High level expression and rational mutagenesis of a designed protein, the minibody. From an insoluble to a soluble molecule.

We recently described the design and chemical synthesis of the minibody, a 61-residue metal binding beta-protein with a novel fold. Characterization of the polypeptide by circular dichroism spectroscopy, size exclusion chromatography, and metal binding studies showed the molecule to be folded, monomeric, globular and able to bind metals. The main obstacle which prevented a more detailed characterization was the very low solubility of the protein in water (about 10 microM). To address this problem, we used two independent approaches: (1) mutagenesis of the beta-sheet framework residues and (2) addition of a solubilizing motif, made of three lysine residues, at the N or C termini. Engineering and production of mutants was facilitated by the achievement of high level expression of the protein in Escherichia coli. Both approaches led to minibody variants with a solubility ranging from tenfold higher up to millimolar levels. For the best-characterized variant obtained so far, the thermodynamic stability calculated from denaturant-induced transition is identical to that of the parent, poorly soluble, molecule.

Coupling protein design and in vitro selection strategies: improving specificity and affinity of a designed beta-protein IL-6 antagonist.

The minibody is a designed small beta-protein conceived to enable the construction of large libraries of minimal discontinuous epitopes displayed on the surface of filamentous phage. The 61 residue molecule consists of three strands from each of the two beta-sheets of the variable domain of immunoglobulins packed face to face, along with the exposed H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on phage, we were able to select a minibody with micromolar affinity for human interleukin-6 that behaves as a selective cytokine antagonist. The minibody exposes a surface composed of two constrained loops, which provides the possibility of improving IL-6 binding and specificity by swapping the hypervariable regions, followed by further selection. We established experimental conditions for "stringent" selection such as monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, we have isolated a minibody with improved antagonistic potency and greater specificity. Furthermore, using hIL-6 mutants carrying amino acid substitutions in distinct surface sites it was possible to carefully define the cytokine region that binds the minibody.

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.

Surface topology of Minibody by selective chemical modifications and mass spectrometry.

The surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A. Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.

High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences.

We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis. We have constructed single-stranded (ss) expression vectors that facilitate this process. These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro. By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods. Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template. We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.

Characterization of human ferritin H chain synthetized in Escherichia coli.

We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2. The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins. All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified. We have shown that the ferritin synthesized in E. coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.

Adhesion molecules in high-grade soft tissue sarcomas: correlation to clinical outcome.

The extracellular matrix (ECM) forms a framework for cell adhesion, but it also regulates growth and differentiation. Normal and malignant cells interact with the ECM via specific receptors, the integrins. To explore the mechanisms of growth and spread in soft tissue sarcomas the expression of the major ECM molecules and their corresponding integrin receptors were studied by immunohistochemistry in high-grade soft tissue sarcomas: malignant fibrous histiocytoma (20 cases), malignant peripheral nerve sheath tumour (17 cases) and synovial sarcoma (21 cases). The expression pattern was compared with cell proliferation and clinical outcome. Integrins were found to be expressed according to histological pattern. In synovial sarcomas, the epithelial component showed a high alpha 2 but negative or minimal detection of alpha 5 expression, while a weak alpha 2 expression and a moderate alpha 5 expression were found in the spindle cell component. No alpha 2 expression was detected in malignant fibrous histiocytoma, and minimal alpha 5 expression was detected in malignant schwannoma. The alpha 6 expression levels were positively correlated with the occurrence of metastases in all types of sarcomas studied. The expression of ECM molecules was downregulated and irregular in most tumours. In conclusion, the divergent integrin expression pattern could be useful in the diagnosis and classification of soft tissue sarcomas. Furthermore, since high laminin receptor expression correlates with occurrence of metastases, it could become a useful prognostic marker.

Idiotype regulation of self responses, autoantibody V regions and neonatal B cell repertoire.

The role of autoantibodies themselves in immune regulation is still unknown. There is evidence that some autoantibody idiotypes (Id) may play a regulatory role in physiologic and possibly pathologic situations of the immune system. Here we present evidence that (1) certain autoantibody Id modulate the immune response to self antigens (regulatory Id), and (2) that the neonatal preimmune repertoire is primarily devoted to self recognition. The humoral and cellular events following immunization with regulatory Id suggest an active participation of T cells in the regulatory events. Based on immunochemical, structural and molecular genetics analysis, it appears that this regulatory Id is of germline origin as it exists within the neonatal preimmune repertoire. B cell hybridomas generated from unstimulated splenocytes of neonatal mice are primarily self-reactive, suggesting that the immune system essentially begins as a self-recognizing system. The findings are discussed with regard to the relationship between germline repertoire, autoantibodies, and regulatory idiotypes.

Autoantibody idiotypy and neonatal B cell repertoire.

During ontogeny, antibody variable (V) regions are subjected to selection events at the level of B-cell clones bearing on their surfaces Ig molecules useful to the developing organism. Antigenic determinants of immunoglobulin V regions (idiotypes) are believed to play an essential role in molecular recognition and immune responsiveness to exogenous and self antigens. Recent data indicate that reactivity with self-antigens is prevalent within the natural neonatal repertoire, suggesting that self-antigens are involved in the selection and shaping of the immunologic repertoire. One implication of the above considerations is that V regions having regulatory idiotypes are borne preferentially on antibodies that react with self-antigens. Here, we report on the molecular characterization of the idiotype 62 expressed by BALB/c autoantibodies to a classic self-antigen, thyroglobulin. We show that under appropriate experimental conditions, Id62 can modulate the autoantibody response through a process requiring the activation of regulatory T cells. We also demonstrate that self--reactive V regions bearing Id62 are present in newborn mice and report on the primary structure of the V-region genes encoding Id62. Lastly, we provide evidence that hybridomas derived from unstimulated splenocytes of normal neonatal BALB/c mice express Ig molecules that react prevalently with self-antigens.

Prognostic significance of cyclin expression in human osteosarcoma.

To evaluate the distribution of cyclin protein expression, in relation to cell proliferation rate and clinical behavior, an immunohistochemical study was performed on 92 tumor samples of patients with high grade osteosarcoma (OS). A large cyclin A- and cyclin E-positive fraction was found respectively in 59% and 47% of the osteosarcomas, while immunostaining for cyclin D1 was weak or absent in most tumor samples. A positive, statistically significant correlation was found between A and E cyclins and Ki67 expression (p<0.001). Disease-free survival (DFS) analysis included 69 of the 92 patients. A significantly higher probability of metastasis was seen in patients lacking cyclin D1 compared to those in which cyclin D1 was positive (p<0.01). Conversely, patients with >40% of cyclin A-positive cells relapsed more frequently than those with <40% of cyclin A-positive cells (p<0.05). The multivariate analysis demonstrated that cyclin A had a lower predective risk in terms of disease-free survival as opposed to the loss of cyclin D1 that is considered a powerful prognostic factor.

Value of immunohistochemical detection of noncollagenous proteins of bone for the diagnosis of bone tumours.

The expression of osteonectin, osteopontin, bone sialoprotein, and osteocalcin was evaluated by immunohistochemistry in 57 cases of osteoid-forming and non-osteoid-forming bone tumours using specific polyclonal antibodies and the avidin-biotin peroxidase complex method. A positive immunostaining was found in all of the osteoid-forming rumours (osteoblastoma and osteosarcoma), both in the cells and in the extracellular matrix. Among non-osteoid-forming tumours, immunoreactivity to noncollagenous proteins was present in the cells but not in the matrix of chondrosarcoma, malignant fibrous histiocytoma, and fibrosarcoma, as well as in the mononuclear component of giant-cell rumours. Contrary to small-cell osteosarcoma, Ewing's sarcoma was always negative for all of the noncollagenous proteins considered. These results suggest that the immunohistochemical evaluation of noncollagenous proteins of bone may be a useful tool for the differential diagnosis of bone neoplasms, particularly among the heterogeneous group of small round cell tumours.

Expression of G1 phase regulators in MG-63 osteosarcoma cell line.

Cyclins and cyclin-dependent kinases (cdks) form complexes that govern transitions during cell cycle phases. In this study we characterized a human osteosarcoma cell line, MG-63, for the expression level of cyclin D1, cyclin E, cdk4, cdk2, and cell cycle inhibitors pRb and p21. To investigate the role of these proteins we treated MG-63 cells with tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Cell proliferation analysis demonstrated an increased proliferation of MG-63 cells with IL-6, while TNF-alpha acted as an anti-proliferative agent. Immunoblotting revealed an increased expression of p21 with TNF-alpha and its complex with cdk2. TNF-alpha reduced the expression of the cyclin E-cdk2 complex. TNF-alpha did not affect the amount of cyclin D1, cyclin E, cdk4, cdk2, and of cyclin D1-cdk4 complex. IL-6 decreased p21 expression and its complex with cdk2, while it increased the cyclin E-cdk2 complex. Cyclin D1 and cdk4 expression and their complex did not change after IL-6 treatment, nor did cyclin E and cdk2 protein expression. Hyperphosphorylated/dephosphorylated Rb protein ratio was reduced with TNF-alpha whereas it increased with IL-6. These results may suggest an important role of p21 and of cyclin E-cdk2 complex in the G1 phase regulation through pRb phosphorylation in MG-63 cells.

Peptide and protein display on the surface of filamentous bacteriophage.

The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics. Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences. Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques. The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines.

Antibodies specific for the neuronal form of the Src protein elicited by an antigenized antibody.

To elicit antibodies directed specifically against the neuron-specific form of the c-src gene product, pp60c-src(+), we used an antigenized antibody comprising a decamer containing the amino acid sequence specific to pp60c-src(+) inserted into the third hypervariable loop of the heavy (H)-chain variable (V)-region. This was used to raise anti-idiotype antibodies reacting with the peptide epitope in rabbits. The antisera reacted with pp60c-src(+), as judged by immune blotting, immunoprecipitation, immune complex kinase assay, and indirect immunofluorescence staining, but did not react with the fibroblast form of the c-src gene product, pp60c-src. Antigenized antibody is a useful approach for producing antibodies able to distinguish between isoforms of the same gene product and specific for the neuronal form of the Src protein.

Of minibody, camel and bacteriophage.

This review describes the design process from conception through realisation and optimisation of a minibody'--a minimised antibody. The result was a proteinaceous molecule of novel fold and metal binding activity. We explain how combinatorial approaches, using phage display libraries, were used to randomise loop regions of the minibody. Variants were then selected for desired activities including in vitro inhibition of human interleukin-6 and the protease of the non-structural protein, NS3, of the hepatitis C virus. One such variant was successfully minimised further to produce a cyclic peptide with similar inhibition properties. Thus the work reviewed provides examples of two important processes in protein design and protein minimisation. We conclude by discussing the role of such studies in medical applications and small molecule drug discovery. We also highlight the potential of our work and similar techniques in the post-genomic era.

Prognostic relevance of C-myc gene expression in giant-cell tumor of bone.

Giant-cell tumor is a primary bone tumor, of uncertain origin, with the potential capacity to metastasize. To study the role of c-myc and c-fos oncogene overexpression in the tumorigenesis and metastatic spread of giant-cell tumors, 32 primary tumors were collected; of these, 19 remained disease-free and 13 metastasized to the lung. Samples of lung metastasis from these 13 patients were also available for study. The expression of c-myc and c-fos mRNA was studied by reverse transcription-polymerase chain reaction and by in situ hybridization. The expression of protein was studied by Western blot analysis and by immunohistochemistry. C-myc mRNA was overexpressed in 12 (38%) of the 32 primary tumors. Thirteen primary tumors metastasized to the lung; in nine (69%) of these, c-myc mRNA was overexpressed. The c-myc protein was overexpressed in seven (54%) of the 13 tumors that metastasized to the lung. C-fos was overexpressed in only one lung metastasis. A strong correlation between the overexpression of c-myc, and the occurrence of metastases was found: thus, c-myc seems a powerful prognosticator in giant-cell tumor. C-myc was overexpressed both in giant cells and in mononuclear cells, suggesting that both cell types are involved in the progression of this tumor.

The management of 138 psoriatic arthritic patients.

Clinical experience obtained in the management of 138 patients of psoriatic arthritis is reported. The correct recognition of arthritic subtype (according to Moll and Wright classification) always resulted essentially in the choice of the therapy. Our programme included rehabilitative, pharmacologic and surgical approaches. Our data suggest that psoriatic arthritis is a mild articular disease when compared to other affections such as rheumatoid arthritis or ankylosing spondylitis. Notwithstanding an accurate therapeutical programme, it is necessary to control atypical cases which not infrequently can occur.

Increased c-myc oncogene expression in Ewing's sarcoma: correlation with Ki67 proliferation index.

AIMS AND BACKGROUND: Ewing's sarcoma is a highly malignant musculoskeletal tumor composed of small round cells. Although important results have been achieved with surgery associated with chemotherapy, recurrent disease is still a major problem. In order to define new prognostic factors useful for therapeutic decision-making, we conducted a study on 38 Ewing's sarcoma samples in which c-myc oncogene expression and Ki67 proliferation index were correlated with clinical outcome. METHODS AND STUDY DESIGN: Nineteen patients developed metastases during follow-up and 10 of these patients died. C-myc and Ki67 protein expression was evaluated by immunohistochemistry performed on 5 microm formalin-fixed and paraffin-embedded sections, while the c-myc mRNA transcript was localized using in situ hybridization. RESULTS: A statistically positive correlation was found between c-myc protein and Ki67 (P = 0.001) and c-myc mRNA and Ki67 expression (P = 0.047). The 38 patients were divided into two groups using as the cutoff 50% of Ki67-positive cells. The disease-free survival and overall survival estimates were 68% and 90%, respectively, in the group of patients with a percentage of Ki67-positive cells <50%, and 25% and 50%, respectively, in the group with a percentage of Ki67-positive cells > or = 50%. The difference between the survival curves was statistically significant (P <0.05 and P <0.01). Furthermore, relapsed patients had a high and uniform expression of c-myc protein and mRNA compared to disease-free patients. CONCLUSION: These results suggest a possible role of the c-myc oncogene and Ki67 antigen in the malignant progression of Ewing's sarcoma.

Bone hemangioendothelioma: an immunohistochemical study related to histological malignancy and proliferative activity (NORs).

AIMS AND BACKGROUND: Forty-four bone hemangioendotheliomas (HEs) of different histological grades were studied to evaluate the expression and distribution of laminin, type IV collagen, cathepsin G and cathepsin D in cell differentiation and malignancy. RESULTS: In poorly-differentiated HEs the discontinuous distribution of laminin and type IV collagen around angioblastic cords, tubes and cavities revealed an irregular and disorganized basement membrane (BM) architecture corresponding to an increased cell proliferation and secretion of cathepsin D and cathepsin G by tumor cells. CONCLUSIONS: The mean nucleolar organizer region (NOR) area, as a measure of cell proliferation, was significantly higher in grade 4 malignancies than in lower grades, revealing novel prognostic parameters.