Epitopes for thyroid stimulating and blocking autoantibodies on the extracellular domain of the human thyrotropin receptor

The majority (97%) of functional epitopes for stimulating thyrotropin receptor (TSHR) antibodies (stimulating TSHRAbs) in a large cohort (n = 59) of Japanese Graves' patients exists on the N-terminal region of the extracellular domain of TSHR, between residues 25 and 165 numbering from the methionine start site. This was determined by measuring the loss of stimulating activity in the Cos-7 cells transfected with TSHR/lutropin-choriogonadotropin receptor (LH-CGR) chimeras wherein TSHR residues 89-165 (Mc2) or 8-165 (Mc1 + 2) are replaced by comparable LH-CGR residues. There is no comparable loss when stimulating TSHRAb activity is measured in an Mc4 chimera, wherein TSHR residues 261 to 370 are replaced. In contrast, immunoglobulin (IgG) preparations from 35 patients with Hashimoto's disease or idiopathic myxedema, who have blocking TSHRAbs causing hypothyroidism, loose blocking TSHRAb activity in the Mc4 chimera, but not the Mc2 or Mc1 + 2 chimeras. Thus, in a large population of Japanese patients with autoimmune thyroid disease caused by TSHR autoantibodies, the major functional epitope for stimulating TSHRAbs is on the N-terminal portion of the TSHR extracellular domain, whereas that for blocking TSHRAbs is on the C-terminal portion of the extracellular domain. To further evaluate the nature of the critical functional epitope between residues 90 to 165, we divided this region approximately in half, creating chimeras Mc2a and Mc2b with, respectively, residues 90-124 or 125-165 replaced by comparable LH-CGR residues. IgGs from all patients tested lost significant stimulating activity using the Mc2a and Mc2b chimeras; however, when present, residual stimulating TSHRAb activity was evident on one or the other half of the region or on both halves, indicating that both segments are required for expression of the stimulating TSHRAb epitope within residues 90-165. Finally, we have identified a complex epitope involving both the N- and C-terminal portion of the extracellular domain that appears to account for the small fraction of stimulating TSHRAbs whose activity is not solely dependent on residues 25 to 165. Thus, using chimeras Mc1 + 2 + 4, with TSHR residues 8-165 and 261-370 substituted, or chimera Mc1 + 2 + 3 + 4, with residues 8-370 substituted, as well as Mc2, Mc1 + 2, and Mc4, we show that the Graves' IgGs which maintain stimulating TSHRAb activity when residues 8-165 of the TSHR are replaced by LH-CGR residues have an epitope involving residues 90-165 and the immunogenic 15mer peptide (YYVFFEEQEDEIIGF), residues, 352-366. Because that peptide can decrease the stimulating TSHRAb activity of these Graves IgGs in assays with the Mc2 chimera alone, we speculate that this complex epitope may be important in an epitope spreading process involved in the formation of stimulating TSHRAbs.     

The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current

Propofol (2,6-diisopropylphenol), an intravenous general anesthetic in active clinical use today, potentiates the action of gamma-aminobutyric acid (GABA) at the type-A receptor and also directly induces current in the absence of GABA. We expressed different combinations of murine GABA(A) receptor alpha1, beta3 and gamma2 subunits in Xenopus oocytes to investigate the subunit dependence of propofol potentiation of pentobarbital-induced current. Pentobarbital induces current in all beta3-subunit-containing receptors, whereas current gating by GABA requires the presence of both alpha1 and beta3 subunits. Therefore, pentobarbital rather than GABA was used to induce current in order to separate the subunit dependence of current gating from the subunit dependence of potentiating action of propofol. alpha1beta3gamma2, alpha1beta3, beta3gamma2, or beta3 subunit combinations all responded to pentobarbital in a dose-dependent manner. True potentiation was defined as the current magnitude to simultaneous application of pentobarbital and propofol exceeding the additive responses to individual drug applications. A dose-dependent propofol potentiation of pentobarbital-induced current was observed in oocytes injected with alpha1beta3 or alpha1beta3gamma2 but not in beta3gamma2 or beta3 subunits, suggesting that the alpha1 subunit was necessary for this modulatory action of propofol. Further examination of the propofol potentiation in chimeras between the alpha1 and beta3 subunits showed that the extracellular amino-terminal half of the alpha1 subunit was sufficient to support propofol potentiation. The different requirements of the receptor structure for the agonistic (gating) and the potentiating actions suggest that these two actions of propofol are distinct processes mediated through its action at distinct sites.     

Mapping human interferon-alpha (IFN-alpha 2) binding determinants of the type I interferon receptor subunit IFNAR-1 with human/bovine IFNAR-1 chimeras

Type I interferons bind to a common receptor (IFNAR), composed of two transmembrane polypeptides, IFNAR-1 and IFNAR-2. Although human IFNAR-1 has a weak intrinsic affinity for human Type I interferons (IFNs), bovine IFNAR-1 binds human Type I IFNs with moderate (nM) affinity, and can be conveniently used to investigate the regions of IFNAR-1 involved in ligand binding. We have constructed 14 bovine/human IFNAR-1 chimeras by exchanging homologous subdomains in the extracellular portion of the receptor. These chimeras were expressed at very high levels on COS cells, and their ability to bind HuIFN-alpha2 was measured. No single bovine subdomain substituted into human IFNAR-1 could confer moderate-affinity ligand binding on the resulting chimera. Simultaneous substitution of bovine IFNAR-1 subdomains 2 and 3 for the homologous human subdomains resulted in a dramatic increase in the binding of IFN-alpha2, suggesting that critical determinants for moderate-affinity ligand binding by BoIFNAR-1 reside in these two subdomains. Bovine subdomains 1 and/or 4 each further enhanced IFN-alpha2 binding in the presence of bovine subdomains 2 and 3. Thus, the binding interactions of BoIFNAR-1 with IFNs appears to be more complex than that of other class II cytokine receptors with their ligands.     

Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain

The specific high-affinity binding of interleukin-4 (IL-4) to its receptor alpha chain is the crucial primary event during IL-4 signalling. Single crystals, suitable for high resolution diffraction studies, have been obtained from a complex between IL-4 and the ectodomain of the receptor alpha chain, also called IL-4-binding protein (IL-4BP). The orthorhombic crystals are in spacegroup P2(1)2(1)2(1) with cell constants a = 5.038 nm, b = 6.841 nm, c = 10.95 nm and diffract to a resolution of at least 0.25 nm when exposed to synchrotron radiation. The volume of the unit cell suggests the presence of a 1:1 IL-4/IL-4BP complex and HPLC analysis of the crystals confirmed that IL-4 and IL-4BP were present in equimolar amounts. An IL-4 variant comprising a total of four methionine residues was generated, labelled with selenomethionine and crystallised in complex with IL-4BP. The crystals are isomorphous to that of the complex with normal IL-4 and therefore can be used to solve the crystallographic phase problem by the method of multiple anomalous diffraction (MAD). The crystal structure of the IL-4/IL-4BP complex will help to understand how IL-4 and other helical cytokines bind and activate their cognate receptor.     

Epitope-specific monoclonal antibodies against human C-terminal procollagen alpha1(III)-propeptide

We have generated monoclonal antibodies against recombinant C-terminal human procollagen alpha1(III) propeptide (PIIICP), produced in E. coli in high yields. The monoclonal antibodies were screened for epitope specificity using recombinant truncated PIIICP. Several antibodies were identified which recognized different regions of the PIIICP molecule. The ability of the antibodies to detect PIIICP antigens in human cell line lysates and supernatants was demonstrated. As PIIICP antigens are a key marker of extracellular matrix metabolism, the monoclonal antibodies described here should be of value for clinical and basic research.     

Increased efficacy of human natural interferon alpha (IFN-alpha n3) versus human recombinant IFN-alpha 2 for inhibition of HIV-1 replication in primary human monocytes

Natural IFN-alpha n3, a purified mixture of many different natural IFN alpha species, was 10- to 100-fold more effective than equal concentrations of human rIFN-alpha 2b or rIFN-alpha 2a for inhibition of HIV replication in primary human monocytes. This difference was highly reproducible in multiple side-by-side experiments using the identical HIV-1 inoculum and the same monocyte target cells: natural IFN-alpha n3 was more effective than rIFN-alpha 2b at lower concentrations for protection against a constant HIV-1 inoculum; cells treated with natural IFN-alpha n3 were protected against a greater HIV-1 challenge than were cells treated with the same concentration of rIFN-alpha 2b. Fractionation of natural IFN-alpha n3 by reversed-phase high-pressure liquid chromatography (RP-HPLC) showed that most antiviral activity for HIV localized to discrete and reproducible peaks. The RP-HPLC peak that contained purified natural IFN-alpha 2b was the least effective fraction. These data suggest heterogeneity among IFN-alpha species for antiviral activity against HIV and may provide a molecular basis for more effective IFN-alpha therapy.     

Mapping immunoreactive epitopes in the human peripheral nervous system using human monoclonal anti-GM1 ganglioside antibodies

A series of monoclonal IgM anti-GM1 ganglioside antibodies has been cloned from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain-Barré syndrome. In solid-phase immunoassay, the antibodies react with GMI, and also in differing degrees to the structurally related glycolipids asialo-GM1 (GA1) and GD1b. Here we describe the binding patterns of six human anti-GM I antibodies to epitopes within the human nervous system. Antibodies were observed to bind to motor neurons and spinal grey matter, dorsal and ventral spinal roots, dorsal root ganglion neurons, nodes of Ranvier, neuromuscular junctions and skeletal muscle. The distribution of immunoreactive epitopes, which included sensory structures, extended beyond those sites conventionally regarded as pathologically affected in anti-GM1 antibody-associated motor nerve syndromes. This undermines a model of disease pathogenesis based solely on antigen distribution. Factors other than the presence or absence of antigen, such as the local ganglioside topography, antibody penetration into, and pathophysiological vulnerability of a particular site may also influence the clinicopathological outcome of anti-GM1 antibody-mediated autoimmune attack.     

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.     

Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2 complexes

The receptor for atrial natriuretic peptide (ANP) is a type-I transmembrane protein containing an extracellular ligand-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. Binding of ANP to the extracellular domain causes activation of the GCase domain by an as yet unknown mechanism. To facilitate studies of the receptor structure and signaling mechanism, we have expressed the extracellular ANP-binding domain of rat ANP receptor (NPR-ECD) in a water-soluble form. NPR-ECD was purified to homogeneity by ANP-affinity chromatography. SDS-PAGE gave a single 61-kDa band, which coincided with a radioactive band obtained by photoaffinity-labeling with N4alpha-azidobenzoyl-125I-ANP(4-28). Edman degradation gave a single amino-terminal sequence expected for the mature protein. Both trifluoromethanesulfonic acid and peptide-N-glycosidase F treatments yielded a 50-kDa band, indicating N-glycosylation. The molecular mass of 57 725 Da determined by mass spectrometry indicates the carbohydrate content at 16%. NPR-ECD bound ANP with an affinity comparable to that of the full-length receptor. The ligand selectivity of NPR-ECD (in the order ANP > brain natriuretic peptide > C-type natriuretic peptide) was also similar to that of the full-length receptor. HPLC gel filtration of NPR-ECD gave a peak with an apparent mass of 74 kDa. Preincubation with ANP generated a new 150-kDa peak with a concomitant decrease of the 74-kDa peak. This shift in peak positions was ANP concentration-dependent and was complete at the NPR-ECD-to-ANP molar ratio of 1:1, indicating equimolar binding. The change in the apparent native molecular weight from 74 to 150 kDa suggests that binding causes dimerization of the NPR-ECD:ANP complex to yield an [NPR-ECD:ANP]2 complex.     

Isolation and characterization of complement receptor type 1 (CR1) storage vesicles from human neutrophils using antibodies to the cytoplasmic tail of CR1

Neutrophil (PMN) activation is associated with increased surface expression of several membrane proteins that are translocated from intracellular pools. Indirect evidence suggests that the intracellular storage pools of complement receptor type 1 (CR1) in resting PMN are distinct from traditional granules and may be the secretory vesicles in which albumin is also stored, but it is not known if this compartment is homogeneous or heterogeneous. To isolate and characterize the CR1-containing vesicles, we used antibodies against unique sequences in the cytoplasmic tail of CR1. Affinity-purified IgG was used to adsorb CR1 storage vesicles from the light membrane fraction (gamma-band) of nitrogen cavitates of resting PMN. The immunoadsorbent could quantitatively remove the CR1-containing vesicles, whereas control adsorbents with nonimmune IgG showed no specific binding of CR1. Immunoblots of specifically isolated vesicles also showed enrichment of albumin, decay-accelerating factor, Fc gammaRIII, and CR3; whereas HLA class I was not detectable in these vesicles. Enzyme assay of specifically isolated vesicles after treatment with Triton X-100 showed that these vesicles contained most of the cells' latent alkaline phosphatase. An additional population of vesicles containing albumin, but not CR1, and that did not bind to anti-CR1 adsorbent was also identified. Immunoelectron microscopy showed that the specifically isolated vesicles had mean diameters of 0.086 to 0.1 microm and stained positive for CR1 and albumin. These results indicate that CR1 storage vesicles can be isolated with antibodies against the cytoplasmic tail of CR1 and show that these vesicles also contain albumin as well as glycosylphosphatidyl inositol-anchored proteins. These results are most compatible with the hypothesis that CR1-containing vesicles have arisen by endocytic retrieval of proteins that had been on the plasma membrane.     

Therapy with monoclonal antibodies. II. The contribution of Fc gamma receptor binding and the influence of C(H)1 and C(H)3 domains on in vivo effector function

An in vivo model is used to define Fc motifs engaged by mAbs to deplete target cells. Human IgG1 and human IgG4 were very potent, and mutations within a motif critical for Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine 234 to valine, and leucine 235 to alanine) completely prevented depletion. Mouse IgG2b was also potent, and mutations to prevent complement activation did not impair depletion with this isotype, as previously shown for human IgG1. In contrast, a mutation that impaired binding to mouse Fc gammaRII (glutamate 318 to alanine) eliminated effector function of mouse IgG2b and also reduced the potency of human IgG4. To reveal potential contributions of domains other than C(H)2, domain switch mutants were created between human IgG1 and rat IgG2a. Two hybrid mAbs were generated with potencies exceeding anything previously seen in this model. While their mechanism of depletion was not defined, their activity appeared dependent upon interdomain interactions in the Fc region.     

Adrenergic receptor and alpha 2 agonist--2: Structure-function relationship of adrenoceptors

Recombinant DNA experiments using chimeric receptors containing portions of alpha 2 and beta 2 adrenoceptors demonstrated structure-function relationships of adrenoceptors. The seventh transmembrane domain determines the subtype ligand binding specificity between alpha 2 and beta 2 adrenoceptors. A further investigation by mutagenesis suggests that a direct interaction between subtype specific ligands and specific amino acids such as Phe (412) and Asn (312) in the seventh transmembrane domain of the alpha 2 and beta 2 adrenoceptors respectively. The third cytoplasmic loop is responsible for determining the specificity of interactions between the receptor and G protein. Recombinant DNA technology also demonstrated that seven transmembrane domains of adrenoceptors have a counterclockwise arrangement when viewed from the outside of the cell.     

Human/mouse chimeric monoclonal antibodies with human IgG1, IgG2, IgG3 and IgG4 constant domains: electron microscopic and hydrodynamic characterization

The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side. Upon addition of bivalent hapten, all of the monoclonal antibodies formed complexes. IgG1, IgG3 and IgG4 formed circular dimers, composed of two antibodies forming a ring-shaped complex, presumably through the binding of two bivalent haptens. IgG2, on the other hand, showed a distribution of complexes which was noticeably different from the other subclasses. Some circular dimers, some linear dimers and a large amount of monomer were seen. This was interpreted in terms of an energy barrier to ring closure arising from the orientation of the Fab arms of IgG2 probably leading to linear dimers as the predominate complex seen with the analytical ultracentrifuge. A substantial number of these dimers probably dissociated upon dilution for examination in the electron microscope. The distribution of the angles between the Fab arms of the monoclonal antibodies forming the circular dimers has been measured for the different subclasses. Most were open at wide angles (> 100 degrees) but some formed very shallow angles, with the Fab arms being nearly parallel to each other. The free energy for this transition was calculated from the ratio of open/closed angles, and it was found to be proportional to the length of the upper hinge of the monoclonal antibody, in agreement with previous nanosecond depolarization results (Dangl et al., Eur. molec. Biol. Org. J. 7, 1989-1994, 1988).     

Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.     

Generation of monoclonal antibodies to the rabbit interleukin-2 receptor alpha chain (CD25) and its distribution in HTLV-1-transformed rabbit T cells

Rabbits can be infected with human retroviruses such as human T-cell leukemia virus-1 (HTLV-1) and human immunodeficiency virus (HIV), and provide useful animal models to study retroviral diseases such as adult T-cell leukemia and HIV. Previously we have succeeded in generating monoclonal antibodies (mAbs) against rabbit CD4, CD5 and CD11a antigens. To make this animal species more amenable to cellular and molecular studies, we have attempted to extend the panel of mAbs against rabbit CD antigens. Here we report on the generation of three neutralizing mAbs against interleukin-2 receptor alpha chain (IL-2R alpha) (CD25), Kei-alpha 1 (IgG2b), Kei-alpha 2 (IgG2a) and Kei-alpha 3 (IgG1). They specifically recognize the rabbit Mr 55,000 IL-2 binding protein, IL-2R alpha, and completely inhibit both high- and low-affinity IL-2 binding to F648b cells that express IL-2R alpha as well as IL-2R beta. The use of mAb Kei-alpha 1 confirmed that the rabbit IL-2R alpha is not only a low-affinity IL-2R on its own but also an essential component of high-affinity IL-2R as found in other animal species, and that rabbit activated T cells including HTLV-1-transformed cell lines express high levels of the IL-2R alpha. Together with mAbs against various rabbit CD antigens that we reported previously, these neutralizing mAbs to IL-2R alpha will be valuable for studies of human retrovirus infections, such as those induced by HTLV-1 or HIV, in rabbits.