Domain-specific monoclonal antibodies to ovotransferrin indicate conservation of determinants involved in avian transferrin receptor recognition

1. Three of five monoclonal antibodies produced to chicken ovotransferrin bound quail ovotransferrin but none of the antibodies bound human, bovine or equine serum transferrin. 2. Equilibrium binding experiments indicate that both quail and chicken ovotransferrin bind to transferrin receptors on chick reticulocytes although the quail protein binds to 40% fewer sites with an affinity which is three times lower than chicken ovotransferrin. 3. The antibodies that recognize quail ovotransferrin block binding of both radiolabelled chicken and quail ovotransferrin to chick reticulocytes. 4. Quail NH2-terminal half-molecule domain appears to be unable to form a functional hybrid holo-ovotransferrin with chicken C-terminal half-molecule domain.     

Differential effect of iodination of ovotransferrin and its two half-molecule domains on binding to transferrin receptors on chick embryo red blood cells.

Iodination of the C-terminal half-molecule domain of ovotransferrin (OTF) causes a significant reduction in binding to transferrin receptors on chick reticulocytes when compared to the binding observed with holo-OTF or the N-terminal half-molecule domain. (In such studies binding of iodinated half-molecule is measured in the presence of equimolar unlabelled complementary half-molecule). In particular iodination of the C-terminal half-molecule domain by the solid-phase reagent Iodogen resulted in half the binding found when ICl was used. The iodinated N-terminal half-molecule domain labelled by either Iodogen or ICl showed consistently higher binding than was observed with the C-terminal half-molecule or Fe2OTF. Although the molecular basis for the reduced binding of these proteins relative to the N-terminal half-molecule has not been definitively established, the implication is that there is a Tyr in the C-terminal domain which is involved in receptor recognition and binding. Addition of one or more bulky iodine atoms to the Tyr interferes with the interaction. Tryptic peptide maps of unlabelled holo-OTF and half-molecule domains and of the half-molecule domains labelled by both ICl and Iodogen are presented. The maps indicate limited access of the tyrosine residues to iodination especially in the C-terminal half-molecule domain. Equilibrium binding experiments have been carried out to compare the Kd (the apparent dissociation constant for the interaction between OTF and the transferrin receptors on chick-embryo red blood cells) with the Bmax, (binding at infinite free-ligand concentration) for Fe2OTF labelled using ICl, Iodogen, Enzymobeads and Chloramine-T. The effect of labelling Fe2OTF by Bolton-Hunter reagent has also been assessed. These studies show that ICl appears to be the reagent of choice for labelling Fe2OTF and its half-molecule domains.     

Epitope and functional specificity of monoclonal antibodies to mouse interferon-gamma: the synthetic peptide approach

Spleen cells from hamsters immunized with recombinant mouse interferon-gamma (IFN-gamma) were fused with mouse myeloma cells, resulting in the production of four anti-IFN-gamma monoclonal antibodies. Binding of 125I-IFN-gamma by these protein A-bound antibodies was specifically blocked by cold IFN-gamma. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFN-gamma, whereas a corresponding C-terminal (95-133) peptide had no effect on binding. The N-terminal specificity of these three antibodies was confirmed by their specific binding of 125I-N-terminal (1-39) peptide. One of the N-terminal specific monoclonal antibodies inhibited both antiviral and macrophage priming (for tumor cell killing) activities of IFN-gamma, whereas the other two had no effect on either biologic function. The selectivity of the inhibition of IFN-gamma function was not due to a differential ability of the N-terminal specific antibodies to bind IFN-gamma. Blocking experiments with cold IFN-gamma and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFN-gamma. An exact determination of the epitope specificity of the monoclonal antibody that inhibited IFN-gamma function could provide insight into the structural basis for the role of the N-terminal domain in the biologic function of IFN-gamma. Polyclonal antibodies to either the N-terminal or the C-terminal peptides also inhibited both the antiviral and the macrophage-priming activities of IFN-gamma. All of the antibodies that inhibited IFN-gamma function also blocked binding of IFN-gamma to membrane receptor on cells, whereas antibodies that did not block function also did not inhibit binding. The data suggest that both the N-terminal and the C-terminal domains of IFN-gamma play an important role in its antiviral and macrophage-priming functions, possibly in a cooperative manner.     

Effects of anion binding on the conformations of the two domains of ovotransferrin

A previous paper (Harris (1985) Biochemistry 24, 7412-7418) reported the occurrence of two classes of anion binding sites in transferrin. To evaluate the locations of the two anion binding sites in relation to the two major domains of transferrin we determined the binding constants of whole ovotransferrin and its two half-molecules by means of the difference UV spectroscopic technique. Anions induced strong negative absorbance at 245 nm in the order: citrate greater than phosphate greater than bicarbonate for whole ovotransferrin and the N-terminal half-molecule; and: phosphate greater than citrate greater than bicarbonate for the C-terminal half-molecule. The anion dissociation constants of the N-terminal half-molecule were consistent with lower dissociation constants, and those of the C-terminal half-molecule, with higher dissociation constants of whole ovotransferrin, indicating that the two classes of anion binding sites correspond to the binding sites in individual structural domains. Anion binding markedly protected the N-terminal half-molecule, but not the C-terminal half-molecule from digestion with trypsin and disulfide reduction with dithiothreitol. As to the far and near ultraviolet CD spectra data, however, there was no significant difference between in the presence and absence of an anion. Therefore, the binding of an anion would induce some conformational changes which were not reflected by the CD spectrum.     

Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.     

The amphipathic alpha-helical repeats of apolipoprotein A-I are responsible for binding of high density lipoproteins to HepG2 cells.

Nine monoclonal antibodies (mAbs) against apoA-I reacting with distinct but overlapping epitopes covering more than 90% of the sequence have been used to block the interaction of 125I-labeled high density lipoprotein (125I-HDL) with HepG2 cells in order to delineate the cell binding domain of apolipoprotein A-I (apoA-I). While 2 mAbs reacting with epitopes exclusively localized in the N-terminal region (residues 1 to 86) enhanced slightly association of 125I-HDL, all other mAbs, which react with epitopes localized in the regions of amphipathic alpha-helical repeats, inhibited that association by 9 to 15%. Although this inhibition is not significant compared to the effect of an irrelevant mAb, combination of these mAbs could significantly inhibit the association of 125I-HDL (32 to 43%) as could polyclonal antibodies (up to 95%). These results are compatible with the concept of HDL binding to these cells via the nonexclusive interaction of each of the amphipathic alpha-helical repeats of apoA-I. When the same approach was applied to block the association of 3H-cholesteryl ether (CE)-labeled HDL to HepG2 cells, each anti-apoA-I could inhibit by 15 to 25% the cellular association of cholesteryl ether while mAbs in combination or polyclonal antibodies could inhibit this association up to 45% or 60%, respectively. The cholesteryl ether radioactivity that remained associated with the cells (40%) in the presence of polyclonal antibodies could be effectively blocked by addition of an antibody against the receptor binding domain of apoE (1D7). Therefore, the differential cellular association of cholesteryl ether compared to apolipoprotein can be explained by the presence of apoE secreted by HepG2 and apoE or apoB/E receptors. Thus, we conclude that the optimum uptake of both cholesteryl ether and apoA-I of HDL by cells requires the accessibility of the entire apoA-I and the cooperative binding of the amphipathic alpha-helical repeats to HepG2 cell membranes. This type of interaction would explain the competitive binding observed for apoA-I, -A-II, and -A-IV by others.     

High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies.

The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha-subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha-protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha-protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha-protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE.     

Binding properties of monoclonal antibody to the cytoplasmic domain of transferrin receptor

Hybridomas secreting monoclonal antibodies to transferrin receptor (TFR) were isolated. One of these antibodies, U-1, recognized the cytoplasmic domain of TFR and the others, N-2 and W-3, recognized its cell surface domains. Only antibody W-3 competed with transferrin (TF) for binding to TFR. Antibody U-1 bound to purified TFR but not to 35S- or 125I-TFR in cell extracts. 125I-Antibody U-1 bound to TFR alone in cell extracts when TFR was bound to antibody N-2-Sepharose 4B, but even in the presense of cell extracts it did not bind to TFR bound to antibody W-3-Sepharose 4B. Antibody W-3 co-precipitated TFR and a protein of about 30 kDa from cell extracts, and also reacted with the 30 kDa protein in cell extracts in the absence of TFR. Based on these results, the existence of two different states of the cytoplasmic domain of TFR is discussed.     

Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU

The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.     

The dimerization of half-molecule fragments of transferrin.

Partial proteolysis was used to prepare half-molecule fragments of hen ovotransferrin. N-Terminal and C-terminal fragments associate to form an N-terminal fragment-C-terminal fragment dimer. Variant forms of the N- and C-terminal fragments can be prepared in which a few amino acid residues are lacking from the C-terminal ends of the fragments. These variant fragments are partially or completely unable to associate; the suggestion that the molecular recognition sites are located in these C-terminal stretches of the N-terminal half-molecule (320-332) and of the C-terminal half-molecule (683-686) is in agreement with X-ray-crystallography data for human lactotransferrin [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1769-1773].     

Production of antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor coupled to protein carriers in different orientations

The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding.     

Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum.

A peptide corresponding to a surface loop in the C-terminal domain of chicken ovotransferrin (residues 570-584) was made by solid-phase synthesis and used to immunize rabbits. A 15-amino acid-residue disulphide-linked loop occurs in both domains of all five transferrins for which the sequence is available and lies on the opposite side of the iron-binding site from the interdomain cleft. Polyclonal antibodies to the peptide were specific for non-reduced holo-ovotransferrin and the C-terminal domain, as shown by e.l.i.s.a. and immunoblotting. The antibody did not inhibit binding of ovotransferrin to receptors on chick-embryo reticulocytes but was able to bind ovotransferrin bound to the cellular receptors at 0 degree C. The loop composed of residues 570-584 appears to be remote from the transferrin receptor-binding site.     

Epitopic regions recognized by monoclonal antibodies against rat brain hexokinase: association with catalytic and regulatory function.

Using direct and competitive epitope mapping methods, 23 monoclonal antibodies (Mabs) against rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were divided into nine groups, each recognizing epitopes within defined surface regions of the N- or C-terminal domains; the latter have been associated with regulatory or catalytic functions, respectively. Reactivity of Mabs with the isolated domains was also studied. Based on the effect of various ligands on immunoreactivity, specific regions involved in ligand-induced conformational changes were identified. Adjacent epitopic regions, designated Regions F and G and located in the N- and C-terminal domains, respectively, were selectively affected by inhibitory hexose 6-phosphates (or analogs), marking these regions as being involved in transmission of the conformational signal from the regulatory N-terminal domain to the catalytic C-terminal domain. Consistent with this, the Ki for inhibition of the enzyme by the glucose 6-phosphate analog, 1,5-anhydroglucitol-6-phosphate, was markedly increased by Mabs binding in these regions, but unaffected by Mabs binding elsewhere in the molecule. Reactivity with Mabs recognizing conformationally sensitive epitopes in Region H of the C-terminal domain was greatly decreased by binding of substrate hexoses that induce closure of a cleft in the catalytic domain; selective recognition of the "open cleft" conformation, thereby preventing closure of the cleft required for progression of the catalytic cycle, can account for the marked decrease in Vmax that results from binding of these Mabs. Reactivity with Mabs binding to Region H was also decreased in the presence of inhibitory hexose 6-phosphates, implying that cleft closure was also induced by the latter; this is consistent with the suggestion that limitation of access to the C-terminal ATP binding site, resulting from cleft closure, is a factor in inhibition of the enzyme.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B.

Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N- and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N- and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the N- and C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.     

Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display

Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the surface of yeast and used to epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for epitope mapping. Using a library of yeast-displayed BoNT/A binding domain (H(C)) mutants and selecting for loss of binding, the fine epitopes of three neutralizing BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of H(C), with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of H(C), in an area not previously thought to be functionally important. Modeling the epitopes shows how all three mAbs could bind BoNT/A simultaneously and may explain, in part, the dramatic synergy observed on in vivo toxin neutralization when these antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody epitopes and the mapping results identify three neutralizing BoNT/A epitopes.     

An extended-X-ray-absorption-fine-structure investigation of diferric transferrins and their iron-binding fragments.

Iron K-edge extended-X-ray-absorption-fine-structure (e.x.a.f.s.) spectra were recorded for diferric human and rabbit serum transferrins and for diferric chicken ovotransferrin in aqueous solution; for ovotransferrin e.x.a.f.s. spectra from the N-terminal and C-terminal domain fragments were also measured. The overall spectral profiles closely resemble one another, indicating similar iron-binding sites. The simulation of the diferric ovotransferrin spectrum suggests a first co-ordination shell consisting of six low-Z ligands (nitrogen/oxygen), two ligands at a distance of approx. 0.185 nm (1.85 A) and four ligands at approx. 0.204 nm (2.04 A). The two shorter distances may correspond to Fe-O (tyrosine), whereas the longer distance is consistent with Fe-N (histidine) and Fe-O (water). Detailed analysis of the spectra of the N-terminal and C-terminal fragments indicates a difference in the short ligand distance.     

Leutropin/beta-adrenergic receptor chimeras bind choriogonadotropin and adrenergic ligands but are not expressed at the cell surface.

In some G-protein-coupled receptors (e.g. beta-adrenergic receptor (beta 2 AR)), the ligand-binding pocket is contained within the hydrophobic transmembrane domain. In others (e.g. luteinizing hormone receptor (LHR)), the relative roles of the extracellular N-terminal domain and the transmembrane region in hormone binding are unknown. To study the roles of these domains, we prepared vectors encoding the rat LHR N-terminal domain alone (L- -), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the vesicular stomatitis virus-G protein (LVV), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the hamster beta 2 AR (LAA), and the beta 2 AR N-terminal domain fused to the transmembrane and C-terminal domains of the rat LHR (ALL). Membrane preparations obtained from COS-7 cells expressing the beta 2 AR or LAA bound the beta-adrenergic antagonist 125I-cyanopindolol with equal affinity, confirming the observation that the beta 2 AR transmembrane domain forms the hormone-binding site. Membranes from COS-7 cells transfected with LHR bound 125I-human choriomic gonadotropin (hCG). However, membranes from LAA-, L(- -)-, and LVV-transfected cells had low capacity to bind 125I-hCG unless they were solubilized with Triton X-100. The affinity of the detergent-solubilized receptors for 125I-hCG was similar to that of the LHR. We were unable to detect binding of 125I-hCG to ALL in the presence or absence of detergent. These observations suggest that, whereas the transmembrane region of the beta 2 AR is sufficient to bind adrenergic ligands, the N-terminal region of the LHR is required for binding of hCG. Although the N terminus of the LHR is sufficient to bind hCG, both the N terminus and the transmembrane domains of the LHR are required for receptor expression on the cell surface.     

Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique.

Phytochromes are red- and far-red light-reversible photoreceptors for photomorphogenesis in plants. Phytochrome A is a dimeric chromopeptide that mediates very low fluence and high irradiance responses. To analyze the surface properties of phytochrome A (phyA), the epitopes of 21 anti-phyA monoclonal antibodies were determined by variously engineered recombinant phyA proteins and the dissociation constants of seven anti-phyA monoclonal antibodies with phyA were measured using a surface plasmon resonance (SPR)-based resonant mirror biosensor (IAsys). Purified oat phyA was immobilized on the sensor surface using a carboxymethyl dextran cuvette in advance, and the interactions of each chosen monoclonal antibody against phyA in either red light absorbing form (Pr) or far-red light absorbing form (Pfr) at different concentrations were monitored. The binding profiles were analyzed using the FAST Fit program of IAsys. The resultant values of dissociation constants clearly demonstrated the differential affinities between the phyA epitopes and the monoclonal antibodies dependent upon Pr vs. Pfr conformations. Monoclonal antibody mAP20 preferentially recognized the epitope at amino acids 653-731 in the Pr form, whereas mAA02, mAP21 and mAR07/mAR08 displayed preferential affinities for the Pfr's surfaces at epitopes 494-601 (the hinge region between the N- and C-terminal domains), 601-653 (hinge in PASI domain), and 772-1128 (C-terminal domain), respectively. The N-terminal extension (1-74) was not recognized by mAP09 and mAP15, suggesting that the N-terminal extreme is not exposed in the native conformation of phyA. On the other hand, the C-terminal domain becomes apparently exposed on Pr-to-Pfr phototransformation, suggesting an inter-domain cross-talk. The use of surface plasmon resonance spectroscopy offers a new approach to study the surface properties of phytochromes associated with the photoreversible structural changes, as well as for the study of protein-protein interactions of phytochromes with their interacting proteins involved in light signaling events in plants.     

Rat and Mouse Monoclonal Antibodies to Human Myelin Basic Protein

BALB/c mice and Lewis rats were immunized with human myelin basic protein and its N- and C-terminal fragments. Mouse X mouse fusions produced seven monoclonal antibodies, all of the IgG class and directed against the N-terminal fragment. Five of the antibodies seemed to be against the same epitope, between amino acid residues 92 and 118. One antibody bound between residues 45 and 91, and the remaining antibody reacted with both peptides 1-44 and 45-91. Three monoclonal antibodies, all of the IgM class, were obtained by rat X rat hybridization. Two monoclonal antibodies, raised against whole myelin basic protein and the C-terminal fragment, respectively, each bound to peptide 118-178. The remaining antibody, raised against the N-terminal fragment, bound to peptide 45-91. These monoclonal antibodies are of interest for use in clinical radioimmunoassays and for immunohistochemical investigation of the structural relationships of the myelin sheath.