Martin Lenter

The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor

E-SELECTIN is an inducible cell-adhesion molecule on endothelial cells, which mediates the binding of neutrophils and functions as a Ca2+-dependent lectin1–3. We have recently identified a 150K glycoprotein as the major ligand for E-selectin on myeloid cells, using a recombinant antibody-like form of mouse E-selectin as an affinity probe4,5. Here we report the isolation of a mouse complementary DNA for this E-selectin ligand (ESL-1). The predicted amino-acid sequence of ESL-1 is 94% identical (over 1,078 amino acids) to the recently identified chicken cysteine-rich fibroblast growth-factor receptor6, except for a unique 70-amino-acid amino-terminal domain of mature ESL-1. Fucosylation of ESL-1 is imperative for affinity isolation with E-selectin-IgG. A fucosylated, recombinant antibody-like form of ESL-1, but not of L-selectin, supports adhesion of E-selectin-transfected Chinese hamster ovary cells. Antibodies against ESL-1 block the binding of mouse myeloid cells to E-selectin. ESL-1, with a structure essentially identical to that of a receptor, thus functions as a cell adhesion ligand of E-selectin.

miR-155 Inhibition Sensitizes CD4+ Th Cells for TREG Mediated Suppression

Background In humans and mice naturally occurring CD4+CD25+ regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance. Principal Findings DNA-Microarray analyses of human as well as murine conventional CD4+ Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4+ Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4+ Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression. Conclusion Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4+ Th cells to nTreg cell-mediated suppression.

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4+ Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4+ T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4+ Th cells upon activation, whereas the ‘standard’ proteasome is up-regulated in Tregs only upon activation.

MicroRNA and proteome expression profiling in early-symptomatic α-synuclein(A30P)-transgenic mice.

The α‐synuclein has been implicated in the pathophysiology of Parkinsons disease (PD), because mutations in the alpha‐synuclein gene cause autosomal‐dominant hereditary PD and fibrillary aggregates of alpha‐synuclein are the major component of Lewy bodies. Since presynaptic accumulation of α‐synuclein aggregates may trigger synaptic dysfunction and degeneration, we have analyzed alterations in synaptosomal proteins in early symptomatic α‐synuclein(A30P)‐transgenic mice by two‐dimensional differential gel electrophoresis. Moreover, we carried out microRNA expression profiling using microfluidic chips, as microRNA have recently been shown to regulate synaptic plasticity in rodents and to modulate polyglutamine‐induced protein aggregation and neurodegeneration in flies. Differentially expressed proteins in α‐synuclein(A30P)‐transgenic mice point to alterations in mitochondrial function, actin dynamics, iron transport, and vesicle exocytosis, thus partially resembling findings in PD patients. Oxygen consumption of isolated brain mitochondria, however, was not reduced in mutant mice. Levels of several microRNA (miR‐10a, ‐10b, ‐212, ‐132, ‐495) were significantly altered. One of them (miR‐132) has been reported to be highly inducible by growth factors and to be a key regulator of neurite outgrowth. Moreover, miR‐132‐recognition sequences were detected in the mRNA transcripts of two differentially expressed proteins. MicroRNA may thus represent novel biomarkers for neuronal malfunction and potential therapeutic targets for human neurodegenerative diseases.

iTRAQ and multiple reaction monitoring as proteomic tools for biomarker search in cerebrospinal fluid of patients with Parkinson's disease dementia

About 30% of patients with Parkinsons disease (PD) develop Parkinsons disease dementia (PDD) in the course of the disease. Until now, diagnosis is based on clinical and neuropsychological examinations, since so far there is no laboratory marker. In this study we aimed to find a neurochemical marker which would allow a risk assessment for the development of a dementia in PD patients. For this purpose, we adopted a gel-free proteomic approach (iTRAQ-method) to identify biomarker-candidates in the cerebrospinal fluid (CSF) of patients with PD, PDD and non-demented controls (NDC). Validation of these candidates was then carried out by multiple-reaction-monitoring (MRM) optimised for CSF. Using the iTRAQ-approach, we were able to identify 16 differentially regulated proteins. Fourteen out of these 16 proteins could then be followed-up simultaneously in our optimised MRM-measurement protocol. However only Tyrosine-kinase-non-receptor-type 13 and Netrin-G1 differed significantly between PDD and NDC cohorts. In addition, a significant difference was found for Golgin-160 and Apolipoprotein B-100 between PD and NDC. Apart from possible pathophysiological considerations, we propose that Tyrosine-kinase non-receptor-type 13 and Netrin G1 are biomarker candidates for the development of a Parkinsons disease dementia. Furthermore we suggest that iTRAQ and MRM are valuable tools for the discovery of biomarker in cerebrospinal fluid. However further validation studies need to be done with larger patient cohorts and other proteins need to be checked as well.

Ubiquitin as potential cerebrospinal fluid marker of Creutzfeldt-Jakob disease.

Until today, a definite diagnosis of Creutzfeldt–Jakob disease (CJD) can only be made neuropathologically. At lifetime the early and differential diagnosis is often a problem. With SELDI we analyzed cerebrospinal fluid (CSF) from 32 CJD patients, 32 patients having other dementive diseases and 31 non‐demented control subjects for diagnosis‐dependent protein pattern differences. In a screening set of patients, peaks that discriminate best between groups were identified. These peaks were subsequently analyzed using an independent validation set of patients. Diagnostic accuracies were compared with established markers like tau protein and 14‐3‐3‐protein. Potential marker proteins were purified and identified by LC‐MS/MS. In the validation set only one peak of 8.6 kDa out of ten in the screening set could be confirmed. This protein was identified to be ubiquitin and increased levels in CSF (but not in serum) of CJD patients were confirmed by Western blot. Ubiquitin allows the correct diagnoses of that CJD cases missed by tau protein or 14‐3‐3‐protein. We conclude that ubiquitin is a promising additional CSF biomarker for diagnosis of CJD, especially in differential diagnostically difficult cases. The selective increase of ubiquitin in CSF of CJD patients might point to an involvement of ubiquitin in pathophysiological process.

Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K

Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O‐glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel‐associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin‐deficient mice have unraveled a critical role of endosialin in site‐specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac‐2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C‐terminal fragment of Mac‐2 BP/90K, which was shown to contain binding sites for galectin‐3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac‐2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up‐regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac‐2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss‐of‐function adhesion experiments of Mac‐2 BP/90K‐expressing tumor cells on endosialin‐expressing fibroblasts revealed a repulsive outcome of the Mac‐2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac‐2 BP/90K on tumor cells.—Becker, R., Lenter, M. C., Vollkommer, T., Boos, A. M., Pfaff, D., Augustin, H. G., Christian, S. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis‐related protein Mac‐2 BP/90K. FASEB J. 22, 3059–3067 (2008)

Crystal Structure of Glucokinase Regulatory Protein.

Glucokinase (GK) plays a major role in the regulation of blood glucose homeostasis in both the liver and the pancreas. In the liver, GK is controlled by the GK regulatory protein (GKRP). GKRP in turn is activated by fructose 6-phosphate (F6P) and inactivated by fructose 1-phosphate (F1P). Disrupting the GK-GKRP complex increases the activity of GK in the cytosol and is considered an attractive concept for the regulation of blood glucose. We have determined the crystal structure of GKRP in its inactive F1P-bound form. The binding site for F1P is located deeply buried at a domain interface, and H-D exchange experiments confirmed that F1P and F6P compete for this site. The structure of the inactive GKRP-F1P complex provides a starting point for understanding the mechanism of fructose phosphate-dependent GK regulation at an atomic level.

Nonviral monocyte chemoattractant protein-1 gene transfer improves arteriogenesis after femoral artery occlusion

Local infusion of recombinant monocyte chemoattractant protein-1 (MCP-1) has been shown to enhance collateral artery formation in rabbit and pig hindlimb models. Owing to clinical disadvantages of protein infusion, a nonviral, liposome-based MCP-1 gene transfer was developed. Collateralization in a porcine hindlimb model served to provide a proof-of-principle for the functional benefit of MCP-1 overexpression. Development of arterial conductance as a measure of functionally relevant collateralization was evaluated in occluded as well as untreated hindlimbs in each animal. At the time of occlusion, MCP-1 and control DNA/DC-30 lipoplexes were transferred to femoral arteries of Goettingen minipigs (two therapeutic MCP-1 groups: 2 and 4 μg and one control group), using the Infiltrator® local drug-delivery device. At 2 weeks following occlusion, collateralization was determined as changes in peripheral haemodynamic conductance, peripheral over aortic blood pressure ratio and angiographically visible morphology of the peripheral vessel tree. Nonviral MCP-1 gene transfer significantly improved peripheral conductance (control 11.69±2.78%, 2 μg 23.81±2.81%, P<0.05 and 4 μg 23.36±3.1%, P<0.05; n=12 per group) as well as the ratio of peripheral over aortic blood pressure (control 0.64±0.03%, 2 μg 0.75±0.02%, P<0.05 and 4 μg 0.75±0.02%, P<0.05; n=12 per group) when compared to the untreated controls 2 weeks after occlusion. Thus, it could be demonstrated for the first time that in situ overexpression of MCP-1 following local nonviral gene transfer is a potential approach to improve peripheral collateralization.

Differential Sialylation of Serpin A1 in the Early Diagnosis of Parkinson’s Disease Dementia

The prevalence of Parkinson’s disease (PD) increases with age. Up to 50% of PD show cognitive decline in terms of a mild cognitive impairment already in early stages that predict the development of dementia, which can occur in up to 80% of PD patients over the long term, called Parkinson’s disease dementia (PDD). So far, diagnosis of PD/PDD is made according to clinical and neuropsychological examinations while laboratory data is only used for exclusion of other diseases. The aim of this study was the identification of possible biomarkers in cerebrospinal fluid (CSF) of PD, PDD and controls (CON) which predict the development of dementia in PD. For this, a proteomic approach optimized for CSF was performed using 18 clinically well characterized patients in a first step with subsequent validation using 84 patients. Here, we detected differentially sialylated isoforms of Serpin A1 as marker for differentiation of PD versus PDD in CSF. Performing 2D-immunoblots, all PDD patients could be identified correctly (sensitivity 100%). Ten out of 24 PD patients showed Serpin A1 isoforms in a similar pattern like PDD, indicating a specificity of 58% for the test-procedure. In control samples, no additional isoform was detected. On the basis of these results, we conclude that differentially sialylated products of Serpin A1 are an interesting biomarker to indicate the development of a dementia during the course of PD.