occur in insects and other invertebrates. 6. Peptides belonging to the tachykinin family have been isolated from molluscan (Octopus) salivary glands and from insect nervous tissue (Locusta migratoria). 7. Invertebrate arginine-vasotocin homologs have been isolated from an insect (Locusta migratoria) and from a mollusc (Conus). 8. In Leucophaea, Locusta and Drosophila, peptides resembling those of the vertebrate gastrin/cholecystokinin family have been identified. 9. As the number of different neuro-/gut peptides with possible function(s) as hormone, neurotransmitter or neuromodulator is now estimated to be of the order of a few hundred, more similarities will probably show up in the near future.     

Physicochemical characterization of lens crystallins from the carp and biochemical comparison with other vertebrate and invertebrate crystallins

Lens crystallins were isolated from the homogenate of carp (Cyprinus carpio) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, immunodiffusion, amino acid analysis, circular dichroism, and protein sequence analysis. Three well-defined fractions corresponding to alpha/beta-, beta-, and gamma-crystallins were obtained in relative weight percentages of 26, 22, and 52%. The native molecular masses of the purified fractions were determined to be 410, 60, and 20 kDa, respectively. The polypeptide compositions as determined by SDS gel electrophoresis revealed the substantial presence of beta-crystallin polypeptides in the alpha-crystallin fraction; this is also evident in the fractionation of amphibian crystallins but is not common in the case of higher classes of vertebrates. The circular dichroism spectra indicate a predominant beta-sheet structure in all three fractions, albeit with some contribution of alpha-helical structure in the gamma-crystallin, the amino acid composition of which bears a resemblance to that of squid crystallin. Sequence comparison of carp gamma-crystallin with frog and calf gamma-crystallins indicates a high degree of homology in their N-terminal segments despite the dissimilarity of amino acid compositions and weak immunological cross-reactivity.     

A lysine rich C-terminal tail is directly involved in the toxicity of CSTX-1, a neurotoxic peptide from the venom of the spider Cupiennius salei

CSTX-1 (74 amino acids, 8,352.62 Da) is a potent neurotoxin from the venom of Cupiennius salei. With the monoclonal antibody 9H3 against CSTX-1, we identified two similar peptides by Western blot analysis. These two peptides were purified by RP-HPLC: CSTX-2a (61 amino acids, 6865.75 Da) and CSTX-2b (60 amino acids, 6709.57 Da). Using ESI-MS analysis and sequencing we verified that CSTX-2a is a truncated version of CSTX-1. CSTX-2b differs from CSTX-2a by the absence of Arg61. Toxicity of CSTX-1, CSTX-2a, and CSTX-2b to Drosophila melanogaster showed that the absence of the last 13 amino acids of CSTX-1 results in a seven-fold activity loss. CSTX-2b, which lacks Arg61 is 190-fold less toxic. We conclude that the C-terminal part of CSTX-1, especially Arg61, is essential for the expression of toxicity. CSTX-1 is degraded to CSTX-2a and CSTX-2b by proteases that are released from venom gland cells by apocrine secretion.     

Cellular refractoriness to the heat-stable enterotoxin peptide is associated with alterations in levels of the differentially glycosylated forms of guanylyl cyclase C.

The heat-stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin binding to its receptor, guanylyl cyclase C (GC-C), results in receptor activation and the production of high intracellular levels of cGMP. GC-C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intracellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose-dependent inability of cells to respond to the peptide in terms of GC-C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST-binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane-associated, hyperglycosylated 145 kDa form of GC-C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST-treated cells. Desensitized cells recovered ST-responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, following processing of the endoplasmic reticulum-associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST-treatment of cells at 4 degrees C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycosylated forms of GC-C can lead to differential ligand-mediated activation of the receptor.     

Luciferase, when fused to an N-terminal signal peptide, is secreted from transfected Plasmodium falciparum and transported to the cytosol of infected erythrocytes

Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a rate-limiting step.     

Ligand binding characteristics of two molecular forms,one equipped with a hydrophobic glycosyl phosphatidylinositol tail,of the folate binding protein purified from human milk

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [³H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.     

Tumor-specific immunity in MUC1.Tg mice induced by immunization with peptide vaccines from the cytoplasmic tail of CD227 (MUC1)

Purpose: CD227 (MUC1), a membrane-associated glycoprotein expressed by many types of ductal epithelia, including pancreas, breast, lung, and gastrointestinal tract, is overexpressed and aberrantly glycosylated by malignant cells. We sought to define epitopes on MUC1 recognized by the different cell-mediated immune responses by an in vivo assay. Epitopes identified by this assay were evaluated for efficacy to protect mice transgenic for human MUC1 (MUC1.Tg) against MUC1-expressing tumor growth. Methods: We investigated contributions of the tandem repeat (TR) and the cytoplasmic tail (CT) of MUC1 to the MUC1-specific immunological rejection of tumor cells. MUC1 cDNA constructs, in which the TR region was deleted or the CT was truncated, were transfected into two different murine tumor cell lines (B16 and Panc02), which were used to challenge mice and evaluate immunological rejection of the tumors. We used tumor rejection in vivo to define epitopes on the TR and CT of MUC1 recognized by T cell–mediated immune responses in a preclinical murine model. Results: Our findings demonstrated that the TR and a portion of the MUC1 CT contributed to CD4⁺ T cell rejection of MUC1-expressing B16 tumor cells, but not rejection of MUC1-expressing Panc02 tumor cells. A separate epitope in the CT of MUC1 was necessary for CD8⁺ T cell rejection of Panc02 tumor cells. Based on these studies, we sought to evaluate the efficacy of immunizing mice transgenic for (and immunologically tolerant to) human MUC1 with peptides derived from the amino acid sequence of the CT of MUC1. Results showed that survival can be significantly prolonged in vaccinated MUC1.Tg mice challenged with MUC1-expressing tumor cells, without induction of autoimmune responses. Conclusions: These studies demonstrated that MUC1 peptides may be utilized as an effective anticancer immunotherapeutic, and confirmed the importance of immunogenic epitopes outside of the TR.Abbreviations aa Amino acid - CT Cytoplasmic tail - IFN- Interferon gamma - MUC1.Tg MUC1 transgenic mice - TR Tandem repeat - wt Wild-type C57BL/6 mice This work was supported by National Institutes of Health grants CA72712 and CA57362 (M.A.H.), National Institutes of Health training grant CA09476 (K.G.K., A.J.G, and M.L.V.), and fellowship awards from the University of Nebraska Medical Center (to K.G.K. and M.L.V).     

Recruitment of MHC class I molecules by tapasin into the transporter associated with antigen processing-associated complex is essential for optimal peptide loading

The ER protein tapasin (Tpn) forms a bridge between MHC class I H chain (HC)/beta(2)-microglobulin and the TAP peptide transporter. The function of this TAP-associated complex was unclear because it was reported that soluble Tpn that has lost TAP interaction would be fully competent in terms of peptide loading and Ag presentation. We found, however, that only wild-type human Tpn (hTpn), but not three soluble hTpn variants, a transmembrane domain point mutant of hTpn (L410-->F), wild-type mouse Tpn, nor a mouse-human Tpn hybrid, fully up-regulated peptide-dependent Bw4 epitopes when expressed in Tpn-deficient.220.B*4402 cells. Consistent with suboptimal peptide loading, the t(1/2) of class I molecules was considerably reduced in the presence of soluble hTpn, hTpn-L410F, and murine Tpn. Furthermore, eluted peptide spectra and the class I-mediated inhibition of NK clones showed distinct differences to the hTpn transfectant. Only wild-type hTpn efficiently recruited HC and calreticulin (Crt) into complexes with TAP and endoplasmic reticulum p57 (ERp57). The L410F mutant was defective in TAP association, but bound to class I molecules, Crt, and ERp57. Mouse Tpn associated with human TAP and ERp57 on the one hand, and with HC and Crt on the other, but failed to recruit normal amounts of HLA class I molecules into the TAP complex. We conclude that the loading with peptides conferring high stability requires the Tpn-mediated introduction of HC into the TAP complex, whereas the mere interaction with Tpn is not sufficient.