Acid Glycan Research Articles

Use of the serum glycan state to predict ovarian cancer patients' clinical response to chemotherapy treatment

Ovarian cancer is the most lethal gynecologic carcinoma; because the tumor often relapses shortly after treatment. Glycosylation plays important roles in cancer drug resistance and could be used as biomarkers to predict the drug response of patients.We used MALDI-QIT-TOF MS to analyze the serum glycomic from patients with different drug responses. Samples were collected before treatment; follow-up visit were performed after 6 months. Forty-eight drug-sensitive patients and 16 drug-resistant patients were enrolled.Compared with drug-sensitive patients, 5 glyco-subclasses and 5 single glycans were significantly altered in drug-resistant patients. Lewis type, α2,3 sialic acid and multibranch glycans were increased, α2,6 sialic acid glycans were decreased. The peak at m/z 2986.44 showed stronger prediction abilities than other single glycans, with an AUC of 0.83. A panel of three increased glycans (m/z 2401.36, H5N4F1S2, a Lewis type biantennary glycan; m/z 2986.44, H6N5S3, a triantennary trisialylated glycan; m/z 3086.39, H6N5F1S3, a Lewis type triantennary glycan) combined with CA125 achieved an AUC value of 0.88, showing a strong discrimination performance.This study provides new insights into N-glycosylation patterns in ovarian cancer patients with different drug response. These altered glycans might serve as biomarkers to reflect patients' drug sensitivity and to guide clinical treatment. SignificanceA large number of ovarian cancer patients experience tumor relapse shortly after initial treatment. Glycosylation plays important roles in cancer drug resistance and could be used as a biomarker to predict the drug response of patients. However, the glycosylation expressed in patients with different drug response have not been elucidated. In the present study, we used MALDI-QIT-TOF MS to analyze the serum glycomic levels of patients with different drug responses. Several glycans were changed significantly between these two groups. A panel of three increased glycans (m/z 2401.36, a Lewis type biantennary glycan, 2986.44, a triantennary trisialylated glycan, and 3086.39, a Lewis type triantennary glycan) combined with CA125 performed better descrimination of these two groups with AUC of 0.88. These altered glycans might serve as biomarkers to reflect patients' drug sensitivity and to guide clinical treatment.

The Cooperative Effect between Polybasic Region (PBR) and Polysialyltransferase Domain (PSTD) within Tumor-Target Polysialyltranseferase ST8Sia II.

ST8Sia II (STX) is a highly homologous mammalian polysialyltransferase (polyST), which is a validated tumor-target in the treatment of cancer metastasis reliant on tumor cell polysialylation. PolyST catalyzes the synthesis of α2,8-polysialic acid (polySia) glycans by carrying out the activated CMP-Neu5Ac (Sia) to N- and O-linked oligosaccharide chains on acceptor glycoproteins. In this review article, we summarized the recent studies about intrinsic correlation of two polybasic domains, Polysialyltransferase domain (PSTD) and Polybasic region (PBR) within ST8Sia II molecule, and suggested that the critical amino acid residues within the PSTD and PBR motifs of ST8Sia II for polysialylation of Neural cell adhesion molecules (NCAM) are related to ST8Sia II activity. In addition, the conformational changes of the PSTD domain due to point mutations in the PBR or PSTD domain verified an intramolecular interaction between the PBR and the PSTD. These findings have been incorporated into Zhou's NCAM polysialylation/cell migration model, which will provide new perspectives on drug research and development related to the tumor-target ST8Sia II.

Toxic effects of arsenic trioxide on spermatogonia are associated with oxidative stress, mitochondrial dysfunction, autophagy and metabolomic alterations.

Arsenic is a toxic metalloid that can cause male reproductive malfunctions and is widely distributed in the environment. The aim of this study was to investigate the cytotoxicity of arsenic trioxide (ATO) induced GC-1 spermatogonial (spg) cells. Our results found that ATO increased the levels of catalase (CAT) and malonaldehyde (MDA) and reactive oxygen species (ROS), while decreasing glutathione (GSH) and the total antioxidant capacity (T-AOC). Therefore, ATO triggered oxidative stress in GC-1 spg cells. In addition, ATO also caused severe mitochondrial dysfunction that included an increase in residual oxygen consumption (ROX), and decreased the routine respiration, maximal and ATP-linked respiration (ATP-L-R), as well as spare respiratory capacity (SRC), and respiratory control rate (RCR); ATO also damaged the mitochondrial structure, including mitochondrial cristae disordered and dissolved, mitochondrial vacuolar degeneration. Moreover, degradation of p62, LC3 conversion, increasing the number of acidic vesicle organelles (AVOs) and autophagosomes and autolysosomes are demonstrated that the cytotoxicity of ATO may be associated with autophagy. Meanwhile, the metabolomics analysis results showed that 20 metabolites (10 increased and 10 decreased) were significantly altered with the ATO exposure, suggesting that maybe there are the perturbations in amino acid metabolism, lipid metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins. We concluded that ATO was toxic to GC-1 spg cells via inducing oxidative stress, mitochondrial dysfunction and autophagy as well as the disruption of normal metabolism. This study will aid our understanding of the mechanisms behind ATO-induced spermatogenic toxicity.

Low Molecular Seleno-Aminopolysaccharides Protect the Intestinal Mucosal Barrier of Rats under Weaning Stress.

Low molecular seleno-aminopolysaccharide (LSA) was synthesized with sodium selenite and low molecular aminopolysaccharide (LA), which is an organic selenium compound. This study is aimed to investigate the protective effect of LSA on the intestinal mucosal barrier in weaning stress rats by detecting the intestinal tissue morphology and function, mucosal thickness and permeability, the structure of MUC2, antioxidant index, the expression level of intracellular transcription factor NF-E2-related factor 2 (Nrf2), and its related factors. The results showed that LSA significantly increased the height of intestinal villi (p < 0.05) and increased the thickness of intestinal mucosa and the number of goblet cells, which indicated that LSA has a protective effect on the intestinal mucosal barrier that is damaged by weaning. Moreover, LSA significantly reduced the level of DAO, D-LA, and LPS compared with the weaning group (p < 0.05), which indicated that LSA reduced the intestinal damage and permeability of weaning rats. In addition, LSA could increase the number and length of glycans chains and the abundance of acid glycans structures in the MUC2 structure, which indicated that LSA alleviated the changes of intestinal mucus protein structure. LSA significantly increased the levels of GSH-Px, SOD, LDH, and CAT, while it decreased the level of MDA in serum and intestinal tissue, which suggested that LSA significantly enhanced the antioxidant capacity and reduced oxidative stress of weaning rats. RT-PCR results showed that LSA significantly increased the expression level of antioxidant genes (GSH-Px, SOD, Nrf2, HO-1), glycosyltransferase genes (GalNT1, GalNT3, GalNT7) and mucin gene (MUC2) in intestinal mucosa (p < 0.05). The results of western blot showed that the LSA activated the Nrf2 signaling pathway by down-regulating the expression of Keap1and up-regulating the expression of Nrf2, and protected the intestinal mucosa from oxidative stress. Overall, LSA could play a protective role in intestinal mucosal barrier of weaning rats by activating the Nrf2 pathway and alleviating the alnormal change of mucin MUC2.

Characterizing the bacterial community across the gastrointestinal tract of goats: Composition and potential function

The composition and function of the microbial community in the gastrointestinal tract (GIT) have increasingly captured the attention of nutritionists because these traits affect the nutrient utilization efficiency and health of host animals. Little information has been reported on these aspects of the goat GIT. This study used 12 female goats (weighing 20.70 ± 1.60 kg and 10 months of age) to examine the composition and function of the microbiota in the rumen, abomasum, jejunum, cecum, and colon. Total genomic DNA was extracted from chyme samples from different sections of the GIT, and the hypervariable region of the 16S rRNA gene was amplified by PCR using bacterial universal primers. The amplicons were sequenced on an Illumina MiSeq platform, and the biological information was analyzed using QIIME software. A total of 857 genera that belonged to 39 phyla were observed across the goat GIT, with Bacteroidetes and Firmicutes dominating. Our results revealed significant differences in the composition, diversity, and species abundance of the bacterial communities in the different sections of the GIT. However, the compositions of the bacterial communities in adjacent GIT segments showed similarities in addition to differences. The study indicated that there were significant differences in microbial function among the GIT regions. In particular, the relative abundances of genes involved in energy metabolism, amino acid metabolism, nucleotide metabolism, and glycan metabolism were overrepresented in samples from the forestomach, and genes related to energy metabolism, amino acid metabolism, and glycan metabolism were mainly enriched in samples from the small intestine. Additionally, the relative abundances of bacteria at the phylum and genus levels were significantly correlated with these metabolic functions. In general, there were significant differences in composition and potential function among the bacterial communities in the goat GIT.

Structural basis for the specificity of renin-mediated angiotensinogen cleavage

The renin–angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å), and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N terminus into a pocket equivalent to a hormone-binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin's active site. Insertion of the N terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin's active-site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT's hormone binding pocket.

Effect of cropping systems after abandoned salinized farmland reclamation on soil bacterial communities in arid northwest China

Cropping systems are extensively used in northwest China, which alter the soil structure and nutrients, causing the changes in the soil bacterial community. Although soil bacterial communities respond differently to altered cropping systems, little is known about possible effect of cropping systems on soil bacterial communities in reclaimed soils. Using pyrosequencing-based analysis of the 16S rRNA gene, we investigated the effect of 5-year cropping systems on bacterial communities in reclaimed soils in northwest China for the first time. The three cropping systems are: maize-wheat rotation (MW), tree intercrop with cotton (TC), and monoculture cotton (MC). We have observed soil properties are obviously changed by cropping systems. The lowest soil electrical conductivity (EC), the highest aggregate stability indices (MWD and GMD) and soil organic matter (SOM) content are found in MW (P < 0.05). The dominant phyla are Firmicutes, Proteobacteria, Chloroflexi and Actinobacteria and these are present across all samples from all treatments. The highest bacterial diversity is observed in MW. The relative abundance of dominant phyla are significantly correlated EC, SOM, Fe, MWD and GMD (P < 0.05), suggesting that EC, SOM, Fe, MWD and GMD are the most important factors in shaping soil bacterial communities. Functional predictions indicate that the abundance of amino acid metabolism, carbon metabolism, glycan biosynthesis, methane metabolism, two-component and fatty acid metabolism are all enriched in MW. Overall, our study reveal that different cropping systems have different impacts not only on bacterial composition and diversity, but also on the main metabolic functions. These findings guide our future work on soil bacterial metabolic functions in different cropping systems and will provide references for salinized farmland reclamation in arid areas.

Increased \u03b12-6 sialylation of endometrial cells contributes to the development of endometriosis

Endometriosis is a disease characterized by implants of endometrial tissue outside the uterine cavity and is strongly associated with infertility. Focal adhesion of endometrial tissue to the peritoneum is an indication of incipient endometriosis. In this study, we examined the effect of various cytokines that are known to be involved in the pathology of endometriosis on endometrial cell adhesion. Among the investigated cytokines, transforming growth factor-β1 (TGF-β1) increased adhesion of endometrial cells to the mesothelium through induction of α2-6 sialylation. The expression levels of β-galactoside α2-6 sialyltransferase (ST6Gal) 1 and ST6Gal2 were increased through activation of TGF-βRI/SMAD2/3 signaling in endometrial cells. In addition, we discovered that terminal sialic acid glycan epitopes of endometrial cells engage with sialic acid-binding immunoglobulin-like lectin-9 expressed on mesothelial cell surfaces. Interestingly, in an in vivo mouse endometriosis model, inhibition of endogenous sialic acid binding by a NeuAcα2-6Galβ1-4GlcNAc injection diminished TGF-β1-induced formation of endometriosis lesions. Based on these results, we suggest that increased sialylation of endometrial cells by TGF-β1 promotes the attachment of endometrium to the peritoneum, encouraging endometriosis outbreaks.

Glycopeptides as a Tool for Early Detection of Cancer.

Protein glycosylation, as one of the most common and complex posttranslational modifications, plays an important role in many biological processes. Along with the intensive progress in MS techniques and development of glycan search tools and databases, glycoproteomics has become a popular subject of studies. The possibility of simultaneous identification of amino acid sequence, glycosylation sites, and glycan composition enabled the monitoring of changes in glycosylation patterns in various pathological states. In this issue, Saraswat etal. describe MS-based investigations of glycopeptide changes in oral cavity squamous cell carcinoma (OSCC). Their findings indicate glycopeptides with changed expression levels and the presence of altered glycan forms observed in four proteins derived from OSCC patients' sera. Proteins carrying this distinctive pattern are in the group of the most abundant components of serum, IgG1, IgG4, HPT, and TRFE, which makes their identification more accessible. Described changes, characteristic for cancer serum samples, may be considered as potential diagnostic indicators of OSCC; however, there is still a need to establish a universal glycopeptide-based biomarkers database, where all glycoproteomic data can be collected from all types of cancer studies and evaluated using meta-analyses. Only then, early diagnosis of patients using MS-based approach will make sense, as those investigations are very convoluted, and all efforts made during sample preparation and analysis will pay off when comprehensive anticancer prevention will be achieved during single measurement.

The HMW2 adhesin of non-typeable Haemophilus influenzae is a human-adapted lectin that mediates high-affinity binding to 2–6 linked N-acetylneuraminic acid glycans

Non-typeable Haemophilus influenzae (NTHi) is a human-adapted bacterial pathogen, responsible for infections of the human respiratory tract. This pathogen expresses a range of adhesins that mediate binding to host cells. Most NTHi strains can express the related adhesins HMW1 and HMW2. Expression of HMW proteins is phase-variable: changes in the length of simple-sequence repeats located in the encoding genes promoter regions results in changes in expression levels of these adhesins. HMW expression is also controlled by epigenetic regulation. HMW1 has been previously demonstrated to bind α 2–3 sialyl-lactosamine, but affinity of this interaction has not been investigated. The host receptor(s) for HMW2 is currently unknown. We hypothesized that host glycans may act as receptors for HMW2-mediated adherence. We examined the glycan-binding activity of HMW2 using glycan arrays and Surface Plasmon Resonance (SPR). These studies demonstrate that HMW2 binds 2–6 linked N-acetylneuraminic acid with high affinity. HMW2 did not bind glycan structures containing the non-human form of sialic acid, N-glycolylneuraminic acid. Thus, the specificity of HMW1 and HMW2 have complementary lectin activities that may allow NTHi distinct niches in the human host.

New Functions and Applications of an Acidic Glycan, Polysialic Acid: Repulsive and Attractive Fields of Polysialic Acid

New Functions and Applications of an Acidic Glycan, Polysialic Acid: Repulsive and Attractive Fields of Polysialic Acid

Glycoengineering HIV-1 Env creates 'supercharged' and 'hybrid' glycans to increase neutralizing antibody potency, breadth and saturation.

The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make some glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE—thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design.

Gut microbiome of Moroccan colorectal cancer patients

Although colorectal cancer is the third leading cause of death in Morocco, there are no studies of the microbiome changes associated with the disease in the Moroccan population. The aim of our study was to compare the stool microbiome of Moroccan cancer patients with healthy individuals. We analyzed the microbiome composition of samples from 11 CRC patients and 12 healthy individuals by 16S rRNA amplicon sequencing. Principal coordinate analysis of samples revealed defined cancer versus healthy clusters. Our findings showed that cancer samples had higher proportions of Firmicutes (T = 50.5%; N = 28.4%; p = 0.04), specifically of Clostridia (T = 48.3%; N = 19.0%; p = 0.002), and Fusobacteria (T = 0.1%; N = 0.0%; p = 0.02), especially of Fusobacteriia (T = 0.1%; N = 0.0%; p = 0.02), while Bacteroidetes were enriched in healthy samples (T = 35.1%; N = 62.8%; p = 0.06), particularly the class Bacteroidia (T = 35.1%; N = 62.6%; p = 0.06). Porphyromonas, Clostridium, Ruminococcus, Selenomonas, and Fusobacterium were significantly overrepresented in diseased patients, similarly to other studies. Predicted functional information showed that bacterial motility proteins, flagellar assembly, and fatty acid biosynthesis metabolism were significantly overrepresented in cancer patients, while amino acid metabolism and glycan biosynthesis were overrepresented in controls. This suggests that involvement of these functional metagenomes is similar and relevant in the carcinogenesis process, independent of the origin of the samples. Results from this study allowed identification of bacterial taxa relevant to the Moroccan population and encourages larger studies to facilitate population-directed therapeutic approaches.

PD56-07 COMPARISON WITH DIAGNOSTIC PERFORMANCE BETWEEN ABERRANT GLYCOSYLATED S2,3PSA TEST AND CONVENTIONAL PSA TESTS

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 2018PD56-07 COMPARISON WITH DIAGNOSTIC PERFORMANCE BETWEEN ABERRANT GLYCOSYLATED S2,3PSA TEST AND CONVENTIONAL PSA TESTS Tohru Yoneyama, Tomokazu Ishikawa, Yuki Tobisawa, Shingo Hatakeyama, Mutsuhiro Date, Kenji Nakamura, Shintaro Narita, Koji Mitsuzuka, Wilhelmina Duivenvoorden, Jehonathan H. Pinthus, Yasuhiro Hashimoto, Takuya Koie, Tomonori Habuchi, Yoichi Arai, and Chikara Ohyama Tohru YoneyamaTohru Yoneyama More articles by this author , Tomokazu IshikawaTomokazu Ishikawa More articles by this author , Yuki TobisawaYuki Tobisawa More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Mutsuhiro DateMutsuhiro Date More articles by this author , Kenji NakamuraKenji Nakamura More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Koji MitsuzukaKoji Mitsuzuka More articles by this author , Wilhelmina DuivenvoordenWilhelmina Duivenvoorden More articles by this author , Jehonathan H. PinthusJehonathan H. Pinthus More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto More articles by this author , Takuya KoieTakuya Koie More articles by this author , Tomonori HabuchiTomonori Habuchi More articles by this author , Yoichi AraiYoichi Arai More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2639AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The overdiagnosis and overtreatment of prostate cancer (PCa) is a major issue worldwide. Maackia amurensis lectin (MAA) preferably binds to a2,3linked sialic acid glycans (S2,3-glycan), and its S2,3-glycan carrying prostate-specific antigen (S2,3PSA) is significantly increase in PCa. The aim of this study to compare the diagnostic performance between serum %S2,3PSA measured by automated micro-total immunoassay systems (μTAS system) and conventional PSA test. We also examined stability of serum S2,3PSA under various storage condition. METHODS Between 2008 and 2018, serum %S2,3PSA levels before prostate biopsy (Pbx) were measured in 175 biopsy-proven benign prostatic hyperplasia patients and 90 PCa patients using μTAS based automated immunoassay. Diagnostic performance of serum S2,3PSA test was evaluated using area under the receiver-operator characteristic curve (AUC). RESULTS The AUC of serum %S2,3PSA (0.7923-0.8090) detecting PCa was much higher than those of PSA (0.5041-0.5481) and F/T ratio (0.7537-0.7321) in any PSA range and the specificity of %S2,3PSA (40.3-42.2%) at 90% sensitivity was much superior to total PSA (17.7-12.2%) and F/T ratio (35.5-36.7%) in any PSA range. The number patients with %S2,3PSA greater than 48% was significantly increased in patients with higher Gleason (more than 4+3) tumor. CONCLUSIONS Although the present results are preliminary, the newly developed μTAS platform for measuring %S2,3PSA can achieve the required assay performance specifications for use in the practical and clinical setting and may improve the accuracy of PCa diagnosis. Additional validation studies are warranted. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1064 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Tohru Yoneyama More articles by this author Tomokazu Ishikawa More articles by this author Yuki Tobisawa More articles by this author Shingo Hatakeyama More articles by this author Mutsuhiro Date More articles by this author Kenji Nakamura More articles by this author Shintaro Narita More articles by this author Koji Mitsuzuka More articles by this author Wilhelmina Duivenvoorden More articles by this author Jehonathan H. Pinthus More articles by this author Yasuhiro Hashimoto More articles by this author Takuya Koie More articles by this author Tomonori Habuchi More articles by this author Yoichi Arai More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Using Chemical Synthesis To Study and Apply Protein Glycosylation.

Protein glycosylation is one of the most common post-translational modifications and can influence many properties of proteins. Abnormal protein glycosylation can lead to protein malfunction and serious disease. While appreciation of glycosylation's importance is growing in the scientific community, especially in recent years, a lack of homogeneous glycoproteins with well-defined glycan structures has made it difficult to understand the correlation between the structure of glycoproteins and their properties at a quantitative level. This has been a significant limitation on rational applications of glycosylation and on optimizing glycoprotein properties. Through the extraordinary efforts of chemists, it is now feasible to use chemical synthesis to produce collections of homogeneous glycoforms with systematic variations in amino acid sequence, glycosidic linkage, anomeric configuration, and glycan structure. Such a technical advance has greatly facilitated the study and application of protein glycosylation. This Perspective highlights some representative work in this research area, with the goal of inspiring and encouraging more scientists to pursue the glycosciences.

Abstract 1557: Longitudinal expression response of glycosylation related genes, regulators, and targets in cancer cell lines treated with eleven anti-tumor agents

Abstract Cancer cells have profound alterations in glycosylation processes, which affects the functioning of malignant cells and their interaction with surrounding environment. We examined the effects of treatment of cancer cell lines with eleven antitumor agents (bortezomib, cisplatin, dasatinib, doxorubicin, erlotinib, gemcitabine, paclitaxel, sirolimus, sorafenib, sunitinib, and topotecan) on transcriptional changes in 179 candidate genes involved in biochemical glycosylation pathways or encoding glycosylation targets, regulators of glycosylation processes, and components of cancer pathways affected by glycosylation. In order to achieve this goal, we analyzed time course gene expression information for the NCI-60 cancer cell line panel using the data from the NCI Transcriptional Pharmacodynamic Workbench (NCI TPW), a resource developed at the National Cancer Institute. Our analysis identified 145 glycosylation related genes that responded to drug treatment in a concerted manner. Many of these genes had a concerted response to multiple agents; however, the direction and the magnitude of concerted response were specific to individual drugs. Expression of multiple genes prior to treatment and/or transcriptional changes in response to treatment were significantly correlated with drug chemosensitivity, measured as log(GI50). Treatment with dasatinib resulted in the largest number of significant correlations of chemosensitivity with gene expression prior to treatment and with gene expression changes in response to treatment. Significant correlations of transcriptional changes after treatment and/or of pre-treatment gene expression levels of glycosylation related genes and glycosylation targets were also observed for other antitumor agents. Chemosensitivity to kinase inhibitors and DNA damaging agents was significantly correlated with expression of genes involved in N- and O-glycosylation, fucosylation, biosynthesis of poly-N-acetyllactosamine, removal of misfolded proteins, binding to hyaluronic acid and other glycans, and cell adhesion. In particular, tumor cell sensitivity or resistance to multiple agents were significantly correlated with changes in expression of C1GALT1C1 (COSMC), FUCA1, SDC1, MUC1, members of the MGAT, GALNT, B4GALT, B3GNT, MAN, and EDEM gene families, and other genes involved in glycosylation processes or encoding glycosylation targets and signaling regulators. We present a catalog of dynamic concerted changes in expression of glycosylation related genes, their ligands, regulators, and targets in response to chemotherapy, and provide a list of genes that influence the strength of response of cancer cells to treatment. These glycosylation related genes may be considered as potential candidates for drug targeting in combination therapy to enhance the effects of treatment. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Julia Krushkal, Yingdong Zhao, Curtis Hose, Anne Monks, James H. Doroshow, Richard Simon. Longitudinal expression response of glycosylation related genes, regulators, and targets in cancer cell lines treated with eleven anti-tumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1557. doi:10.1158/1538-7445.AM2017-1557

ADAMTS‐13 glycans and conformation‐dependent activity

Background ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.

Identification of the Consistently Altered Metabolic Targets in Human Hepatocellular Carcinoma.

Background & AimsCancer cells rely on metabolic alterations to enhance proliferation and survival. Metabolic gene alterations that repeatedly occur in liver cancer are largely unknown. We aimed to identify metabolic genes that are consistently deregulated, and are of potential clinical significance in human hepatocellular carcinoma (HCC).MethodsWe studied the expression of 2,761 metabolic genes in 8 microarray datasets comprising 521 human HCC tissues. Genes exclusively up-regulated or down-regulated in 6 or more datasets were defined as consistently deregulated. The consistent genes that correlated with tumor progression markers (ECM2 and MMP9) (Pearson correlation P < .05) were used for Kaplan-Meier overall survival analysis in a patient cohort. We further compared proteomic expression of metabolic genes in 19 tumors vs adjacent normal liver tissues.ResultsWe identified 634 consistent metabolic genes, ∼60% of which are not yet described in HCC. The down-regulated genes (n = 350) are mostly involved in physiologic hepatocyte metabolic functions (eg, xenobiotic, fatty acid, and amino acid metabolism). In contrast, among consistently up-regulated metabolic genes (n = 284) are those involved in glycolysis, pentose phosphate pathway, nucleotide biosynthesis, tricarboxylic acid cycle, oxidative phosphorylation, proton transport, membrane lipid, and glycan metabolism. Several metabolic genes (n = 434) correlated with progression markers, and of these, 201 predicted overall survival outcome in the patient cohort analyzed. Over 90% of the metabolic targets significantly altered at the protein level were similarly up- or down-regulated as in genomic profile.ConclusionsWe provide the first exposition of the consistently altered metabolic genes in HCC and show that these genes are potentially relevant targets for onward studies in preclinical and clinical contexts.

Physiological and proteomic response of Limonium bicolor to salinity

Medicinal halophyte Limonium bicolor (Bag.) Kuntze was treated with 200 mM NaCl, the physiological parameters including fresh weight, dry weight, salt gland number, MDA levels and root activity were investigated, comparative proteomic analyses of leaf and root were carried out using 2D-PAGE combined with MALDI-TOF/TOF-MS. The results showed that L. bicolor performed a positive effect on its growth under NaCl stress since increase in different extent of fr wt, dry wt, salt gland number, MDA level and SOD activity of leaves and roots, as well as root activity. More than 460 protein spots in leaves and 600 protein spots in roots were visualized and matched in 2D gels, the results of proteomic analyses showed that 45 proteins were identified in leaves and 49 proteins in roots to be differentially expressed. Based on their functions, these proteins were grouped into categories covering a wide range of molecular processes, including carbohydrate, energy, lipid, nucleotide and amino acid metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, biosynthesis of secondary metabolites, redox homeostasis, transcription, cytoskeleton and transport, as well as folding, sorting and degradation. The upregulating and downregulating of differentially- expressed proteins with different physiological function were also reflected in the physiological parameters. These results are helpful for further research work on salt response mechanisms and medicinal utilization of L. bicolor.

Longitudinal Transcriptional Response of Glycosylation-Related Genes, Regulators, and Targets in Cancer Cell Lines Treated With 11 Antitumor Agents.

Cellular glycosylation processes are vital to cell functioning. In malignant cells, they are profoundly altered. We used time-course gene expression data from the NCI-60 cancer cell lines treated with 11 antitumor agents to analyze expression changes of genes involved in glycosylation pathways, genes encoding glycosylation targets or regulators, and members of cancer pathways affected by glycosylation. We also identified glycosylation genes for which pretreatment expression levels or changes after treatment were correlated with drug sensitivity. Their products are involved in N-glycosylation and O-glycosylation, fucosylation, biosynthesis of poly-N-acetyllactosamine, removal of misfolded proteins, binding to hyaluronic acid and other glycans, and cell adhesion. Tumor cell sensitivity to multiple agents was correlated with transcriptional response of C1GALT1C1, FUCA1, SDC1, MUC1; members of the MGAT, GALNT, B4GALT, B3GNT, MAN, and EDEM families; and other genes. These genes may be considered as potential candidates for drug targeting in combination therapy to enhance treatment response.