Jarema Peter Kochan

Autoantibodies against the High-Affinity IgE Receptor as a Cause of Histamine Release in Chronic Urticaria

BACKGROUND Most urticarias are induced by vasoactive mediators such as histamine released from mast cells. Although mast cells are activated by allergens through cross-linking of cell-surface--bound IgE, this mechanism does not appear to explain most cases of chronic urticaria, which, when allergic, infectious, drug-induced, or physical causes cannot be identified, are classified as idiopathic. METHODS We recruited 26 patients with chronic idiopathic urticaria, in whom intradermal injection of autologous serum caused a wheal-and-flare response. Serum from four patients that induced marked histamine release from basophils from a donor with very low serum IgE levels was studied with respect to the IgE dependence of the histamine release, the activity of the IgG fractions, and the neutralizing effect of a recombinant preparation of the soluble extracellular domain of the alpha subunit of the high-affinity IgE receptor (sFc epsilon RI alpha). RESULTS The histamine-releasing activity of the serum was abolished by passive sensitization of basophils with myeloma IgE, enhanced after dissociation of IgE by treatment with lactic acid, and induced by IgG fractions from the serum of all four patients. Preincubation of the serum and isolated IgG with sFc epsilon RI alpha resulted in almost complete neutralization. CONCLUSIONS Histamine-releasing IgG autoantibodies against the alpha subunit of the high-affinity IgE receptor are present in the circulation of some patients with chronic urticaria. Autoantibody-induced cross-linking of IgE receptors may be an important mechanism in the pathogenesis of chronic urticaria and other diseases mediated by mast cells.

Leptin Receptor Long-form Splice-variant Protein Expression in Neuron Cell Bodies of the Brain and Co-localization with Neuropeptide Y mRNA in the Arcuate Nucleus

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Localization of a susceptibility gene for type 2 diabetes to chromosome 5q34-q35.2.

We report a genomewide linkage study of type 2 diabetes (T2D [MIM 125853]) in the Icelandic population. A list of type 2 diabetics was cross-matched with a computerized genealogical database clustering 763 type 2 diabetics into 227 families. The diabetic patients and their relatives were genotyped with 906 microsatellite markers. A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 2.90, P=1.29 x 10(-4)) in all diabetics. Since obesity, here defined as body mass index (BMI) > or =30 kg/m(2), is a key risk factor for the development of T2D, we studied the data either independently of BMI or by stratifying the patient group as obese (BMI > or =30) or nonobese (BMI <30). A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 3.64, P=2.12 x (10)-5) in the nonobese diabetics. No linkage was observed in this region for the obese diabetics. Linkage analysis conditioning on maternal transmission to the nonobese diabetics resulted in a LOD score of 3.48 (P=3.12 x 10(-5)) in the same region, whereas conditioning on paternal transmission led to a substantial drop in the LOD score. Finally, we observed potential interactions between the 5q locus and two T2D susceptibility loci, previously mapped in other populations.

The Inositol 5′-Phosphatase SHIP Binds to Immunoreceptor Signaling Motifs and Responds to High Affinity IgE Receptor Aggregation

Immunoreceptors such as the high affinity IgE receptor, FcεRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcεRI ITAM-containing subunits β and γ. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the FcεRI β or γ ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor ζ chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5′-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. FcεRI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.

Interaction of Phosphorylated FcϵRIγ Immunoglobulin Receptor Tyrosine Activation Motif-based Peptides with Dual and Single SH2 Domains of p72syk ASSESSMENT OF BINDING PARAMETERS AND REAL TIME BINDING KINETICS

To examine the characteristics of the interaction of the FcεRIγ ITAM with the SH2 domains of p72syk, the binding of an 125I-labeled dual phosphorylated FcεRIγ ITAM-based peptide to the p72syk SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72syk SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcεRIγ ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

Splice Variants of the OB Receptor Gene are Differentially Expressed in Brain and Peripheral Tissues of Mice

A high affinity receptor for OB protein was recently cloned from the choroid plexus of mice. At least six alternatively spliced forms of the OB receptor (OB-R) gene have been described, all of which encode proteins containing the OB-R extracellular domain. One splice variant encodes a receptor with a long intracellular domain, OB-RL, that has been implicated in OB-R signaling. Here, we have used in situ hybridization to examine the localization of OB-R splice variants in brain and peripheral tissues of adult and newborn mice. Using a probe hybridizing with all known splice variants, we confirmed that OB-R mRNA was widely distributed in the adult tissues. In the CNS, choroid plexus was the major site of expression. We now demonstrate that OB-R mRNA is expressed in peripheral tissues; primarily associated with connective tissues. In addition, OB-R mRNA was detected at higher levels in peripheral tissues of newborn mice than in adult mice. With a probe specific for OB-RL, we confirmed that high mRNA expression was detected in hypothalamic nuclei, while low levels were observed in choroid plexus. We now report that in peripheral tissues of adult mice, OB-RL mRNA expression was either very low or undetectable. In newborn mice, the pattern of OB-RL message expression in the CNS was similar to that of adult mice, while bone was the site of highest OB-RL message expression in the peripheral tissue. These data suggest different biological roles for OB-R splice variants encoding the short and long forms of OB-R. The localization of OB-RL to hypothalamic nuclei supports the idea that OB-RL is the brain receptor that mediates OB protein signaling and actions. In addition, the expression of OB-R message in newborn mice also suggests a biological role of OB-R during development in mice.

Role of glycosylation sites in the IgE Fc molecule.

The Fc region of immunoglobulin E (IgE) comprising the C epsilon 3 and C epsilon 4 domains (residues 329-547) is sufficient for binding to the high-affinity IgE Fc receptor (Fc epsilon RI alpha). Three potential N-linked glycosylation sites are present within the C epsilon 3 domain. To determine the effect of the glycosylation sites on IgE Fc synthesis and on Fc epsilon RI alpha binding, site-directed mutagenesis was performed. Mutant IgE Fc constructs were expressed in COS cells and analyzed for protein synthesis and secretion, and Fc epsilon RI alpha binding activity. We find that only N371 and N394 are glycosylated, and that the residues surrounding the glycosylation site at N394 are required for Fc epsilon RI alpha binding activity.

Emerging Picture of the Receptor with High Affinity for IgE

The cDNAs for two of the subunits of the receptor with high affinity for IgE have been isolated and sequenced. That for the IgE-binding alpha subunit predicts a polypeptide with a single transmembrane segment. The predicted extracellular amino terminal portion, comprised of 180 residues, is homologous to the corresponding region of Fc gamma receptors and contains two truncated immunoglobulin-like domains. The cDNA for beta predicts a peptide with four transmembrane segments, but its sequence is unrelated to known proteins. Experiments to isolate the cDNA for the gamma subunit have begun; when completed, cotransfection of the three genes will be attempted to get expression, a process that has not so far been successful for the individual subunits. Such expression will permit further analysis of the receptors function and ultimately may provide information on how best to intervene clinically in the activation of mast cells and basophils.

A novel strategy for secreting proteins : use of phosphatidylinositol-glycan-specific phospholipase D to release chimeric phosphatidylinositol-glycan anchored proteins

Phosphatidylinositol–glycan–specific phospholipase D (PI–G PLD) specifically hydrolyzes the inositol–phosphate linkage in phosphatidylinositol–glycan (PI–G) anchored proteins. We recently deduced the primary structure of this enzyme and demonstrated specific enzymatic activity in transfected cells. Co–transfection of PI–G PLD with a natural PI–G anchored protein resulted in the secretion of the PI–G anchored protein via a PI–G PLD specific mechanism. We have taken advantage of these observations to develop an alternative system that may be useful for expressing and secreting proteins not amenable to secretion by conventional methods. Chimeric PI–G anchored proteins were constructed by transferring the COOH–terminal signal peptide for PI–G anchor attachment from placental alkaline phosphatase or from the low affinity IgG receptor, FcGRIIIB, to proteins that are not normally PI–G anchored. This process facilitates the cell surface expression of several proteins including the high affinity IgE receptor α subunit, FcERIα, which otherwise requires at least one other subunit for surface expression. Co–expression of these chimeric PI–G anchored proteins with PI–G PLD resulted in their secretion via a PI–G PLD specific mechanism.

Discovery of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors

Through high throughput screening and subsequent hit identification and optimization, we synthesized a series of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as the first reported potent and reversible GFAT inhibitors. SAR studies of this class of compounds indicated significant impact on GFAT inhibition potency by substitutions on the A-ring and C-ring. The ketone group was found to be necessary for high potency. Compound 28 (RO0509347) demonstrated potent GFAT inhibition (IC(50)=1μM) with a desirable pharmacokinetic profile in rats, and showed significant efficacy in reducing the glucose excursion in an OGTT test in ob/ob mice.