Abstracts

Chemical biology of antigen presentation by MHC molecules
van Kasteren, S.I.; Overkleeft, H.S.; Ovaa, H.; Neefjes, J.J.

MHC class I and MHC class II molecules present peptides to the immune system to drive proper T cell responses. Pharmacological modulation of T-cell responses can offer treatment options for a range of immune-related diseases. Pharmacological downregulation of MHC molecules may find application in treatment of auto-immunity and transplantation rejection while pharmacological activation of antigen presentation would support immune responses to infection and cancer. Since the cell biology of MHC class I and MHC class II antigen presentation is understood in great detail, many potential targets for manipulation have been defined over the years. Here, we discuss how antigen presentation by MHC molecules can be modulated by pharmacological agents and how chemistry can further support the study of antigen presentation in general. The chemical biology of antigen presentation by MHC molecules shows surprising options for immune modulation and the development of future therapies.

Limits of miniaturization: Assessing ITP performance in sub-micron and nanochannels
Janssen, Kjeld G. H.; Li, Jiajie; Hoang, Hanh T.; Vulto, Paul; van den Berg, Richard J. B. H. N.; Overkleeft, Herman S.; Eijkel, Jan C. T.; Tas, Niels R.; van der Linden, Heiko J.; Hankemeier, Thomas

The feasibility of isotachophoresis in channels of sub micrometer and nanometer dimension is investigated. A sample injection volume of 0.4 pL is focused and separated in a 330 nm deep channel. The sample consists of a biomatrix containing the fluorescently-labeled amino acids glutamate and phenylalanine, 20 attomoles of each. Isotachophoretic focusing is successfully demonstrated in a 50 nm deep channel. Separation of the two amino acids in the 50 nm deep channel however, could not be performed as the maximum applicable voltage was insufficient. This limit is imposed by bubble formation that we contribute to cavitation as a result of the mismatch in electro-osmotic flow, so called electrocavitation. This represents an unexpected limit on the miniaturization of ITP. Nonetheless, we report the smallest isotachophoretic separation and focusing experiment to date, both in terms of controlled sample injection volume and channel height.

Synthesis of Eight 1-Deoxynojirimycin Isomers from a Single Chiral Cyanohydrin
van den Nieuwendijk, Adrianus M. C. H.; van den Berg, Richard J. B. H. N.; Ruben, Mark; Witte, Martin D.; Brussee, Johannes; Boot, Rolf G.; van der Marel, Gijsbert A.; Aerts, Johannes M. F. G.; Overkleeft, Herman S.

Eight configurational 1-deoxynojirimycin isomers have been synthesized starting from a chiral cyanohydrin as the common precursor. The cyanohydrin chiral pool building block is easily accessible in large quantities by using almond hydroxynitrile lyase as the chiral catalyst in condensing hydrogen cyanide and crotonaldehyde. Our work complements the large body of literature on the synthesis of 1-deoxynojirimycin derivatives with the distinguishing feature that eight stereoisomers of this important class of glycosidase inhibitors can be derived from a common precursor in an efficient manner.

Identification of glucose kinase-dependent and -independent pathways for carbon control of primary metabolism, development and antibiotic production in Streptomyces coelicolor by quantitative proteomics
Gubbens, J.; Janus, M.; Florea, B. I.; Overkleeft, H. S.; van Wezel, G. P.

Members of the soil-dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient availability is a major determinant for the switch to development and antibiotic production in streptomycetes. Carbon catabolite repression (CCR), a main signalling pathway underlying this phenomenon, was so far considered fully dependent on the glycolytic enzyme glucose kinase (Glk). Here we provide evidence of a novel Glk-independent pathway in Streptomyces coelicolor, using advanced proteomics that allowed the comparison of the expression of some 2000 proteins, including virtually all enzymes for central metabolism. While CCR and inducer exclusion of enzymes for primary and secondary metabolism and precursor supply for natural products is mostly mediated via Glk, enzymes for the urea cycle, as well as for biosynthesis of the ?-butyrolactone Scb1 and the responsive cryptic polyketide Cpk are subject to Glk-independent CCR. Deletion of glkA led to strong downregulation of biosynthetic proteins for prodigionins and calcium-dependent antibiotic (CDA) in mannitol-grown cultures. Repression of bldB, bldN, and its target bldM may explain the poor development of S.?coelicolor on solid-grown cultures containing glucose. A new model for carbon catabolite repression in streptomycetes is presented.

Identification and isolation of lantibiotics from culture: a bioorthogonal chemistry approach
Li, J.; Girard, G.; Florea, B. I.; Geurink, P. P.; Li, N.; van der Marel, G. A.; Overhand, M.; Overkleeft, H. S.; van Wezel, G. P.

A distinguishing feature of the lantibiotic family of cyclic peptides is the presence of thioethers. Treatment of a lantibiotic with an alkaline solution at high pH gives rise to a beta-elimination reaction yielding the corresponding ring opened precursor, containing a dehydro-amino acid residue. We here reveal in a proof-of-concept study that a ring opened lantibiotic (mersacidin) can be captured for pull-down from a culture broth, subsequently released and identified by mass spectrometry.

A pH-Sensitive, Colorful, Lanthanide-Chelating Paramagnetic NMR Probe
Liu, W. M.; Keizers, P. H. J.; Hass, M. A. S.; Blok, A.; Tirnmer, M.; Sarris, A. J. C.; Overhand, M.; Ubbink, M.

Paramagnetic lanthanides ions are broadly used in NMR spectroscopy. The effects of unpaired electrons on NMR spectral parameters provide a powerful tool for the characterization of macromolecular structures and dynamics. Here, a new lanthanide-chelating NMR probe, Caged Lanthanide NMR Probe-7 (CLaNP-7), is presented. It can be attached to protein surfaces via two disulfide bridges, yielding a probe that is rigid relative to the protein backbone. CLaNP-7 extends the application range of available probes. It has a yellow color, which is helpful for sample preparation. Its effects are comparable to those of CLaNP-5, but its charge is two units lower (+1) than that of CLaNP-5 (+3), reducing the change in surface potential after probe attachment. It also has a different magnetic susceptibility tensor, so by using both tags, two sets of structural restraints can be obtained per engineered cysteine pair. Moreover, it was found that the orientation of the magnetic susceptibility tensor is pH dependent (pK(a) approximate to 7) when a histidine residue is located in the neighborhood of the probe attachment site. The results show that the His imidazole group interacts with the CLaNP-7 tag. It is proposed that the histidine residue forms a hydrogen bond to a water/hydroxyl molecule that occupies the ninth coordination position on the lanthanide, thus breaking the two-fold symmetry of the CLaNP tag in a pH-dependent way.

The Antimalarial Natural Product Symplostatin 4 Is a Nanomolar Inhibitor of the Food Vacuole Falcipains
Stolze, S. C.; Deu, E.; Kaschani, F.; Li, N.; Florea, B. I.; Richau, K. H.; Colby, T.; van der Hoom, R. A. L.; Overkleeft, H. S.; Bogyo, M.; Kaiser, M.

The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4's mode of action. Finally, we reveal a critical influence of the unusual methyl-methoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors.

Biomarkers in the diagnosis of lysosomal storage disorders: proteins, lipids, and inhibodies
Aerts, J. M.; Kallemeijn, W. W.; Wegdam, W.; Joao Ferraz, M.; van Breemen, M. J.; Dekker, N.; Kramer, G.; Poorthuis, B. J.; Groener, J. E.; Cox-Brinkman, J.; Rombach, S. M.; Hollak, C. E.; Linthorst, G. E.; Witte, M. D.; Gold, H.; van der Marel, G. A.; Overkleeft, H. S.; Boot, R. G.

A biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. In the case of lysosomal storage disorders (LSDs), primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Clinical applications of biomarkers are found in improved diagnosis, monitoring disease progression, and assessing therapeutic correction. These are illustrated by reviewing the discovery and use of biomarkers for Gaucher disease and Fabry disease. In addition, recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described. Such probes, coined inhibodies, offer entirely new possibilities for more sophisticated molecular diagnosis, enzyme replacement therapy monitoring, and fundamental research.

Novel protecting groups in carbohydrate chemistry
Codee, J. D. C.; Ali, A.; Overkleeft, H. S.; van der Marel, G. A.

Protecting groups play a pivotal role in carbohydrate chemistry. This review describes a selection of protecting groups and protecting group strategies that have been introduced since the beginning of this century. (C) 2010 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Glycosphingolipids and Insulin Resistance
Aerts, J. M.; Boot, R. G.; van Eijk, M.; Groener, J.; Bijl, N.; Lombardo, E.; Bietrix, F. M.; Dekker, N.; Groen, A. K.; Ottenhoff, R.; van Roomen, C.; Aten, J.; Serlie, M.; Langeveld, M.; Wennekes, T.; Overkleeft, H. S.

Glycosphingolipids are structural membrane components, residing largely in the plasma membrane with their sugar-moieties exposed at the cell's surface. In recent times a crucial role for glycosphingolipids in insulin resistance has been proposed. A chronic state of insulin resistance is a rapidly increasing disease condition in Western and developing countries. It is considered to be the major underlying cause of the metabolic syndrome, a combination of metabolic abnormalities that increases the risk for an individual to develop Type 2 diabetes, obesity, cardiovascular disease, polycystic ovary syndrome and nonalcoholic fatty liver disease. As discussed in this chapter, the evidence for a direct regulatory interaction of glycosphingolipids with insulin signaling is still largely indirect. However, the recent finding in animal models that pharmacological reduction of glycosphingolipid biosynthesis ameliorates insulin resistance and prevents some manifestations of metabolic syndrome, supports the view that somehow glycosphingolipids act as critical regulators, Importantly, since reductions in glycosphingolipid biosynthesis have been found to be well tolerated, such approaches may have a therapeutic potential.