Galectin-3-binding protein inhibits extracellular heparan 6-O-endosulfatase Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). In spite of its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Aspartate ß-Hydroxylase Is Upregulated in Head and Neck Squamous Cell Carcinoma and Regulates Invasiveness in Cancer Cell Models.

Aspartate ß-hydroxylase (ASPH) is a protein associated with malignancy in a wide range of tumors. We hypothesize that inhibition of ASPH activity could have anti-tumor properties in patients with head and neck cancer. In this study, we screened tumor tissues of 155 head and neck squamous cell carcinoma (HNSCC) patients for the expression of ASPH using immunohistochemistry. We used an ASPH inhibitor, MO-I-1151, known to inhibit the catalytic activity of ASPH in the endoplasmic reticulum, to show its inhibitory effect on the migration of SCC35 head and neck cancer cells in cell monolayers and in matrix-embedded spheroid co-cultures with primary cancer-associated fibroblast (CAF) CAF 61137 of head and neck origin. We also studied a combined effect of MO-I-1151 and HfFucCS, an inhibitor of invasion-blocking heparan 6-O-endosulfatase activity. We found ASPH was upregulated in HNSCC tumors compared to the adjacent normal tissues. ASPH was uniformly high in expression, irrespective of tumor stage. High expression of ASPH in tumors led us to consider it as a therapeutic target in cell line models. ASPH inhibitor MO-I-1151 had significant effects on reducing migration and invasion of head and neck cancer cells, both in monolayers and matrix-embedded spheroids. The combination of the two enzyme inhibitors showed an additive effect on restricting invasion in the HNSCC cell monolayers and in the CAF-containing co-culture spheroids. We identify ASPH as an abundant protein in HNSCC tumors. Targeting ASPH with inhibitor MO-I-1151 effectively reduces CAF-mediated cellular invasion in cancer cell models. We propose that the additive effect of MO-I-1151 with HfFucCS, an inhibitor of heparan 6-O-endosulfatases, on HNSCC cells could improve interventions and needs to be further explored.

Heparan-6-O-Endosulfatase 2 Promotes Invasiveness of Head and Neck Squamous Carcinoma Cell Lines in Co-Cultures with Cancer-Associated Fibroblasts.

Local invasiveness of head and neck squamous cell carcinoma (HNSCC) is a complex phenomenon supported by interaction of the cancer cells with the tumor microenvironment (TME). We and others have shown that cancer-associated fibroblasts (CAFs) are a component of the TME that can promote local invasion in HNSCC and other cancers. Here we report that the secretory enzyme heparan-6-O-endosulfatase 2 (Sulf-2) directly affects the CAF-supported invasion of the HNSCC cell lines SCC35 and Cal33 into Matrigel. The Sulf-2 knockout (KO) cells differ from their wild type counterparts in their spheroid growth and formation, and the Sulf-2-KO leads to decreased invasion in a spheroid co-culture model with the CAF. Next, we investigated whether a fucosylated chondroitin sulfate isolated from the sea cucumber Holothuria floridana (HfFucCS) affects the activity of the Sulf-2 enzyme. Our results show that HfFucCS not only efficiently inhibits the Sulf-2 enzymatic activity but, like the Sulf-2 knockout, inhibits Matrigel invasion of SCC35 and Cal33 cells co-cultured with primary HNSCC CAF. These findings suggest that the heparan-6-O-endosulfatases regulate local invasion and could be therapeutically targeted with the inhibitory activity of a marine glycosaminoglycan.

A 6-O-endosulfatase activity assay based on synthetic heparan sulfate oligomers.

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the postsynthetic editing of heparan sulfate (HS), which regulates many important biological processes. The activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well-characterized synthetic substrates for the probing of its catalytic activity. We introduce synthetic HS oligosaccharides to fill this gap, and we use our recombinant Sulf-2 protein to show that a paranitrophenol (pNP)-labeled synthetic oligosaccharide allows a reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange high-pressure liquid chromatography and quantified by UV absorbance. This simple assay allows the detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that the N-glycosylation of the Sulf-2 enzyme affects the activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan.

A multiplexed microflow LC-MS/MS-PRM assay for serologic quantification of IgG N- and HPX O- glycoforms in liver fibrosis.

Targeted quantification of glycoproteins has not reached its full potential because of limitations of the existing analytical workflows. In this study, we introduce a targeted microflow LC-MS/MS-PRM method for the quantification of multiple glycopeptides in unfractionated serum samples. The entire preparation of 16 samples in a batch is completed within 3 h, and the LC-MS quantification of all the glycoforms in a sample is completed in 15 min in triplicate, including online capture and desalting. We demonstrate applicability of the workflow on a multiplexed quantification of eight N-glycoforms of immunoglobulin G (IgG) together with two O-glycoforms of hemopexin (HPX). We applied the assay to a serologic study of fibrotic liver disease in patients of HCV etiology. The results document that specific IgG- and HPX-glycoforms detect efficiently fibrotic disease of different degree, and suggest that the LC-MS/MS-PRM assays may provide rapid and reproducible biomarker assay targeting simultaneously the N- and O-glycoforms of the peptides. We propose that such high throughput multiplexed methods may advance the clinical use of the LC-MS/MS assays.

Galectin-3-binding protein inhibits extracellular heparan 6-O-endosulfatse Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). In spite of its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the secretome of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP has functional relevance, and may regulate physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Extracellular Heparan 6-O-Endosulfatases SULF1 and SULF2 in Head and Neck Squamous Cell Carcinoma and Other Malignancies.

Pan-cancer analysis of TCGA and CPTAC (proteomics) data shows that SULF1 and SULF2 are oncogenic in a number of human malignancies and associated with poor survival outcomes. Our studies document a consistent upregulation of SULF1 and SULF2 in HNSC which is associated with poor survival outcomes. These heparan sulfate editing enzymes were considered largely functional redundant but single-cell RNAseq (scRNAseq) shows that SULF1 is secreted by cancer-associated fibroblasts in contrast to the SULF2 derived from tumor cells. Our RNAScope and patient-derived xenograft (PDX) analysis of the HNSC tissues fully confirm the stromal source of SULF1 and explain the uniform impact of this enzyme on the biology of multiple malignancies. In summary, SULF2 expression increases in multiple malignancies but less consistently than SULF1, which uniformly increases in the tumor tissues and negatively impacts survival in several types of cancer even though its expression in cancer cells is low. This paradigm is common to multiple malignancies and suggests a potential for diagnostic and therapeutic targeting of the heparan sulfatases in cancer diseases.

A Rapid LC-MS/MS-PRM Assay for Serologic Quantification of Sialylated O-HPX Glycoforms in Patients with Liver Fibrosis.

Development of high throughput robust methods is a prerequisite for a successful clinical use of LC-MS/MS assays. In earlier studies, we reported that nLC-MS/MS measurement of the O-glycoforms of HPX is an indicator of liver fibrosis. In this study, we show that a microflow LC-MS/MS method using a single column setup for capture of the analytes, desalting, fast gradient elution, and on-line mass spectrometry measurements, is robust, substantially faster, and even more sensitive than our nLC setup. We demonstrate applicability of the workflow on the quantification of the O-HPX glycoforms in unfractionated serum samples of control and liver disease patients. The assay requires microliter volumes of serum samples, and the platform is amenable to one hundred sample injections per day, providing a valuable tool for biomarker validation and screening studies.

Glycoforms of human prostate-specific membrane antigen (PSMA) in human cells and prostate tissue.

INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

PD-L1 Glycosylation and Its Impact on Binding to Clinical Antibodies.

Immune checkpoint inhibitors, including PD-L1/PD-1, are key regulators of the immune response and promising targets in cancer immunotherapy. N-glycosylation of PD-L1 affects its interaction with PD-1, but little is known about the distribution of glycoforms at its four NXS/T sequons. We optimized LC-MS/MS methods using collision energy modulation for the site-specific resolution of specific glycan motifs. We demonstrate that PD-L1 on the surface of breast cancer cell line carries mostly complex glycans with a high proportion of polyLacNAc structures at the N219 sequon. Contrary to the full-length protein, the secreted form of PD-L1 expressed in breast MDA-MB-231 or HEK293 cells demonstrated minimum N219 occupancy and low contribution of the polyLacNAc structures. Molecular modeling of PD-L1/PD-1 interaction with N-glycans suggests that glycans at the N219 site of PD-L1 and N74 and N116 of PD-1 may be involved in glycan-glycan interactions, but the impact of this potential interaction on the protein function remains at this point unknown. The interaction of PD-L1 with clinical antibodies is also affected by glycosylation. In conclusion, PD-L1 expressed in the MDA-MB-231 breast cancer cell line carries polyLacNAc glycans mostly at the N219 sequon, which displays the highest variability in occupancy and is most likely to influence the interaction with PD-1.

Low Collision Energy Fragmentation in Structure-Specific Glycoproteomics Analysis.

Glycosylation is a major post-translational modification of proteins that regulates many biological processes including protein folding, structure stability, receptor activation, and immune responses. The glycans attached to proteins represent an important determinant of the protein interaction-specificity and maintain the 3D structure of proteins. Mass spectrometry (MS) is one of the most efficient tools used in the current studies of glycoproteins and structure of their glycoforms. Collision energy (CE) is a crucial instrument parameter that can be exploited to improve structural resolution because different linkages of glycan units show different stabilities under CID/HCD fragmentation. Here we report the utility of CE modulation for qualitative and quantitative analysis of site- and structure-specific glycoforms of proteins. Using CE modulation, we were able to break selectively specific glycan linkages on intact glycopeptides and get, to some degree, structure-specific mass spectrometric signals. Structure- and CE-specific oxonium ions provide sufficient information for the resolution of outer arm structure motifs with recognized biological functions. The complementary Y-ions, generated under optimized low CE (soft) conditions, provide additional structural information including features specific to the chitobiose core. This methodology of multiple CE fragmentation without merging spectral information can significantly improve confidence of glycopeptide identification and structural resolution by providing additional information to the established glycopeptide-search algorithms and tools.

N-Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures.

Brain-derived neurotrophic factor (BDNF) is generated by proteolytic cleavage of a prodomain from the proBDNF precursor either intracellularly by furin-like proteases or extracellularly by plasmin or matrix metalloproteinases. ProBDNF carries a single N-glycosylation sequon (Asn-127) that remains virtually unstudied despite being located in a highly conserved region proximal to the proteolytic site. To study the proBDNF structure and function, here we expressed the protein and its nonglycosylated N127Q mutant in HEK293F cells. We found that mutation of the Asn-127 prevents intracellular maturation and secretion, an effect reproduced in WT proBDNF by tunicamycin-induced inhibition of N-glycosylation. Absence of the N-glycan did not affect the kinetics of proBDNF cleavage by furin in vitro, indicating that effects other than a direct furin-proBDNF interaction may regulate proBDNF maturation. Using an optimized LC-MS/MS workflow, we demonstrate that secreted proBDNF is fully glycosylated and carries rare N-glycans terminated by GalNAcß1-4GlcNAcß1-R (LacdiNAc) extensively modified by terminal sulfation. We and others noted that this type of glycosylation is protein-specific, extends to proBDNF expressed in PC12 cells, and implies the presence of interacting partners that recognize this glycan epitope. The findings of our study reveal that proBDNF carries an unusual type of N-glycans important for its processing and secretion. Our results open new opportunities for functional studies of these protein glycoforms in different cells and tissues.

Quantitative Analysis of Sex-Hormone-Binding Globulin Glycosylation in Liver Diseases by Liquid Chromatography-Mass Spectrometry Parallel Reaction Monitoring.

Sex-hormone-binding globulin (SHBG) is a liver-secreted glycoprotein and a major regulator of steroid distribution. It has been reported that the serum concentration of SHBG changes in liver disease. To explore the involvement of SHBG in liver disease of different etiologies in greater detail, we developed a sensitive and selective liquid chromatography-mass spectrometry parallel reaction monitoring workflow to achieve quantitative analysis of SHBG glycosylation microheterogeneity. The method uses energy-optimized "soft" fragmentation to extract informative Y ions for maximal coverage of glycoforms and their quantitative comparisons. A total of 15 N-glycoforms of two N-glycosites and 3 O-glycoforms of 1 O-glycosite of this low-abundance serum protein were simultaneously analyzed in the complex samples. At the same time, we were able to partially resolve linkage isoforms of the fucosylated glycoforms and to identify and quantify SHBG N-glycoforms that were not previously reported. The results show that both core and outer-arm fucosylation of the N-glycoforms increases with liver cirrhosis but that a further increase of fucosylation is not observed with hepatocellular carcinoma (HCC). In contrast, the α-2-6 sialylated glycoform of the O-glycopeptide of SHBG increases in liver cirrhosis, and a significant 2-fold further increase is observed in HCC. In general, we do not find a significant contribution of different liver disease etiologies to the observed changes in glycosylation; however, elevation of the newly reported HexNAc(4)Hex(6) N-glycoform is associated with alcoholic liver disease.

Increased sialylation of site specific O-glycoforms of hemopexin in liver disease.

BACKGROUND: Non-invasive monitoring of liver disease remains an important health issue. Liver secreted glycoproteins reflect pathophysiological states of the organ and represent a rational target for serologic monitoring. In this study, we describe sialylated O-glycoforms of liver-secreted hemopexin (HPX) and quantify them as a ratio of disialylated to monosialylated form (S-HPX). METHODS: We measured S-HPX in serum of participants of the HALT-C trial using a LC-MS/MS-MRM assay. RESULTS: Repeated measurements of S-HPX in the samples of 23 disease-free controls, collected at four different time points, show that the ratio remains stable in the healthy controls but increases with the progression of liver disease. The results of measurement of S-HPX in serum of participants of the HALT-C trial show that it increased significantly (Kruskal-Wallis test, p < 0.01) in liver disease as the stage of fibrosis progressed in liver biopsies. We observed a 1.7-fold increase in fibrosis defined as Ishak score 3-4 (24.9 + 14.2, n = 22) and 4.7-fold increase in cirrhosis defined as Ishak score 5-6 (68.6 + 38.5; n = 24) compared to disease-free controls (14.7 + 6.7, n = 23). S-HPX is correlated with AFP, bilirubin, INR, ALT, and AST while inversely correlated with platelet count and albumin. In an independent verification set of samples, S-HPX separated the Ishak 5-6 (n = 15) from the Ishak 3-4 (n = 15) participants with AuROC 0.84; at the same time, the Ishak 3-4 group was separated from disease-free controls (n = 15) with AuROC 0.82. CONCLUSION: S-HPX, a measure of sialylated O-glycoforms of hemopexin, progressively increases in fibrotic and cirrhotic patient of HCV etiology and can be quantified by an LC-MS/MS-MRM assay in unfractionated serum of patients. Quantification of sialylated O-glycoforms of this liver secreted glycoprotein represents a novel measure of the stage of liver disease that could have a role in monitoring the progression of liver pathology.

Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease.

Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

Protein and site specificity of fucosylation in liver-secreted glycoproteins.

Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC-MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15-20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains.

Telmisartan ameliorates glutamate-induced neurotoxicity: roles of AT(1) receptor blockade and PPARγ activation.

Sartans (Angiotensin II AT(1) Receptor Blockers, ARBs) are powerful neuroprotective agents in vivo and protect against IL-1ß neurotoxicity in vitro. The purpose of this research was to determine the extent of sartan neuroprotection against glutamate excitotoxicity, a common cause of neuronal injury and apoptosis. The results show that sartans are neuroprotective, significantly reducing glutamate-induced neuronal injury and apoptosis in cultured rat primary cerebellar granule cells (CGCs). Telmisartan was the most potent sartan studied, with an order of potency telmisartan > candesartan > losartan > valsartan. Mechanisms involved reduction of pro-apoptotic caspase-3 activation, protection of the survival PI3K/Akt/GSK-3ß pathway and prevention of glutamate-induced ERK1/2 activation. NMDA receptor stimulation was essential for glutamate-induced cell injury and apoptosis. Participation of AT(1A) receptor was supported by glutamate-induced upregulation of AT(1A) gene expression and AT(1) receptor binding. Conversely, AT(1B) or AT(2) receptors played no role. Glutamate-induced neuronal injury and the neuroprotective effect of telmisartan were decreased, but not abolished, in CGCs obtained from AT(1A) knock-out mice. This indicates that although AT(1) receptors are necessary for glutamate to exert its full neurotoxic potential, part of the neuroprotective effect of telmisartan is independent of AT(1) receptor blockade. PPARγ activation was also involved in the neuroprotective effects of telmisartan, as telmisartan enhanced PPARγ nuclear translocation and the PPARγ antagonist GW9662 partially reversed the neuroprotective effects of telmisartan. The present results substantiate the therapeutic use of sartans, in particular telmisartan, in neurodegenerative diseases and traumatic brain disorders where glutamate neurotoxicity plays a significant role.

LC-MS3 quantification of O-glycopeptides in human serum.

Quantitative analysis of site-specific glycosylation of proteins is a challenging part of glycoproteomic research. Multiple enrichment steps are typically required in the analytical workflows to achieve adequate characterization of the site-specific glycoforms. In spite of recent advances, quantitative workflows need further development. Here, we report a selective and sensitive MS2 followed by further fragmentation in the linear IT-MS analyzer (MS3) multiple reaction monitoring workflow mass spectrometric method for direct analysis of O-glycopeptides in difficult matrix such as serum. Method optimization was performed using two serum glycoproteins, hemopexin (HPX) and sex hormone binding globulin. With the optimized MS3 workflow, we were able to analyze major glycoforms of HPX directly in human serum. Quantification of the minor glycoforms of HPX and glycoforms of sex hormone binding globulin required enrichment of the protein because these analytes were below the sensitivity of the 4000 quadrupole ion trap hybrid mass spectrometer in the complex serum background. In conclusion, we present a quantitative method for site-specific analysis of O-glycosylation with general applicability to mucin-type glycoproteins. Our results document reliable application of the optimized MS3 multiple reaction monitoring workflow to the relative quantification of O-glycosylation microheterogeneity of HPX in human serum. Introduction of isotopically labeled standards would be desirable to achieve absolute quantification of the analytes. The possibility to analyze serum samples directly represents a significant improvement of the quantitative glycopeptide workflows with the potential for use in clinical applications.

Six commercially available angiotensin II AT1 receptor antibodies are non-specific.

Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization.

Telmisartan directly ameliorates the neuronal inflammatory response to IL-1ß partly through the JNK/c-Jun and NADPH oxidase pathways.

BACKGROUND: Blockade of angiotensin II type 1 (AT1) receptors ameliorates brain inflammation, and reduces excessive brain interleukin-1 beta (IL-1ß) production and release from cortical microglia. The aim of this study was to determine whether, in addition, AT1 receptor blockade directly attenuates IL-1ß-induced inflammatory responses in neuronal cultures. METHODS: SK-N-SH human neuroblasts and primary rat cortical neurons were pretreated with telmisartan followed by exposure to IL-1ß. Gene expression was determined by reverse transcriptase (RT)-PCR, protein expression and kinase activation by western blotting, NADPH oxidase activity by the lucigenin method, prostaglandin E2 (PGE2) release by enzyme immunoassay, reactive oxygen species (ROS) generation by the dichlorodihydrofluorescein diacetate fluorescent probe assay, and peroxisome proliferator-activated receptor gamma (PPARγ) involvement was assessed with the antagonists GW9662 and T0070907, the agonist pioglitazone and the expression of PPARγ target genes ABCG1 and CD36. RESULTS: We found that SK-N-SH neuroblasts expressed AT1 but not AT2 receptor mRNA. Telmisartan reduced IL-1ß-induced cyclooxygenase-2 (COX-2) expression and PGE2 release more potently than did candesartan and losartan. Telmisartan reduced the IL-1ß-induced increase in IL-1R1 receptor and NADPH oxidase-4 (NOX-4) mRNA expression, NADPH oxidase activity, and ROS generation, and reduced hydrogen peroxide-induced COX-2 gene expression. Telmisartan did not modify IL-1ß-induced ERK1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation or nuclear factor-κB activation but significantly decreased IL-1ß-induced c-Jun N-terminal kinase (JNK) and c-Jun activation. The JNK inhibitor SP600125 decreased IL-1ß-induced PGE2 release with a potency similar to that of telmisartan. The PPARγ agonist pioglitazone reduced IL-1ß-induced inflammatory reaction, whereas telmisartan did not activate PPARγ, as shown by its failure to enhance the expression of the PPARγ target genes ABCG1 and CD36, and the inability of the PPARγ antagonists GW9662 and T0070907 to modify the effect of telmisartan on COX-2 induction. The effect of telmisartan on IL-1ß-stimulated COX-2 and IL-1R1 mRNA expression and ROS production was replicated in primary rat cortical neurons. CONCLUSIONS: Telmisartan directly ameliorates IL-1ß-induced neuronal inflammatory response by inhibition of oxidative stress and the JNK/c-Jun pathway. Our results support the hypothesis that AT1 receptor blockers are directly neuroprotective, and should be considered for the treatment of inflammatory conditions of the brain.