Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both... Show moreGlycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody. Show less
Leeuwen, T. van; Araman, C.; Pournara, L.P.; Kampstra, A.S.B.; Bakkum, T.; Marqvorsen, M.H.S.; ... ; Kasteren, S.I. van 2021
Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and... Show moreProteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation. Show less
Ende, T.C. van den; Heuts, J.M.M.; Gential, G.P.P.; Visser, M.; Graaff, M.J. van de; Ho, N.I.; ... ; Filippov, D.V. 2020
Synthetic vaccines, based on antigenic peptides that comprise MHC−I and MHC‐II T‐cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity,... Show moreSynthetic vaccines, based on antigenic peptides that comprise MHC−I and MHC‐II T‐cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll‐like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3CysSK4. A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini‐UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T‐cell therapy. Homogeneous mini‐UPam‐SP conjugates have been prepared in good yields by stepwise solid‐phase synthesis that employed a mini‐UPam building block pre‐prepared in solution and the standard set of Fmoc‐amino acids. The immunogenicity of the novel mini‐UPam‐SP conjugates was demonstrated by using the cancer patient's T‐cells. Show less
Smeden, J. van; Al-Khakany, H.; Wang, Y.; Visscher, D.; Stephens, N.; Absalah, S.; ... ; Bouwstra, J.A. 2020
Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we... Show moreIndividuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) stratum corneum ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active, epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in stratum corneum ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered stratum corneum ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS. Show less
Boer, D.E.C.; Smeden, J. van; Al-Khakany, H.; Melnik, E.; Dijk, R. van; Absalah, S.; ... ; Bouwstra, J.A. 2020
Patients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin... Show morePatients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin barrier function. In this study we analyzed the localization and activity of lipid enzymes β-glucocerebrosidase (GBA) and acid sphingomyelinase (ASM) in the skin of AD patients and controls. Localization of both the expression and activity of GBA and ASM in the epidermis of AD patients was altered, particularly at lesional skin sites. These changes aligned with the altered SC lipid composition. More specifically, abnormal localization of GBA and ASM related to an increase in specific ceramide subclasses [AS] and [NS]. Moreover we related the localization of the enzymes to the amounts of SC ceramide subclasses and free fatty acids (FFAs). We report a correlation between altered localization of active GBA and ASM and a disturbed SC lipid composition. Localization of antimicrobial peptide beta-defensin-3 (HBD-3) and AD biomarker Thymus and Activation Regulated Chemokine (TARC) also appeared to be diverging in AD skin compared to control. This research highlights the relation between correct localization of expressed and active lipid enzymes and a normal SC lipid composition for a proper skin barrier. Show less
Esbroeck, A.C.M. van; Kantae, V.; Di, X.; Wel, T. van der; Dulk, J. den; Stevens, A.F.; ... ; Stelt, M. van der 2019
The endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid... Show moreThe endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid (RA)-induced neurite outgrowth of Neuro-2a cells. First, we confirmed that RA stimulation of Neuro-2a cells increases 2-AG production and neurite outgrowth. The diacylglycerol lipase (DAGL) inhibitor DH376 blocked 2-AG production and reduced neuronal differentiation. Surprisingly, CRISPR/Cas9-mediated knockdown of DAGLα and DAGLβ in Neuro-2a cells did not reduce 2-AG levels, suggesting another enzyme capable of producing 2-AG in this cell line. Chemical proteomics revealed DAGLβ and α,β-hydrolase domain containing protein (ABHD6) as the only targets of DH376 in Neuro-2a cells. Biochemical, genetic and lipidomic studies demonstrated that ABHD6 possesses DAGL activity in conjunction with its previously reported monoacylglycerol lipase activity. RA treatment of Neuro-2a cells increased by three-fold the amount of active ABHD6. Our study shows that ABHD6 exhibits significant DAG lipase activity in Neuro-2a cells in addition to its known MAG lipase activity and suggest it is involved in neuronal differentiation. Show less
Koenders, S.T.A.; Wijaya, L.S.; Erkelens, M.N.; Bakker, A.T.; Noord, V.E. van der; Rooden, E.J. van; ... ; Stelt, M. van der 2019
Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of... Show moreRetinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts. Show less
Esbroeck, A.C.M. van; Kantae, V.; Di, X.; Wel, T. van der; Dulk, J. den; Stevens, A.F.; ... ; Stelt, M. van der 2019
The endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid... Show moreThe endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid (RA)-induced neurite outgrowth of Neuro-2a cells. First, we confirmed that RA stimulation of Neuro-2a cells increases 2-AG production and neurite outgrowth. The diacylglycerol lipase (DAGL) inhibitor DH376 blocked 2-AG production and reduced neuronal differentiation. Surprisingly, CRISPR/Cas9-mediated knockdown of DAGL alpha and DAGL beta in Neuro-2a cells did not reduce 2-AG levels, suggesting another enzyme capable of producing 2-AG in this cell line. Chemical proteomics revealed DAGL beta and alpha,beta-hydrolase domain containing protein (ABHD6) as the only targets of DH376 in Neuro-2a cells. Biochemical, genetic and lipidomic studies demonstrated that ABHD6 possesses DAGL activity in conjunction with its previously reported monoacylglycerol lipase activity. RA treatment of Neuro-2a cells increased by three-fold the amount of active ABHD6. Our study shows that ABHD6 exhibits significant DAG lipase activity in Neuro-2a cells in addition to its known MAG lipase activity and suggest it is involved in neuronal differentiation. Show less
Mieremet, A.; Boiten, W.A.; Dijk, R. van; Gooris, G.S.; Overkleeft, H.S.; Aerts, J.M.F.G.; ... ; El Ghalbzouri, A. 2019
Relative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of... Show moreRelative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of the cell-culture medium. However, skin functionality is directly influenced by environmental RH. As the barrier formation in HSEs is different, there is a need to better understand the role of cell-culture conditions during the generation of HSEs. In this study, we aim to investigate the effects of RH on epidermal morphogenesis and lipid barrier formation in HSEs. Therefore, two types of HSEs were developed at 90% or at 60% RH. Assessments were performed to determine epidermal morphogenesis by immunohistochemical analyses, ceramide composition by lipidomic analysis, and lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction. We show that reduction of RH mainly affected the uppermost viable epidermal layers in the HSEs, including an enlargement of the granular cells and induction of epidermal cell activation. Neither the composition nor the organization of the lipids in the intercorneocyte space were substantially altered at reduced RH. In addition, lipid processing from glucosylceramides to ceramides was not affected by reduced RH in HSEs as shown by enzyme expression, enzyme activity, and substrate-to-product ratio. Our results demonstrate that RH directly influences epidermal morphogenesis, albeit the in vitro lipid barrier formation is comparable at 90% and 60% RH. Show less
Grimm, S.H.; Gagestein, B.; Keijzer, J.F.; Liu, N.; Wijdeven, R.H.; Lenselink, E.B.; ... ; Stelt, M. van der 2019
Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in... Show moreAcute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors. Show less
Zhou, J.; Mock, E.D.; Martella, A.; Kantae, V.; Di, X.; Burggraaff, L.; ... ; Stelt, M. van der 2019
Phospholipase A2, group XVI (PLA2G16) is a thiol-hydrolase from the HRASLS family that regulates lipolysis in adipose tissue and has been identified as a host factor enabling the cellular entry of... Show morePhospholipase A2, group XVI (PLA2G16) is a thiol-hydrolase from the HRASLS family that regulates lipolysis in adipose tissue and has been identified as a host factor enabling the cellular entry of picornaviruses. Chemical tools are essential to visualize and control PLA2G16 activity, but have not been reported to date. Here we show that MB064, a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16. Competitive activity-based protein profiling (ABPP) using MB064 enabled the discovery of α-ketoamides as the first selective PLA2G16 inhibitors. LEI110 was identified as a potent PLA2G16 inhibitor (Ki = 20 nM) that reduces cellular arachidonic acid levels and oleic acid-induced lipolysis in human HepG2 cells. Gel-based ABPP and chemical proteomics showed that LEI110 is a selective pan-inhibitor of the HRASLS-family of thiol hydrolases (i.e. PLA2G16, HRASLS2, RARRES3 and iNAT). Molecular dynamic simulations of LEI110 in the reported crystal structure of PLA2G16 provided insight in the potential ligand-protein interactions to explain its binding mode. In conclusion, we have developed the first selective inhibitor that can be used to study the cellular role of PLA2G16. Show less
Es, D. van der; Berni, F.; Hogendorf, W.F.J.; Meeuwenoord, N.; Laverde, D.; Diepen, A. van; ... ; Codée, J.D.C. 2018
Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as... Show moreChemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photo-click chemistry and hold promise as biomarkers in translational drug discovery. Show less
Smeden, J. van; Dijkhoff, I.M.; Helder, R.W.J.; Al-Khakany, H.; Boer, D.E.C.; Schreuder, A.; ... ; Bouwstra, J.A. 2017
A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although... Show moreA recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system. Show less
Es, D. van der; Groenia, N.A.; Laverde, D.; Overkleeft, H.S.; Huebner, J.; Marel, G.A. van der; Codee, J.D.C. 2016
The first synthesis of different Enterococcus faecium wall teichoic acid (WTA) fragments is presented. The structure of these major cell wall components was elucidated recently and it was shown... Show moreThe first synthesis of different Enterococcus faecium wall teichoic acid (WTA) fragments is presented. The structure of these major cell wall components was elucidated recently and it was shown that these glycerolphosphate (GroP) based polymers are built up from -6-(GalNAc-α(1-3)-GalNAc-β(1-2)-GroP)- repeating units. We assembled WTA fragments up to three repeating units in length, in two series that differ in the stereochemistry of the glycerolphosphate moiety. The key GalNAc-GalNAc-GroP synthons, required for the synthesis, were generated from galactosazide building blocks that were employed in highly stereoselective glycosylation reactions to furnish both the α- and β-configured linkages. By comparing the NMR spectra of the synthesized fragments with the isolated material it appears that the hereto undefined stereochemistry of the glycerol phosphate moiety is sn-glycerol-3-phosphate. The generated fragments will be valuable tools to study their immunological activity at the molecular level. Show less
Baggelaar, M.P.; Chameau, P.J.P.; Kantae, V.; Hummel, J.; Hsu, K.L.; Janssen, F.; ... ; Stelt, M. van der 2015
