Horst Ahorn

Identification of tumor antigens in renal cell carcinoma by serological proteome analysis.

We have investigated the suitability of proteomics for identification of tumor‐associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two‐dimensional gel electrophoresis (2‐DE) and silver staining. Protein patterns were markedly different (∼800 spots in RCCs versus ∼1400 spots in kidney). 2‐DE immunoblotting revealed five RCC‐specific spots, reproducibly reactive with RCC‐patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2‐DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22‐alpha (SM22‐α), is an actin‐binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22‐α is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22‐α were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor‐associated antigens.

Biochemical Characterization of Pru a 2, a 23-kD Thaumatin-Like Protein Representing a Potential Major Allergen in Cherry (Prunus avium)

Background: The prevalence of allergy to fruits and vegetables increased with pollinosis over the last 10 years. So far, clusters of hypersensitivity have been established and corroborated by the molecular characterization of individual cross-reacting allergens. Several case studies demonstrated the existence of allergic reactions to fruits of the subfamily Prunoideae (apricots, cherries, plums and peaches). Here, we present the characterization of a major allergen in cherry. Methods: Characterization was performed using IgE immunoblotting and immunoblot inhibition, N-terminal sequencing, mass spectroscopy analysis and PCR-based cDNA cloning. Results: A 23-kD protein was identified as IgE-binding component. As all cherry-extract-reactive sera displayed IgE-binding to this band, it was designated a major allergen from Prunus avium (Pru a 2). Sequencing the corresponding cDNA identified Pru a 2 as a thaumatin-like protein belonging to the group 5 of pathogenesis-related proteins. Conclusions: A thaumatin-like protein in cherry has been identified as a major allergen (Pru a 2). Homologous proteins from the thaumatin family share sequence similarities and should therefore be checked for the capability to elicit an IgE-mediated allergic reaction.

Crystal Structure of Bisphosphorylated IGF-1 Receptor Kinase: Insight into Domain Movements upon Kinase Activation

BACKGROUND The insulin-like growth-factor-1 (IGF-1) receptor, which is widely expressed in cells that have undergone oncogenic transformation, is emerging as a novel target in cancer therapy. IGF-1-induced receptor activation results in autophosphorylation of cytoplasmic kinase domains and enhances their capability to phosphorylate downstream substrates. Structures of the homologous insulin receptor kinase (IRK) exist in an open, unphosphorylated form and a closed, trisphosphorylated form. RESULTS We have determined the 2.1 A crystal structure of the IGF-1 receptor protein tyrosine kinase domain phosphorylated at two tyrosine residues within the activation loop (IGF-1RK2P) and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. Compared to the homologous insulin receptor kinase, IGF-1RK2P is trapped in a half-closed, previously unobserved conformation. Observed domain movements can be dissected into two orthogonal rotational components. CONCLUSIONS Conformational changes upon kinase activation are triggered by the degree of phosphorylation and are crucially dependent on the conformation of the proximal end of the kinase activation loop. This IGF-1RK structure will provide a molecular basis for the design of selective antioncogenic therapeutic agents.

Correlation of clinical data with proteomics profiles in 24 patients with B-cell chronic lymphocytic leukemia.

The development of human cancer is caused by complex molecular perturbations leading to variable clinical behavior often even in single disease entities. To prove that expression profiling on the protein level can be correlated with clinical data we systematically compared in a pilot study the protein expression patterns obtained by 2‐dimensional gel electrophoresis with clinical features in human B‐cell chronic lymphocytic leukemia (B‐CLL), a disease characterized by broad clinical variability. Statistical methods were devised to analyze the spot pattern from 24 patient samples. This analysis allowed the identification of proteins that clearly discriminated between the patient groups with defined chromosomal characteristics or whose expression levels did correlate with clinical parameters such as patient survival. This report demonstrates that the correlation of large‐scale protein expression profiles with clinical data can be used to gain new insights into molecular aspects of a disease. The data described here show that B‐CLL patient populations with shorter survival times exhibit changed levels of redox enzymes, heat shock protein 27 and protein disulfide isomerase. These molecules may be potentially involved in drug resistance.

N- myc oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angiogenesis and suppresses neuroblastoma tumor growth

Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies. Tumor angiogenesis appears to be governed by alterations of tumor suppressor or oncogenes operant in a broad range of tumors. We have addressed this issue in neuroblastoma, a malignancy characterized by the near-exclusive amplification and overexpression of the N-Myc oncogene. Here, we report that N-Myc overexpression results in down-regulation of interleukin-6 (IL-6) and that IL-6 is an inhibitor of endothelial cell proliferation and VEGF-induced rabbit corneal angiogenesis. STAT3 is instrumental for IL-6 activity as infection with adenoviruses expressing a phosphorylation deficient STAT3 mutant renders endothelial cells insensitive to the antiproliferative action of IL-6. Finally, though IL-6 does not influence neuroblastoma cell growth, IL-6-expressing xenograft tumors in mice exhibit reduced neovascularization and suppressed growth. Our data shed new light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastomas.

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas.

Abstract Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (Kd = 1.7×10–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans.

Isolation and Cloning of Bet v 1-Homologous Food Allergens from Celeriac (Api gl) and Apple (Mal dl)

Up to 70% of tree pollen allergic patients tend to display allergic reactions to a broad range of fruits and vegetables such as hazelnuts, apples, celeriac and carrots. At least part of this food intolerance is due to Bet v 1 (major birch pollen allergen) - related allergens from different plant species, as it was shown in IgE-immunoblots (1–3).

MYCN Oncogene and Angiogenesis: Down-Regulation of Endothelial Growth Inhibitors in Human Neuroblastoma Cells

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates three inhibitors of endothelial cell proliferation. One of them was identified by amino acid sequencing as being identical with activin A, a developmentally-regulated protein. Down-regulation involves interaction of the N-myc protein with the activin A promoter. Work is ongoing to characterize the other two endothelial cell inhibitors. We suggest that the N-myc induced down-regulation of angiogenesis inhibitors could contribute to tumor angiogenesis.

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcεRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcεRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(α2–3)Gal(β1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only α-mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.

Complete1H- and13C-NMR spectroscopic assignment of two pentadecapeptides by reverse C,H correlation techniques

SummaryTwo oligopeptides representing a wild type and a modified version of the polyprotein cleavage region for proteinase2 A of the human rhinovirus type 2 (HRV 22 A) have been investigated using homo- and heterocorrelated 2 D NMR spectroscopy. Proton-detected C,H correlation techniques turned out to be extremely useful for sequential resonance assignment. In one case, the complete amino acid backbone could be analyzed without using NOESY spectra, thus avoiding ambiguities inherent to this method. No defined tertiary structure of the cleavage region could be detected in either molecule. However, one of the oligopeptides is present to a very low extent in a conformation different from therandom-coil arrangement.ZusammenfassungZwei Oligopeptide, die die native Spaltregion für die Proteinase2 A des humanen Rhinovirus von Serotyp 2 (HRV 22 A) beziehungsweise eine modifizierte Version repräsentieren, wurden mit Hilfe von homo- und heterokorrelierter 2 D-NMR-Spektroskopie untersucht. Protondetektierte C,H-Korrelationstechniken erwiesen sich als außerordentlich hilfreich bei der sequenziellen Zuordnung. In einem Fall konnte das gesamte Aminosäureskelett ohne Zuhilfenahme von NOESY-Spektren analysiert und damit die dieser Methode inhärenten Unsicherheiten bei der Zuordnung vermieden werden. In keinem der beiden Moleküle konnte eine definierte Tertiärstruktur der Spaltregion ermittelt werden. Eines der beiden Oligopeptide liegt jedoch zu einem sehr geringen Prozentsatz in einer von derrandom-coil Anordnung abweichenden Konformation vor.