Bernd Weigle

Structural and functional insights into RAGE activation by multimeric S100B

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF‐hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X‐ray structure of human Ca2+‐loaded S100B was determined at 1.9 Å resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size‐exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V‐domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.

Identification of SOX2 as a novel glioma-associated antigen and potential target for T cell-based immunotherapy.

Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A*0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8+ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.

N-Glycans on the receptor for advanced glycation end products influence amphoterin binding and neurite outgrowth

In this study we show that embryonic neurite growth‐promoting protein amphoterin binds to carboxylated N‐glycans previously identified on mammalian endothelial cells. Since amphoterin is a ligand for the receptor for advanced glycation end products (RAGE), and the ligand‐binding V‐domain of the receptor contains two potential N‐glycosylation sites, we hypothesized that N‐glycans on RAGE may mediate its interactions with amphoterin. In support of this, anti‐carboxylate antibody mAbGB3.1 immunoprecipitates bovine RAGE, and PNGase F treatment reduces its molecular mass by 4.5 kDa, suggesting that the native receptor is a glycoprotein. The binding potential of amphoterin to RAGE decreases significantly in presence of soluble carboxylated glycans or when the receptor is deglycosylated. Oligosaccharide analysis shows that RAGE contains complex type anionic N‐glycans with non‐sialic acid carboxylate groups, but not the HNK‐1 (3‐sulfoglucuronyl β1–3 galactoside) epitope. Consistent with the functional localization of RAGE and amphoterin at the leading edges of developing neurons, mAbGB3.1 stains axons and growth cones of mouse embryonic cortical neurons, and inhibits neurite outgrowth on amphoterin matrix. The carboxylated glycans themselves promote neurite outgrowth in embryonic neurons and RAGE‐transfected neuroblastoma cells. This outgrowth requires full‐length, signalling‐competent RAGE, as cells expressing cytoplasmic domain‐deleted RAGE are unresponsive. These results indicate that carboxylated N‐glycans on RAGE play an important functional role in amphoterin‐RAGE‐mediated signalling.

Tumoricidal Potential of Native Blood Dendritic Cells: Direct Tumor Cell Killing and Activation of NK Cell-Mediated Cytotoxicity

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity for DC-based cancer vaccination protocols. Novel findings reveal that besides their role as potent inducers of tumor-specific T cells, human DCs display additional antitumor effects. Most of these data were obtained with monocyte-derived DCs, whereas studies investigating native blood DCs are limited. In the present study, we analyze the tumoricidal capacity of M-DC8+ DCs, which represent a major subpopulation of human blood DCs. We demonstrate that IFN-γ-stimulated M-DC8+ DCs lyse different tumor cell lines but not normal cells. In addition, we show that tumor cells markedly enhance the production of TNF-α by M-DC8+ DCs via cell-to-cell contact and that this molecule essentially contributes to the killing activity of M-DC8+ DCs. Furthermore, we illustrate the ability of M-DC8+ DCs to promote proliferation, IFN-γ production, and tumor-directed cytotoxicity of NK cells. The M-DC8+ DC-mediated enhancement of the tumoricidal potential of NK cells is mainly dependent on cell-to-cell contact. These results reveal that, in addition to their crucial role in activating tumor-specific T cells, blood DCs exhibit direct tumor cell killing and enhance the tumoricidal activity of NK cells. These findings point to the pivotal role of DCs in triggering innate and adaptive immune responses against tumors.

Prostate stem cell antigen: Identification of immunogenic peptides and assessment of reactive CD8+ T cells in prostate cancer patients

Identification of TAAs recognized by CD8+ CTLs paved the way for new concepts in cancer therapy. In view of the heterogeneity of tumors and their diverse escape mechanisms, CTL‐based cancer therapy largely depends on an appropriate number of TAAs. In prostate cancer, the number of antigens defined as suitable targets of CTLs remains rather limited. PSCA is widely distributed in prostate cancer. In this report, we define immunogenic peptides of PSCA which are recognized by circulating CD8+ T cells from prostate cancer patients and able to activate CTLs in vitro. Screening the amino acid sequence of PSCA for peptides containing a binding motif for HLA‐A*0201 resulted in 8 candidate peptides. Specificity and affinity of peptide binding were verified in a competition assay. Frequencies of CD8+ T lymphocytes reactive against selected epitopes were determined in the blood of prostate cancer patients using the ELISPOT assay. Increased frequencies were revealed for CD8+ T cells recognizing the peptides ALQPGTALL and AILALLPAL. CTLs from prostate cancer patients were raised against these 2 peptides in vitro when presented by autologous DCs. They specifically recognized peptide‐pulsed T2 target cells and prostate cancer cells that were HLA‐A*0201‐ and PSCA‐positive, indicating that these peptides were naturally generated by tumor cells. These data suggest that PSCA is a promising target for the immunotherapy of prostate cancer.

RAGE-mediated MAPK activation by food-derived AGE and non-AGE products

Investigating the cellular effects of food compounds formed by heat treatment during processing, we recently demonstrated the expression of the receptor for advanced glycation endproducts (RAGE) and the p44/42 MAP kinase activation by casein-N(epsilon )-(carboxymethyl)lysine (casein-CML), a food-derived AGE, in the intestinal cell line Caco-2. In this work, we report a Caco-2 p44/42 MAP kinase activation by bread crust and coffee extract. After identification, quantification, and synthesis of two key compounds formed in association with the process-induced heat impact applied to bread dough and coffee beans, those compounds, namely the AGE pronyl-glycine and the non-AGE N-methylpyridinium, were also demonstrated for the first time to activate the p44/42 MAP kinase through binding to RAGE in Caco-2 cells. Blocking of RAGE by an antagonistic antibody and expression of C-terminally truncated RAGE resulted in a reduced Caco-2- and HEK-293-MAP kinase activation. These findings unequivocally point to a RAGE-mediated activating effect of chemically defined food-derived, thermally generated products, both, AGEs and non-AGEs, on cellular signal transduction pathways involved in inflammatory response and cellular proliferation.

A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr)

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5×108 IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I–restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4+ and CD8+ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.

Identification of a Novel Mammary-Restricted Cytochrome P450, CYP4Z1, with Overexpression in Breast Carcinoma

By screening a transcriptome database for expressed sequence tags that are specifically expressed in mammary gland and breast carcinoma, we identified a new human cytochrome P450 (CYP), termed CYP4Z1. The cDNA was cloned from the breast carcinoma line SK-BR-3 and codes for a protein of 505 amino acids. Moreover, a transcribed pseudogene CYP4Z2P that codes for a truncated CYP protein (340 amino acids) with 96% identity to CYP4Z1 was found in SK-BR-3. CYP4Z1 and CYP4Z2P genes consisting of 12 exons are localized in head-to-head orientation on chromosome 1p33. Tissue-specific expression was investigated using real-time reverse transcription PCR with normalized cDNA from 18 different human tissues. CYP4Z1 mRNA was preferentially detected in breast carcinoma tissue and mammary gland, whereas only marginal expression was found in all other tested tissues. Investigation of cDNA pairs from tumor/normal tissues obtained from 241 patients, including 50 breast carcinomas, confirmed the breast-restricted expression and showed a clear overexpression in 52% of breast cancer samples. The expression profile of CYP4Z2P was similar to that of CYP4Z1 with preference in breast carcinoma and mammary gland but a lower expression level in general. Immunoblot analyses with a specific antiserum for CYP4Z1 clearly demonstrated protein expression in mammary gland and breast carcinoma tissue specimens as well as in CYP4Z1-transduced cell lines. Confocal laser-scanning microscopy of MCF-7 cells transfected with a fluorescent fusion protein CYP4Z1-enhanced green fluorescent protein and a subcellular fractionation showed localization to the endoplasmic reticulum as an integral membrane protein concordant for microsomal CYP enzymes.

Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway

Signal transduction via the endothelial receptor for advanced glycation end products (RAGE) plays a key role in vascular inflammation. Recent observations have shown that the myeloperoxidase‐H2O2‐chloride system of activated phagocytes is highly up‐regulated under inflammatory conditions where hypochlorous acid (HOCl) is formed as the major oxidant. Albumin, an in vivo carrier for myeloperoxi‐dase is highly vulnerable to oxidation and a major representative of circulating advanced oxidized proteins during inflammatory diseases. Immunohistochem‐ical studies performed in the present study revealed marked colocalization of HOCl‐modified epitopes with RAGE and albumin in sections of human atheroma, mainly at the endothelial lining. We show that albumin modified with physiologically relevant concentrations of HOCl, added as reagent or generated by the myelo‐peroxidase‐H2O2‐chloride system, is a high affinity li‐gand for RAGE. Albumin, modified by HOCl in the absence of free amino acids/carbohydrates/lipids to exclude formation of AGE‐like structures, induced a rapid, RAGE‐dependent activation of extracellular signal‐regulated kinase 1/2 and up‐regulation of the proin‐flammatory mediator monocyte chemoattractant pro‐tein‐1. Cellular activation could be blocked either by a specific polyclonal anti‐RAGE IgG and/or a specific mitogen‐activated protein‐kinase kinase inhibitor. The present study demonstrates that HOCl‐modified albumin acts as a ligand for RAGE and promotes RAGEmediated inflammatory complications.—Marsche, G., Semlitsch, M., Hammer, A., Frank, S., Weigle, B., Demling, N., Schmidt, K., Windischhofer, W., Waeg, G., Sattler, W., Malle, E. Hypochlorite‐modified albumin colocalizes with RAGE in the artery wall and promotes MCP‐1 expression via the RAGE‐Erk1/2 MAP‐kinase pathway. FASEB J. 21, 1145–1152 (2007)

Age-dependent changes of glyoxalase I expression in human brain

Increased modification and crosslinking of proteins by advanced glycation end products (AGEs) is a characteristic feature of aging, and contributes to the formation of many of the lesions of neurodegenerative diseases including neurofibrillary tangles and amyloid plaques in Alzheimers disease. Therefore, defense mechanisms against AGE formation or detoxification of their precursors such as the glyoxalase system are of particular interest in aging research. Thus, we investigated the age-dependent protein expression, the activity as well as the RNA level of glyoxalase I in Brodmann area 22 (auditory association area of superior temporal gyrus) of the human cerebral cortex. Our immunohistochemical results demonstrate the localization of glyoxalase I in neurons, predominantly pyramidal cells, as well as in astroglia, located predominantly in the subpial region. The number of glyoxalase I expressing neurons and astroglia increases with age, with a peak at approximately 55 years, and progressively decreases thereafter. These results were confirmed by biochemical investigations in total brain tissue, where the RNA, the protein level as well as the activity of glyoxalase I enzyme were analyzed in different age groups. In conclusion, the increase in glyoxalase I expression up to the age of 55 may be a compensatory mechanism against high oxoaldyde levels and the accumulation of AGEs. However, the decline of glyoxalase expression and activity in old age, possibly caused by impairment in transcription or/and translation, may subsequently lead to increased levels of reactive carbonyl compounds, followed by protein crosslinking, inflammation, oxidative stress and neuronal degeneration.