GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.     

Microencapsulation technology: A novel method for monoclonal antibody production

Monoclonal antibodies have been envisioned as useful agents for human therapeutic and diagnostic applications in vivo. Recent results from human clinical trials suggest that this potential is becoming a reality. Attention is now shifting to the development of methods to produce monoclonal antibodies of a quality acceptable for widespread human use and in sufficient quantity to be a commercially viable product. Microencapsulation technology has been demonstrated to be suited to the large-scale production of both human and murine monoclonal antibodies of high purity and activity, for use in applications both in vitro and in vivo.     

A novel monoclonal antibody specific for lymphatic endothelium

The difficulty of identifying and differentiating lymphatic and blood microvessels in tissue sections can be overcome by a monoclonal antibody specific for lymphatic endothelium. Unfortunately, the only known antibody also reacts with the endothelium of some blood vessels. The technique of double immunization (passive, with an antiserum to blood endothelium, and active, with a suspension of lymphatic endothelial cells) was, therefore, used to increase the chances of recognizing specific lymphatic antigens by the mouse immune system. The monoclonal antibody obtained, LyMAb, a G₁ immunoglobulin, reacted strongly with the endothelium of bovine thoracic duct, mesenteric collecting vessels and lymphatic vessels of gall-bladder and lymph nodes and moderately with those of the intestinal wall. Blood vessels (intercostal arteries, azygos vein and blood microvessels of all organs tested) were consistently negative. The antibody was species-specific and did not react with formalin-fixed, paraffin-embedded sections. Cross-reactivity was limited to some connective tissue fibres and scattered cells in the lymph node parenchyma, intestinal villi and hepatic lobules.     

A monoclonal antibody for G protein-coupled receptor crystallography

G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.     

Neutralization of NGF-TrkA receptor interaction by the novel antagonistic anti-TrkA monoclonal antibody MNAC13: a structural insight

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.     

A highly charged sequence of chick hsp90: a good candidate for interaction with steroid receptors

The sequence of the entire chick 90 kDa heat shock protein (hsp90), the non hormone binding component of the heterooligomeric form of steroid receptors, is reported. A comparison of the amino acid sequence of the chick hsp90 to that of the homologous hsp90 from yeast to man, reveals 64-96% identity respectively, and even with E. coli hsp90 an identity of 44% is observed. Analysis of the sequence and a secondary structure prediction of chick hsp90 suggest that two hydrophilic regions A and B, predicted in alpha-helix may play a role in the interaction of hsp90 with other proteins such as steroid hormone receptors. While there are regions of the sequences completely conserved in all hsps90, the most negatively charged hydrophilic region (A) is absent in the E. coli protein.     

A monoclonal antibody to human insulin receptor

A murine hybridoma secreting antibody against human insulin receptor was produced by fusing FO myeloma cells with spleen and lymph node cells from a mouse that had been immunized with insulin receptor purified from human placenta. The secreted antibody was an IgG₁ (κ), designated αIR-1. Like the previously described rabbit polyclonal antibody, αIR-1 did not inhibit insulin binding. It specifically immunoprecipitated ¹²⁵I-insulin-receptor complexes as well as unoccupied receptor previously labeled directly with lactoperoxidase. Thus, αIR-1 interacts with the receptor at a site distinct from the insulin binding site. Unlike previously described anti-insulin receptor antibodies, αIR-1 exhibits strong tissue and species specificity.     

Epitope mapping for the anti-rabbit cholesteryl ester transfer protein monoclonal antibody that selectively inhibits triglyceride transfer.

Among the monoclonal antibodies (Mab) against rabbit plasma cholesteryl ester transfer protein (CETP), Mab 14-8F cross-reacted with human CETP and selectively inhibited triglyceride transfer but not cholesteryl ester transfer (Ko, K. W. S., T. Ohnishi, and S. Yokoyama. 1994. J. Biol. Chem. 269: 28206;-28213). The epitope of this antibody was studied by using synthetic fragment peptides of rabbit and human CETP. Mab 14-8F reacted with the peptide R451-Q473 of human CETP near the carboxyl-terminal and not with the peptides representing any other regions, and inhibited the binding of human CETP to the goat antibody against its carboxyl-terminal peptide R451-S476. The experiments with a series of the fragment peptides in this region revealed that the epitope requires the segment 465-473 (EHLLVDFLQ) of human CETP or 485-493 (KHLLVDFLQ) of rabbit CETP (core epitope) though neither peptide by itself binds to the antibody. Both peptides needed extension at least by one residue beyond either amino- or carboxyl-end in order to show the reactivity to the antibody, but the effect was not highly residue-specific at least at the amino-end. Circular dichroism analysis demonstrated the increase of helical conformation by the extension of the "core epitope" peptides to either direction. Thus, the epitope is dependent on conformation of the core epitope induced by the presence of an additional residue(s) in either end. The core epitope occupies the central 64% of the reported linear epitope of Mab TP2, a widely used anti-human CETP monoclonal antibody that inhibits both cholesteryl ester and triglyceride transfer.Therefore, we conclude that the limited interaction of Mab with a common lipid interaction site causes selective inhibition of the transfer of triglyceride that has presumably lower priority than cholesteryl ester for the CETP reaction.     

Monovalent cation selectivity for ATP-dependent association of the glucocorticoid receptor with hsp70 and hsp90.

We have reported previously that incubation of the immunopurified transformed hormone-free glucocorticoid receptor with rabbit reticulocyte lysate reconstitutes the receptor complex with hsp90 and that reconstitution is accompanied by concomitant repression of DNA binding activity and regeneration of the steroid binding conformation (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). In this work we further characterize this system by defining the small M(r) components of reticulocyte lysate required for both structural and functional reconstitution of the receptor-hsp90 complex. Reconstitution is ATP-dependent and there is a direct relationship between the extent of hsp90 binding to the receptor and the number of specific steroid binding sites that are generated. Dialysis of reticulocyte lysate inactivates its reconstituting activity. Addition of an ATP-regenerating system or readdition of small M(r) lysate components (in the form of a Centricon C30 filtrate) has little effect, but the presence of both restores full reconstituting activity to dialyzed lysate, as assayed by steroid binding activity and by the binding of hsp90 and hsp70 to the receptor. The small M(r) activity is heat-stable, and it can be completely replaced by NH+4, K+, and Rb+, with K+ producing a maximal effect at the concentration normally present in undialyzed lysate. Na+ and Li+ have no reconstituting activity. This ion selectivity demonstrates that a monovalent cation binding site is involved in receptor heterocomplex reconstitution. It is intriguing that the protein unfoldase (e.g. clathrin uncoating ATPase) activity of hsp70 is known to have a similar monovalent cation dependence, and that under all conditions where hsp90 becomes bound to the receptor, we find that hsp70 is also bound.     

Epitope mapping of anti-human transferrin monoclonal antibodies: potential uses for transferrin-transferrin receptor interaction studies

Human transferrin (hTf) is an 80 kDa glycoprotein involved in iron transport from the absorption sites to the sites of storage and utilization. Additionally, transferrin also plays a relevant role as a bacteriostatic agent preventing uncontrolled bacterial growth in the host. In this work we describe a well-characterized Mabs panel in terms of precise epitope localization and estimate affinity for the two major hTf isoforms. We found at least four antigenic regions in the hTf molecule, narrowed down the interacting antigen residues within three of such regions, and located them on a molecular model of hTf. Two of the antigenic regions partially overlap with previously described transferrin-binding sites for both human receptor (antigenic region I: containing amino acid residues from Asp-69 to Asn-76 at the N-lobe) and bacterial receptors from two pathogenic species (antigenic region III: amino acid residues from Leu-665 to Ser-672 at the C-lobe). Hence, such monoclonal antibodies (Mabs) could be used as an additional tool for conformational studies and/or the characterization of the interaction between hTf and both types of receptor molecules.     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

A monoclonal anti-peptide antibody recognizes the adrenocorticotropic receptor

We have produced a monoclonal antibody that specifically recognizes the adrenocorticotropic receptor on rat adrenal cells. The immunogen was designed from an RNA sequence complementary to the mRNA coding for ACTH1-24. This complementary peptide, termed HTCA, has been shown to specifically bind ACTH and was proposed to mimic the ACTH binding site of the hormone receptor. The monoclonal anti-HTCA antibody recognized a restricted domain of the HTCA peptide, bound to Y-1 adrenal cells with a KD of 1.8 nM, and blocked the binding of 125I-ACTH to rat adrenal cells. These findings show that anti-HTCA competes with ACTH for binding to the ACTH receptor.     

一种新的抗人角蛋白单克隆抗体

In this paper, we reported a novel monoclonal antibody against human keratins, R 6-2-14. The antigen used for immunization was derived from human callus, keratins in which traditionally are classified as "Soft" keratins. However, when we studied the tissue specificity of this antibody, it was found that it only reacted strongly with "Hard" keratins of various mammalian species, but no detectable cross-reactivity with any of the "Soft" keratins. This antibody may provide a useful tool for the study of hair regeneration, nail regeneration, corn pathology and differentiation of mammalian epidermal derivatives.     

Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.     

A novel method for monitoring monoclonal antibody production during cell culture

We describe a new format for surface-based fluoroimmunoassays that allows detection of biomolecule interactions without separation steps. The bioactive layer was immobilized on the surface of a glass substrate covered with silver islands that provide optical amplification of the distinctive fluorescence signal from bound probes when compared to unbound probes. The technique used was phase-modulation fluorometry that allows sensitive detection of bound probes with a very short lifetime in the presence of excess free probes in solution. The new method was applied to assay monoclonal antibody production during cell culture. Excellent agreement was found between the new method and ELISA analysis of hybridoma cell culture samples. It is predicted that the near real time monitoring of protein products during bioprocessing will be possible with the described technology.     

A monoclonal antibody which inhibits the factor Va:factor Xa interaction

An immunoprecipitation technique has been used to determine the subunit specificity of two of the monoclonal antibodies to bovine Factor V(Va) developed by this laboratory. One of the antibodies is specific for the 74,000-dalton subunit (the E chain) of Factor Va, and the other antibody is specific for the 94,000-dalton subunit (the D chain). The binding of Factor Va to phospholipid was studied by light scattering, and the interaction of Factor Xa with phospholipid-bound Factor Va was examined using 5-dimethylaminonaphthalene-1-sulfonyl-glutamyl-glycyl-arginyl-Xa (Dns-EGR-Xa). Neither the antibody specific for the E chain nor the antibody specific for the D chain inhibit the binding of Factor Va to phospholipid vesicles. The antibody specific for the E chain blocks the increase in fluorescence polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid vesicles and calcium. This antibody also inhibits the association of Dns-EGR-Xa with phospholipid-bound Factor Va as determined by gel-exclusion high pressure liquid chromatography. The antibody specific for the D chain of Factor Va does not block the increase in polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid, and calcium. It was concluded that the antibody specific for the E chain of Factor Va binds at or near the Factor Xa-binding site on the E chain and that the Factor Va E chain plays a significant role in binding Factor Xa.     

A novel human IgA monoclonal antibody protects against tuberculosis

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.     

Characterization of a novel interaction between the secretory Na+-K+-Cl- cotransporter and the chaperone hsp90

The first isoform of the Na(+)-K(+)-Cl(-) cotransporter (NKCC1) is of central importance for the control of cellular ion concentration and epithelium-mediated salt secretion. Several studies have established that a change in intracellular [Cl(-)] (Cl(-)(i)) represents a key signaling mechanism by which NKCC1-induced Cl(-) movement is autoregulated and by which Cl(-) entry and exit on opposite sides of polarized cells are coordinated. Although this signaling mechanism is coupled to a pathway that leads to post-translational modification of the carrier, no unifying model currently accounts for the ion dependence of NKCC1 regulation. In this paper, evidence is presented for the first time that hsp90 associates with the cytosolic C terminus of NKCC1, probably when the carrier is predominantly in its unfolded form during early biogenesis. Evidence is also presented that the Cl(-)(i)-dependent regulatory pathway can be activated by a thermal stress but that it is no longer operational if NKCC1-expressing cells are pretreated with geldanamycin, an antibiotic that inhibits hsp90, albeit nonspecifically. Taken together, our data indicate that binding of hsp90 to NKCC1 may be required for Na(+)-K(+)-Cl(-) cotransport to occur at the cell surface and that it could play an important role in ion-dependent signaling mechanisms, insofar as the maneuvers that were used to alter the expression or activity of the chaperone do not exert their main effect by inducing other cellular events such as the unfolded protein response. Further studies will be required to elucidate the functional relevance of this novel interaction.