Diversification of the functions of proglucagon and glucagon receptor genes in fish.

The teleost fish-specific genome duplication gave rise to a great number of species inhabiting diverse environments with different access to nutrients and life histories. This event produced duplicated gcg genes, gcga and gcgb, for proglucagon-derived peptides, glucagon and GLP-1 and duplicated gcgr receptor genes, gcgra and gcgrb, which play key roles connecting the consumption of nutrients with glucose metabolism. We conducted a systematic survey of the genomes from 28 species of fish (24 bony (Superclass Osteichthyes), 1 lobe-finned (Class Sarcoperygii), 1 cartilaginous (Superclass Chondrichthyes), and 2 jawless (Superclass Agnatha)) and find that almost all surveyed ray-finned fish contain gcga and gcgb genes with different coding potential and duplicated gcgr genes, gcgra and gcgrb that form two separate clades in the phylogenetic tree consistent with the accepted species phylogeny. All gcgb genes encoded only glucagon and GLP-1 and gcga genes encoded glucagon, GLP-1, and GLP-2, indicating that gcga was subfunctionalized to produce GLP-2. We find a single glp2r, but no glp1r suggesting that duplicated gcgrb was neofunctionalized to bind GLP-1, as demonstrated for the zebrafish gcgrb (Oren et al., 2016). In functional experiments with zebrafish gcgrb and GLP-1 from diverse fish we find that anglerfish GLP-1a, encoded by gcga, is less biologically active than the gcgb anglerfish GLP-1b paralog. But some other fish (zebrafish, salmon, and catfish) gcga GLP-1a display similar biological activities, indicating that the regulation of glucose metabolism by GLP-1 in ray-finned fish is species-specific. Searches of genomes in cartilaginous fish identified a proglucagon gene that encodes a novel GLP-3 peptide in addition to glucagon, GLP-1, and GLP-2, as well as a single gcgr, glp2r, and a new glucagon receptor-like receptor whose identity still needs to be confirmed. The sequence of the shark GLP-1 contained an N-terminal mammalian-like extension that in mammals undergoes a proteolytic cleavage to release biologically active GLP-1. Our results indicate that early in vertebrate evolution diverse regulatory mechanisms emerged for the control of glucose metabolism by proglucagon-derived peptides and their receptors and that in ray-finned fish they included subfunctionalization and neofunctionalization of these genes.

Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice.

Most treatments that prevent autoimmune diabetes in nonobese diabetic (NOD) mice require intervention at early pathogenic stages, when insulitis is first developing. We tested whether dendritic cell (DC)-expanded, islet antigen-specific CD4+ CD25+ suppressor T cells could treat diabetes at later stages of disease, when most of the insulin-producing islet beta cells had been destroyed by infiltrating lymphocytes. CD4+ CD25+ CD62L+ regulatory T cells (T reg cells) from BDC2.5 T cell receptor transgenic mice were expanded with antigen-pulsed DCs and IL-2, and were then injected into NOD mice. A single dose of as few as 5x10(4) of these islet-specific T reg cells blocked diabetes development in prediabetic 13-wk-old NOD mice. The T reg cells also induced long-lasting reversal of hyperglycemia in 50% of mice in which overt diabetes had developed. Successfully treated diabetic mice had similar responses to glucose challenge compared with nondiabetic NOD mice. The successfully treated mice retained diabetogenic T cells, but also had substantially increased Foxp3+ cells in draining pancreatic lymph nodes. However, these Foxp3+ cells were derived from the recipient mice and not the injected T reg cells, suggesting a role for endogenous T reg cells in maintaining tolerance after treatment. Therefore, inoculation of DC-expanded, antigen-specific suppressor T cells has considerable efficacy in ameliorating ongoing diabetes in NOD mice.

Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells.

Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen-presenting cells and display short bound peptide fragments derived from self- and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self-antigens and initiate the CD4(+) T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5 × 10(8) splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endolysosomal (12%), nuclear (14%), and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/µL to 12 pmol/µL or from approximately 13 to 2 × 10(5) copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.

Structural Mapping and Functional Characterization of Zebrafish Class B G-Protein Coupled Receptor (GPCR) with Dual Ligand Selectivity towards GLP-1 and Glucagon.

GLP-1 and glucagon regulate glucose metabolism through a network of metabolic pathways initiated upon binding to their specific receptors that belong to class B G-protein coupled receptors (GPCRs). The therapeutic potential of glucagon is currently being evaluated, while GLP-1 is already used in the treatment of type 2 diabetes and obesity. Development of a second generation of GLP-1 based therapeutics depends on a molecular and structural understanding of the interactions between the GLP-1 receptor (GLP-1R) and its ligand GLP-1. There is considerable sequence conservation between GLP-1 and glucagon and between the hGLP-1R and human glucagon receptor (hGCGR), yet each receptor recognizes only its own specific ligand. Glucagon receptors in fish and frogs also exhibit ligand selectivity only towards glucagon and not GLP-1. Based on competitive binding experiments and assays of increase in intracellular cAMP, we demonstrate here that a GPCR in zebrafish (Danio rerio) exhibits dual ligand selectivity towards GLP-1 and glucagon, a characteristic not found in mammals. Further, many structural features found in hGLP-1R and hGCGR are also found in this zebrafish GPCR (zfGPCR). We show this by mapping of its sequence and structural features onto the hGLP-1R and hGCGR based on their partial and complementary crystal structures. Thus, we propose that zfGPCR represents a dual GLP-1R/GCGR. The main differences between the three receptors are in their stalk regions that connect their N-terminal extracellular domains (NECDs) with their transmembrane domains and the absence of loop 3 in the NECD in zfGLP-1R/GCGR. These observations suggest that the interactions between GLP-1 and glucagon with loop 3 and the stalk regions may induce different conformational changes in hGLP-1R and hGCGR upon ligand binding and activation that lead to selective recognition of their native ligands.

Isolation and structure-function studies of a glucagon-like peptide 1 receptor from goldfish Carassius auratus: identification of three charged residues in extracellular domains critical for receptor function.

A better understanding of the molecular mechanism of ligand-receptor interaction of glucagon-like peptide 1 (GLP-1) receptors (GLP-1Rs) is useful for the design of potent GLP-1 analogs that could potentially be used as a treatment for diabetic patients. Changes in the ligand and receptor sequences during evolution provide invaluable clues to evaluate the functional motifs of the receptor that are responsible for ligand interaction. For these reasons, in the present study, we have isolated and functionally characterized a GLP-1R from goldfish. Its amino acid sequence shows 50.8% and 52.3% identity with the human glucagon (hGLU) and GLP-1Rs, respectively, and 84.1% with the zebrafish GLP-1R (the only other GLP-1R isolated from teleost fish). Peptides that are structurally different from goldfish (gf)GLP-1, such as gfGLU and hGLU and human GLP-1 (7-36)amide, are also capable of stimulating this receptor, albeit with lower potencies than gfGLP-1. gfGLP-1 stimulates the formation of cAMP through the recombinant gfGLP-1R with EC(50) = 0.18 nM, whereas EC(50) values for gfGLU, human GLP-1 (7-36)amide, and hGLU are 0.53 nM, 0.9 nM, and 1.2 nM, respectively. These results indicate that the gfGLP-1R is structurally more flexible than its mammalian counterpart and that its binding pocket can accommodate a wider spectrum of peptide ligands. Previous studies demonstrated that the charged residues in the extracellular domains of mammalian GLP-1R, particularly those found in the N-terminal domain and the first exoloop, are important for ligand binding. We investigated the roles of the conserved charged residues in the function of the gfGLP-1R. Eleven mutant receptors were constructed, and the effects of mutations were determined by functional assays. Our results demonstrated that three charged residues (D(113), R(197), and D(205)) present in the extracellular domains are critical for receptor function.

Identification and characterization of a glucagon receptor from the goldfish Carassius auratus: implications for the evolution of the ligand specificity of glucagon receptors in vertebrates.

The structural basis of ligand selectivity of G protein-coupled receptors for metabolic hormones has been an area of intense investigation, and yet it remains unresolved. One approach to delineating the mechanism of ligand-receptor interactions is to compare the ligand specificities of receptors expressed in species that emerged at different times within vertebrate evolution. In this paper we describe the isolation, functional, and phylogenetic characterization of the glucagon receptor from the goldfish Carassius auratus (Teleostei, order Cypriniformes), and compare its ligand specificity with that of the homologous rat receptor. Goldfish (gf) glucagon stimulated glucose production in a dose-dependent manner from isolated goldfish hepatocytes, resulting in 5-fold increase at 1 microm. The goldfish glucagon receptor (gfGlucR) shares 56, 51, 50, and 52% amino acid identities with frog Rana tigrina regulosa, mouse, rat, and human glucagon receptors, respectively. In competitive binding experiments, the recombinant gfGlucR displays high affinity toward goldfish, zebrafish, and human glucagons (IC(50) = 0.6, 9, and 13 nm, respectively) but not toward goldfish glucagon-like peptide-1 or human glucagon-like peptide-1 (7-36) amide. Whereas both goldfish and human glucagons stimulated dose-dependent increases in intracellular cAMP through the recombinant gfGlucR, the recombinant rat GlucR interacted only with human glucagon, analogous to the specificity of the previously characterized glucagon receptor from the frog R. tigrina regulosa. Our results demonstrate that the binding pocket of gfGlucR can accommodate a broad range of glucagon structures and that in the frogs and mammals, there is a structural switch to a more restrictive conformation of glucagon receptors.

Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.

Expression of a functional eotaxin (CC chemokine ligand 11) receptor CCR3 by human dendritic cells.

Critical to the function of Ag-presenting dendritic cells (DCs) is their capacity to migrate to lymphoid organs and to sites of inflammation. A final stage of development, termed maturation, yields DCs that are strong stimulators of T cell-mediated immunity and is associated with a remodeling of the cell surface that includes a change in the levels of expression of many molecules, including chemokine receptors. We show in this study that CCR3, a chemokine receptor initially discovered on eosinophils, is also expressed by human DCs that differentiate from blood monocytes, DCs that emigrate from skin (epidermal and dermal DCs), and DCs derived from CD34+ hemopoietic precursors in bone marrow, umbilical cord blood, and cytokine-elicited peripheral blood leukapheresis. Unlike other chemokine receptors, such as CCR5 and CCR7, the expression of CCR3 is not dependent on the state of maturation. All DC subsets contain a large intracellular pool of CCR3. The surface expression of CCR3 is not modulated following uptake of particulate substances such as zymosan or latex beads. CCR3 mediates in vitro chemotactic responses to the known ligands, eotaxin and eotaxin-2, because the DC response to these chemokines is inhibited by CCR3-specific mAbs. We postulate that expression of CCR3 may underlie situations where both DCs and eosinophils accumulate in vivo, such as the lesions of patients with Langerhans cell granulomatosis.