Biochemical characterization and bacterial expression of an odorant-binding protein from <Emphasis Type="Italic">Locusta migratoria</Emphasis>

Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera.     

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K _D of CA52?CDARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs?? potentialities to target, to purify, and to modulate the function of cellular markers.     

The role of glycosylation in protein antigenic properties

Glycosylation of proteins is a common event and contributes to protein antigenic properties. Most data have been obtained from model studies on glycoprotens with well-defined structure or synthetic glycopeptides and their respective monoclonal antibodies. Antibodies raised against glycoprotein antigens may be specific for their carbohydrate units which are recognized irrespective of the protein carrier (carbohydrate epitopes), or in the context of the adjacent amino acid residues (glycopeptidic epitopes). Conformation or proper exposure of peptidic epitopes of glycoproteins is also frequently modulated by glycosylation due to intramolecular carbohydrate-protein interactions. The effects of glycosylation are broad: glycosylation may 'inactivate' the peptidic epitope or may be required for its reactivity with the antibody, depending on the structure of the antigenic site and antibody fine specificity. Evidence is increasing that similar effects of glycosylation pertain to T cell-dependent cellular immune responses. Glycosylated peptides can be bound and presented by MHC class I or II molecules and elicit glycopeptide-specific T cell clones. Received 5 July 2001; received after revision 9 October 2001; accepted 11 October 2001     

K-Lymphocyte cytotoxicity: role of human blood group ABO, rhesus and P alloantibodies]

Using human erythrocytes of known antigenic density, sensitized by ABO and Rhesus (D) alloantibodies, it is shown that K cell cytotoxicity mediated by peripheral blood lymphocytes is directly correlated with the number of IgG molecules specifically bound to the target cell surface. The lytic sensitivity of P1k but not P2 or p erythrocytes coated with anti-Tja (anti-P + P1 + Pk) antibodies, demonstrates that effector K cells are mainly triggered through the IgG ANTI-Pk component of such sera.     

Ligand-dependent localization and function of ORP–VAP complexes at membrane contact sites

Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP–VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP–VAP association, alters the subcellular targeting of ORP–VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP–VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER–lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP–VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.     

Studies on chemically induced tumors in rats: I. Heterogeneity of tumor cells and establishment of syngeneic, tumor-specific cytotoxic T cell clones

Sarcoma P1 was induced in DA rats by DMBA. Anti-P1 antibodies were produced in DA rats, purified via fixed tumor cells and used to induce anti-idiotypic antibodies in syngeneic rats. The anti-idiotypic antibodies were used to generate cytotoxic, P1 specific DA T cells in vitro. These cytotoxic T cells and P1 tumor cells were cloned by limiting dilution. Using the DA anti-P1 specific cytotoxic T cell clones, we were able to characterize 2 types of P1 tumor cell clones, namely those which were susceptible and those which were resistant to the P1 specific cytotoxic T cells. Cytotoxic T cell injected i.v. into syngeneic DA rats could not prevent the development of lethal P1 tumors.     

The humoral immune response to heat shock proteins.

Humoral immune reactions to heat shock proteins (hsp) from microorganisms are one aspect of microbial infections in humans. The production of antibodies which are specific to epitopes present on procaryotic hsp leads also to the appearance of cross-reactive serum antibodies in the host organism that react with human hsp. This article discusses the consequences of such autoreactive antibodies for the host in context with the development of immune tolerance and autoimmune diseases, especially rheumatoid arthritis (RA), and in experimental animal models for arthritis such as adjuvant arthritis in rats. On the basis of epitope cross-reactivity between hsp and other host proteins, a hypothesis is presented for the development of autoimmune disease following the production of hsp-specific antibodies.     

The humoral immune response to heat shock proteins

Humoral immune reactions to heat shock proteins (hsp) from microorganisms are one aspect of microbial infections in humans. The production of antibodies which are specific to epitopes present on procaryotic hsp leads also to the appearance of cross-reactive serum antibodies in the host organism that react with human hsp. This article discusses the consequences of such autoreactive antibodies for the host in context with the development of immune tolerance and autoimmune diseases, especially rheumatoid arthritis (RA), and in experimental animal models for arthritis such as adjuvant arthritis in rats. On the basis of epitope cross-reactivity between hsp and other host proteins, a hypothesis is presented for the development of autoimmune disease following the production of hsp-specific antibodies.     

Cross-reactive monoclonal antibodies to alcohol dehydrogenases

Summary Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.     

Cross-reactive monoclonal antibodies to alcohol dehydrogenases

Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.     

Mapping the antigenicity of copper-treated cellular prion protein with the scrapie isoform

When recombinant and cellular prion protein (PrP(C)) binds copper, it acquires properties resembling the scrapie isoform (PrP(Sc)), namely protease resistance, detergent insolubility and increased beta sheet content. However, whether the conformations of PrP(C) induced by copper and PrP(Sc) are similar has not been studied in great detail. Here, we use a panel of seven monoclonal antibodies to decipher the epitopes on full-length mouse PrP(C) that are affected by exogenous copper, and to compare the antigenicity of the copper-treated full-length PrP(C) with the full-length PrP(Sc) present in scrapie-infected mouse brains. In the presence of copper, we found that epitopes along residues 115-130 and 153-165 become more accessible on PrP(C). These regions correspond to the two beta sheet strands in recombinant PrP and they were proposed to be important for prion conversion. However, when we compared the antibody-binding patterns between full-length PrP(C) with full-length PrP(Sc) and between copper-treated full-length PrP(C) with full-length PrP(Sc), antibody binding to residues 143-155 and 175-185 was consistently increased on PrP(Sc). Collectively, our results suggest that copper-treated full-length PrP(C) does not resemble full-length PrP(Sc), despite acquiring PrP(Sc)-like properties. In addition, since each full-length protein reacts distinctively to some of the antibodies, this binding pattern could discriminate between PrP(C) and PrP(Sc).     

Conformational dynamics of the beta2-microglobulin C terminal in the cell-membrane-anchored major histocompatibility complex type I

We have recently described an anti-beta2-microglobulin (beta2-m) monoclonal antibody (mAb 14H3) capable of recognizing the epitope 92-99 of the protein in the monomeric native state as well as in the fibrillar polymeric state, but not in the major histocompatibility complex type I (MHCI) anchored to the cell membrane. In the present study, we investigated the molecular basis for the inaccessibility of the C-terminal end of beta2-m in the MHCI complex, and demonstrated that mAb 14H3 binds the soluble fraction of the MHCI complex with a Kd of 0.3 microM. An interaction between the complex and the membrane protects beta2-m from immunological recognition at the MHCI level. This protection from antibody recognition can be weakened by procedures such as heat shock or gamma irradiation that perturb the membrane structure and commit the cell to the apoptotic pathway. mAb 14H3 can recognize MHCI in a transient state that most likely precedes beta2-m shedding and may be proposed as a useful tool for dynamic analysis of MHCI conformational modifications.     

Soluble proteins of chemical communication in the social wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis>

Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.Received 14 May 2003; received after revision 8 June 2003; accepted 12 June 2003     

Quaternary structure-dependent idiotope and antigen binding of a monoclonal antibody specific for conformational epitope on type II collagen

We previously generated a monoclonal antibody (mAb) against a putative pathogenic epitope on native type II collagen (CII) for the induction of collagen-induced arthritis in mice (mAb1), and an anti-idiotypic mAb which appears to possess the internal image of the CII epitope (mAb2). In the present study, the structural basis of the antigen/mAb1 and mAb1/mAb2 interactions was examined. When partially SH-reduced mAb1 was analysed on Western blots, only fragments containing both heavy (H) and light (L) chains were recognized by mAb2. When mAb2 was partially SH-reduced, only fragments containing both H and L chains were recognized by mAb1. H and L chains were separated from mAb1 in a reduced, denatured condition, and each chain and a mixture of the two were refolded. mAb2 reacted specifically to the renatured whole IgG molecule of mAb1, but not to the refolded L or to H chains. Recombinant single chain Fv (scFv) generated from mAb1 and mAb2 had properties of the original mAbs, whereas genetical ly constructed chimeric scFvs, consisting of V_H from mAb1 and an irrelevant V_L , or V_L of mAb1 and an irrelevant V_H , did not react either to CII or to mAb2. Thus, interactions among CII, mAb1 and mAb2 appear to depend on quaternary structures containing different protein subunits. These observations support the internal image property of the mAb2. In addition, this dependency on quaternary structure for recognition of proteins may also be relevant to other protein-protein interactions. Received 29 July 1996; received after revision 13 September 1996; accepted 18 October 1996     

Immunochemical similarities among the hemolymph juvenile hormone binding proteins of moths

The hemolymph from various species of moths was analyzed for cross-reactivity with a panel of six monoclonal antibodies made against the hemolymph juvenile hormone binding protein ofManduca sexta. With the exception of one antibody, the immunoreactivity was limited to the sphingid family. One monoclonal antibody cross-reacted with a number of lepidopteran species; however, families such as Noctuidae and Pyralidae, known to have high affinity, low molecular weight juvenile hormone binding proteins, did not cross-react. Immunological cross-reactivity withManduca sexta juvenile hormone binding protein in several primitive moth families supports the current model of phylogenetic relationships in the order Lepidoptera.     

Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody.

The monoclonal antibody (mAB) L1, which binds to the nucleocapsid protein of canine distemper virus (CDV), was shown to bind to avirulent CDV obtained after serial passages in Vero cells, but not to two different virulent demyelinating CDV-strains propagated in dog glial cell cultures. However, when both virulent CDV-strains were passaged through Vero cells they expressed, after a number of passages, an epitope recognized by mAB L1. The occurrence of the L1 epitope appeared to coincide with loss of virulence in animal inoculation experiments.     

Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody

The monoclonal antibody (mAB) L1, which binds to the nucleocapsid protein of canine distemper virus (CDV), was shown to bind to avirulent CDV obtained after serial passages in Vero cells, but not to two different virulent demyelinating CDV-strains propagated in dog glial cell cultures. However, when both virulent CDV-strains were passaged through Vero cells they expressed, after a number of passages, an epitope recognized by mAB L1. The occurrence of the L1 epitope appeared to coincide with loss of virulence in animal inoculation experiments.     

Protein intrinsic disorder as a flexible armor and a weapon of HIV-1

Many proteins and protein regions are disordered in their native, biologically active states. These proteins/regions are abundant in different organisms and carry out important biological functions that complement the functional repertoire of ordered proteins. Viruses, with their highly compact genomes, small proteomes, and high adaptability for fast change in their biological and physical environment utilize many of the advantages of intrinsic disorder. In fact, viral proteins are generally rich in intrinsic disorder, and intrinsically disordered regions are commonly used by viruses to invade the host organisms, to hijack various host systems, and to help viruses in accommodation to their hostile habitats and to manage their economic usage of genetic material. In this review, we focus on the structural peculiarities of HIV-1 proteins, on the abundance of intrinsic disorder in viral proteins, and on the role of intrinsic disorder in their functions.     

Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3

Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.     

Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils

Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6-24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48-120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.