Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves' patients' sera

Previous attempts to generate autoantibody-reactive, secreted thyrotropin receptor (TSHR) ectodomain in mammalian cells have failed because of retention within the cell of material with immature carbohydrate. We have overcome this difficulty by performing progressive carboxyl-terminal truncations of the human TSHR ectodomain (418 amino acid residues including signal peptide). Three ectodomain variants (TSHR-261, TSHR-289, and TSHR-309) were truncated at residues 261, 289, and 309, respectively. Unlike the full ectodomain, ectodomain variants were secreted with an efficiency inversely proportional to their size. Secreted ectodomain variants contained approximately 20 kDa of complex carbohydrate. TSHR-261 was chosen for further study because it was secreted very efficiently and neutralized autoantibodies in Graves' patients' sera. This ectodomain variant was partially purified using sequential lectin and nickel-chelate chromatography, permitting the first direct visualization and quantitation of the mammalian TSHR. Most important, very small (nanogram) quantities of this material neutralized 70-100% of TSHR autoantibody activity in all 18 Graves' sera studied. In summary, carboxyl-terminal truncation of the human TSHR ectodomain generates a secreted protein with complex carbohydrate that neutralizes autoantibodies in Graves' patients' sera. Antigenically active TSHR will be valuable for future studies on the diagnosis, pathogenesis, and immunotherapy of Graves' disease.     

Thyrotropin receptor cleavage at site 1 does not involve a specific amino acid motif but instead depends on the presence of the unique, 50 amino acid insertion

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intramolecular cleavage of the single chain receptor at two separate sites. The region involved in cleavage at Site 2 has been identified, but previous mutagenesis studies failed to identify Site 1. We now report fortuitous observations on the effect of trypsin on the TSHR that localizes a small region harboring Site 1. Thus, as detected by immunoblotting and by 125I-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearing a glycan moiety from the C terminus of the A subunit. Based on the TSHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglycosylated A subunit relative to a series of C-terminal truncated TSHR ectodomain variants, places cleavage Site 1 in the vicinity of, or closely upstream to, residue 317. Remarkably, mutagenesis of every amino acid residue between residues 298-316 (present study) and 317-362 (previous data) did not prevent cleavage at Site 1. However, cleavage at this site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor family. In summary, these data provide novel insight into TSHR intramolecular cleavage. Cleavage at Site 1 does not depend on a specific amino acid motif and differs from cleavage at Site 2 by involvement of a mechanism requiring the presence of the enigmatic TSHR 50-amino acid "insertion."     

Thyrotropin receptor cleavage at site 1 involves two discontinuous segments at each end of the unique 50-amino acid insertion

Among the glycoprotein hormone receptors, only the thyrotropin receptor (TSHR) cleaves (at two sites) into disulfide-linked A and B subunits. A 50-amino acid insertion unique to the TSHR ectodomain (residues 317-366) plays no role in ligand binding or signal transduction, but its deletion abrogates cleavage at Site 1, closely upstream of the insertion. We sought to define the region within the 50-amino acid tract involved in TSHR cleavage at Site 1. Mutation of small segments within this region previously failed to prevent cleavage at Site 1. We, therefore, divided the 50-amino acid insertion into quartiles and deleted each one individually (TSHR residues 317-327, 328-338, 339-350, and 351-362). As determined by covalent cross-linking of 125I-TSH to intact cells expressing the mutant receptors, none of these deletions prevented TSHR cleavage at Site 1. Neither did larger deletions of quartiles 1 + 2, 2 + 3, and 3 + 4. However, qualitative differences in the extent of receptor cleavage suggested that quartiles 1 and 4 were playing a greater role in cleavage at Site 1 than were the middle two quartiles. In support of this hypothesis, deletion of these two discontinuous segments almost completely eliminated TSHR cleavage at Site 1. In conclusion, intramolecular cleavage at Site 1 requires the presence of the N-terminal and C-terminal quartiles of the 50-amino acid insertion unique to the TSHR. Taken together with previous observations, our data suggest that this tract may provide a discontinuous binding site for a protease that clips the TSHR at Site 1.     

An N-linked glycosylation motif from the noncleaving luteinizing hormone receptor substituted for the homologous region (Gly367 to Glu369) of the thyrotropin receptor prevents cleavage at its second, downstream site

The thyrotropin receptor (TSHR) exists in two forms (single polypeptide and two subunits), whereas the lutropin/chorionic gonadotropin receptor (LH/CGR) is a single chain. Recent data suggest that the TSHR cleaves at two sites. We mutagenized selected chimeric TSH-LH/CGR to localize the cleavage sites in the TSHR. All 23 receptors mutated in the estimated vicinity of the upstream site cleaved into two subunits as determined by 125I-TSH cross-linking to intact cells. In contrast, in a series of mutations homologous to the noncleaving LH/CGR, the downstream TSHR cleavage site localized to three amino acids (GQE367-369). Remarkably, group substitution of these residues, but not substitution of individual residues, abolished cleavage. Moreover, the mutation that prevented cleavage (GQE367-369NET) transposed a motif (NET291-293) that is glycosylated in the LH/CGR. TSHR cleavage or noncleavage after substitution of GQE367-369 with other triplets (AAA, NQE, and NQT) was consistent with a role for N-linked glycosylation at this site. In summary, our data (i) support the concept that the TSHR cleaves at two sites, (ii) relate TSHR residues GQE367-369 to cleavage at the second, downstream site, and (iii) suggest that cleavage or noncleavage at site two is related to N-linked glycosylation. These findings provide new insight into the evolutionary divergence of two closely related receptors.     

Delineation of tyrosine-containing epitopes within the beta subunit of bovine thyrotropin

The epitopes recognised by two monoclonal antibodies (mAb 279 and mAb 299), specific for the beta subunit of bovine thyroid-stimulating hormone (bTSH), have been localised using a technique in which the tyrosine residues in the bTSH beta subunit were subjected to modification when the bTSH beta subunit was complexed with either mAb or in the free, unbound state. The epitope recognised by mAb 279 was localised to the C-terminal region of bTSH beta with the tyrosine residue Tyr104 protected from modification by the presence of this mAb. In addition, the experimental results indicate that the tyrosine residues Tyr18 and/or Tyr112 are also involved in the mAb 279 epitope. The epitope recognised by mAb 299 was localised to the region 59-74 of bTSH beta as both Tyr59 and Tyr74 were protected from modification by the presence of this mAb. Since both mAbs have been previously found to inhibit receptor binding, the sequence regions/amino acid positions recognised by these mAbs are likely to represent determinants for receptor binding. Moreover, these data indicate that the identified amino acid residues are located on the surface of the molecule, consistent with predictions of the tertiary structure of the bTSH beta subunit based on the recently elucidated X-ray crystal structure of human chorionic gonadotropin.     

Cerebrolysin ameliorates performance deficits, and neuronal damage in apolipoprotein E-deficient mice

We report a method for the purification and radioactive labeling of human TSH receptor (TSHR). The method is based on the construction of a fusion TSHR (TSHR-Xa-BIO) which consists of the N-terminal 725 amino acids of human TSHR linked to the 4-amino acid Xa protease cleavage site and the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase (the C-terminal domain directs the efficient posttranslational biotinylation of the protein). TSHR-Xa-BIO was produced in HeLa cells using recombinant vaccinia virus. The expressed protein was fully functional and was biotinylated with an efficiency of about 90%. Streptavidin-agarose-immobilized TSHR-Xa-BIO was labeled with 125I using the chloramine T oxidation procedure and specifically eluted from the solid phase after cleavage with protease Xa. Isolated native radiochemically pure 125I-labeled TSHR specifically interacted with pathological autoantibodies in the sera of patients with Graves' disease, and thus could be useful for the detection of these autoantibodies by immunoprecipitation analysis.     

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). I. Phagocytes

When western blotting analysis of Japanese eel neutrophil lysate was performed using antibody against the synthetic peptide corresponding to the carboxyl terminal region of human cytochrome b558 large subunit, a broad band was specifically detected at approximately 90 kDa. The antibody recognized the epitope present in eel neutrophils only after the cells were permeabilized with detergent. These results indicate that the large subunit of cytochrome b exists in fish neutrophils and the epitope is exposed to the cytoplasmic side of membrane, and suggest that cytochrome b is conserved across species.     

Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.     

Differential effects of antibodies to vascular cell adhesion molecule-1 and distinct epitopes of the alpha4 integrin in HgCl2-induced nephritis in Brown Norway rats

Four distinct epitopes (A, B1, B2, and C) have been functionally defined on the human alpha4 integrin. In this study, two cross-reactive antihuman alpha4 monoclonal antibodies (mAb) (HP2/1 and HP2/4 specific for epitopes B1 and B2, respectively) were used to functionally characterize the rat VLA-4 subunit and to define similar functional epitopes in this rodent species. It was found that B1 and B2 anti-alpha4 mAb completely block adhesion to fibronectin, but the inhibition of adhesion to vascular cell adhesion molecule-1 (VCAM-1) with HP2/1 mAb was lower than with HP2/4 mAb. It was also observed that epitope B2 HP2/4 mAb induced homotypic aggregation in rat lymphocytes, whereas epitope B1 HP2/1 mAb did not. Using the HgCl2 model of nephritis, this study shows the protective effect of both anti-alpha4 mAb against infiltration of the renal interstitium by leukocytes. Nevertheless, HP2/1 mAb, but not HP2/4 mAb, virtually abolished the anti-glomerular basement membrane antibody synthesis and glomerular deposits. These findings indicate the dual but independent role played by alpha4 integrins in both extravasation of leukocytes and in the production of antibodies. Finally, this study demonstrates that anti-rat VCAM-1 mAb showed a positive reactivity of the renal vascular endothelium and, most importantly, that administration of anti-VCAM-1 antibodies completely abrogated the interstitial cell infiltrates without affecting anti-glomerular basement membrane antibody production. These results confirm the important role played by VLA-4/VCAM-1 pathway in leukocyte infiltration, and further support the dual and independent role of alpha4 integrins in both renal infiltration and autoantibody synthesis in this model of renal disease.     

A novel LFA-1 activation epitope maps to the I domain

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA-1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM-1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA-1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.     

A phase II study of interleukin-2 in 49 patients with relapsed or refractory acute leukemia

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation.     

Processing and activation of pro-interleukin-16 by caspase-3

Interleukin-16, a proinflammatory cytokine produced in CD8(+) lymphocytes, is synthesized as a precursor protein (pro-IL-16). It is postulated that the C-terminal region of pro-IL-16 is cleaved, releasing bioactive IL-16. To characterize IL-16 cleavage, we transfected COS cells with a cDNA encoding a approximately 50-kDa form of pro-IL-16. Transfected COS cells released a approximately 20-kDa IL-16 cleavage product shown to consist of the 121 C-terminal residues of pro-IL-16 by immunoblotting and amino acid sequencing. Cleaved IL-16, but not pro-IL-16, exhibited lymphocyte chemoattractant activity. A C-terminal approximately 20-kDa IL-16 polypeptide was also released when pro-IL-16 was treated with concanavalin A-stimulated CD8(+) lymphocyte lysate. Cleavage occurred after an Asp, suggesting involvement of a caspase (interleukin-1beta-converting enzyme/CED-3) family protease. Using recombinant caspases and granzyme B, we determined that pro-IL-16 cleavage is mediated only by caspase-3. Relevance to pro-IL-16 processing in primary lymphocytes was supported by identifying the p20 subunit of activated caspase-3 in stimulated CD8(+) lymphocytes and by inhibition of CD8(+) lymphocyte lysate-mediated cleavage with Ac-DEVD-CHO. Pro-IL-16 is a substrate for caspase-3, and cleavage by this enzyme releases biologically active IL-16 from its inactive precursor.     

Pharmacodynamic profile of prolonged etoposide administration in patients with small cell lung cancer and non-Hodgkin''s lymphoma

The thyrotropin (TSH) receptor belongs to a subfamily of G protein-coupled receptors, which also includes luteinizing hormone and follicle-stimulating hormone receptors. The TSH receptor (TSHR) differs from the latter by the presence of an additional specific segment in the C-terminal part of its ectodomain. We show here that this insertion is excised in the majority of receptor molecules. Preparation of specific monoclonal antibodies to this region, microsequencing, enzyme-linked immunosorbent assay, and immunoblot studies have provided insight into the mechanisms of this excision. In the human thyroid gland, N termini of the transmembrane receptor beta subunit were found to be phenylalanine 366 and leucines 370 and 378. In transfected L cells a variety of other more proximal N termini were found, probably corresponding to incomplete excisions. The most extreme N terminus was observed to lie at Ser-314. These observations suggest that after initial cleavage at Ser-314 the inserted fragment of TSHR is progressively clipped out by a series of cleavage reactions progressing up to amino acids 366-378. The impossibility of recovering the excised fragment from purified receptor, cell membranes, or culture medium supports this interpretation. The cleavage enzyme has previously been shown to be inhibited by BB-2116, an inhibitor of matrix metalloproteases. However, we show here that it is unaffected by tissue inhibitors of metalloproteases. The cleavage enzyme is very similar to TACE (tumor necrosis factor alpha-converting enzyme) in both these characteristics. However, incubation of the TSH receptor with the purified recombinant catalytic domain of TACE, co-transfection of cells with TACE and TSHR expression vectors, and the use of mutated Chinese hamster ovary cells in which TACE is inactive suggested that the TSHR cleavage enzyme is different from TACE. TACE and TSHR cleavage enzyme may thus possibly be related but different members of the adamalysin family of metzincin metalloproteases.     

Growth-regulated formation of heteromeric complexes of the centromere and promoter factor, Cbf1p, in yeast

The gastrointestinal pathogen Aeromonas hydrophila strain A186 produces a collagen-binding protein (CNBP) which is found extracellularly and loosely associated with the cell surface. The cell-associated CNBP was purified by sequential ammonium sulphate precipitation, size-exclusion chromatography and ion-exchange chromatography, or by sequential ammonium sulphate precipitation and affinity chromatography with collagen-Sepharose. The purified CNBP was homogeneous in SDS-PAGE, and had a mol. wt of c. 98 kDa. Cyanogen bromide cleavage of the CNBP destroyed collagen-binding activity; however, enzymic digestion with Staphylococcus aureus V8 protease generated > 10 polypeptide fragments, from which a 30-kDa polypeptide contained the strongest collagen-binding activity. Binding of collagen by the CNBP was restricted to the alpha1 (I) chain of the collagen molecule and binding seemed to involve both the carbohydrate moieties and certain peptide sequences on the collagen. Collagen-saccharides generated by alkaline hydrolysis inhibited collagen binding by A. hydrophila. Also, glycosidase digestion and chemical alteration of the carbohydrate residues of collagen reduced its ability to be bound by the CNBP. Collagen-homologous synthetic peptides inhibited binding of 125I-collagen by the bacteria.     

Induction of the 2B9 antigen/dipeptidyl peptidase IV/CD26 on human natural killer cells by IL-2, IL-12 or IL-15

Activation of human natural killer (NK) cells involves sequential events including cytokine production and induction of cell surface molecules, resulting in the enhancement of cytolytic activity. To delineate the activation process of NK cells, we generated murine monoclonal antibodies (mAbs) against YT, a human large granular lymphocyte/natural killer (LGL/NK) cell line. Among the mAbs reactive with YT cells, one mAb, termed 2B9, was noted because of the lack of reactivity with most of the human T- and B-cell lines tested. In fresh peripheral blood mononuclear cells (PBMC), however, the majority of cells expressing this antigen (Ag) were T cells but not CD16+ nor CD56+ NK cells. Since YT cells showed an activated phenotype expressing interleukin-2 (IL-2) receptor alpha chain, we examined whether 2B9 Ag could be induced on normal human peripheral blood NK cells by cytokines known to activate NK cells. The 2B9 Ag was induced on NK cells by IL-2, IL-12 or IL-15 while no induction was observed by interferon-gamma (IFN-gamma). Biochemical analysis showed that anti-2B9 mAb recognized a 115 kDa molecule in YT cells. A cDNA clone encoding the 2B9 Ag was isolated from a cDNA expression library of YT cells and its sequence was identical to CD26 cDNA although it was not of full length. Transient expression of the 2B9 cDNA on COS-7 cells revealed that this cDNA encodes the antigenic epitope(s) recognized by anti-2B9 mAb as well as Ta1, an anti-CD26 mAb. These results showed that the 2B9 Ag is identical to CD26, and demonstrated that CD26 is an activation antigen on CD16+ CD56+ NK cells inducible by IL-2, IL-12 or IL-15.     

Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

Identification of amino acid residues contributing to the pore of a P2X receptor

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.     

Accessibility of epitopes on UvrB protein in intermediates generated during incision of UV-irradiated DNA by the Escherichia coli Uvr(A)BC endonuclease

Structural intermediates generated during incision of damaged DNA by the Uvr(A)BC endonuclease were probed with monoclonal antibodies (mAbs) raised against the Escherichia coli UvrB protein. It was found that the epitope of B2C5 mAb, mapped at amino acids (aa) 171-278 of UvrB, is not accessible in any of the preformed Uvr intermediates. Preformed B2C5-UvrB immunocomplexes, however, inhibited formation of those intermediates. B2C5 mAb seems to interfere with the formation of the UvrA-UvrB complex due to overlapping of its epitope and the UvrA binding region of UvrB. Conversely, the epitope of B3C1 mAb (aa 1-7 and/or 62-170) was accessible in all Uvr intermediates. The epitope of B*2E3 mAb (aa 171-278) was not accessible in any of the nucleoprotein intermediates preceding UvrB-DNA preincision complex. However, B*2E3 was able to immunoprecipitate this complex and to inhibit overall incision. B2A1 mAb (aa 8-61) inhibited formation of those Uvr intermediates requiring ATP binding and/or hydrolysis by UvrB. B*2B9 mAb (aa 473-630) inhibited Uvr nucleoprotein complexes involving UvrB. B*2B9 seems to prevent the binding of the UvrA-UvrB complex to DNA. The epitope of the B*3E11 mAb (aa 379-472) was not accessible in Uvr complexes formed at damaged sites. These results are discussed in terms of structure-functional mapping of UvrB protein.