Oxygen-dependent regulation of F-box proteins in Toxoplasma gondii is mediated by Skp1 glycosylation.

A dynamic proteome is required for cellular adaption to changing environments including levels of O2, and the SKP1/CULLIN-1/F-box protein/RBX1 (SCF) family of E3 ubiquitin ligases contributes importantly to proteasome-mediated degradation. We examine, in the apicomplexan parasite Toxoplasma gondii, the influence on the interactome of SKP1 by its novel glycan attached to a hydroxyproline generated by PHYa, the likely ortholog of the HIFα PHD2 oxygen-sensor of human host cells. Strikingly, the representation of several putative F-box proteins (FBPs) is substantially reduced in PHYaΔ parasites grown in fibroblasts. One, FBXO13, is a predicted lysyl hydroxylase related to the human JmjD6 oncogene except for its F-box domain. The abundance of FBXO13, epitope-tagged at its genetic locus, was reduced in PHYaΔ parasites thus explaining its diminished presence in the SKP1 interactome. A similar effect was observed for FBXO14, a cytoplasmic protein of unknown function that may have co-evolved with PHYa in apicomplexans. Similar findings in glycosylation-mutant cells, rescue by proteasomal inhibitors, and unchanged transcript levels, suggested the involvement of the SCF in their degradation. The effect was selective, because FBXO1 was not affected by loss of PHYa. These findings are physiologically significant because the effects were phenocopied in parasites reared at 0.5% O2. Modest impact on steady-state SKP1 modification levels suggests that effects are mediated during a lag phase in hydroxylation of nascent SKP1. The dependence of FBP abundance on O2-dependent SKP1 modification likely contributes to the reduced virulence of PHYaΔ parasites owing to impaired ability to sense O2 as an environmental signal.

Proteoglycan 4 (lubricin) is a highly sialylated glycoprotein associated with cardiac valve damage in animal models of infective endocarditis.

Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of tooth surfaces and are generally associated with oral health, but can also cause infective endocarditis (IE). These species express "Siglec-like" adhesins that bind sialylated glycans on host glycoproteins, which can aid the formation of infected platelet-fibrin thrombi (vegetations) on cardiac valve surfaces. We previously determined that the ability of S. gordonii to bind sialyl T-antigen (sTa) increased pathogenicity, relative to recognition of sialylated core 2 O-glycan structures, in an animal model of IE. However, it is unclear when and where the sTa structure is displayed, and which sTa-modified host factors promote valve colonization. In this study, we identified sialylated glycoproteins in the aortic valve vegetations and plasma of rat and rabbit models of this disease. Glycoproteins that display sTa vs. core 2 O-glycan structures were identified by using recombinant forms of the streptococcal Siglec-like adhesins for lectin blotting and affinity capture, and the O-linked glycans were profiled by mass spectrometry. Proteoglycan 4 (PRG4), also known as lubricin, was a major carrier of sTa in the infected vegetations. Moreover, plasma PRG4 levels were significantly higher in animals with damaged or infected valves, as compared with healthy animals. The combined results demonstrate that, in addition to platelet GPIbα, PRG4 is a highly sialylated mucin-like glycoprotein found in aortic valve vegetations and may contribute to the persistence of oral streptococci in this protected endovascular niche. Moreover, plasma PRG4 could serve as a biomarker for endocardial injury and infection.

O-glycosylated clusterin as a sensitive marker for diagnosing early stages of prostate cancer.

BACKGROUND: Prostate-specific antigen (PSA) has been the most popular diagnostic marker for prostate cancer. The frequent occurrence of low PSA values (<10 ng/ml) in patients with highly suspicious prostate cancer, however, has undermined the accuracy of clinical examinations. The aim of this study was to develop a better resolution for diagnosing prostate cancer to overcome the disadvantage of PSA. METHODS: We focused on the glycosylation status of patients' serum proteins and conducted comprehensive lectin microarray analyses to characterize N- and O-glycans using sera from prostate cancer and benign prostatic diseases. Next, we retrieved candidate serum proteins with characteristic glycan structures using lectin-immobilized beads and identified them by quantitative mass spectrometry using a technique referred to as isobaric tag for relative and absolute quantitation (iTRAQ) labeling. Finally, we constructed a new assay to quantify a candidate glycoprotein with the newly identified glycans. RESULTS: Lectin microarray analyses revealed that sera from patients with prostate cancer had a higher affinity for Jacalin, Amaranthus caudatus (ACA) lectin, and Maclura pomifera (MPA) lectin, compared with that from patients with benign prostatic diseases and normal subjects, suggesting that O-glycosylated proteins are more abundant in sera from patients with prostate cancer. Then, serum glycoproteins preferentially adsorbed onto Jacalin-Agarose as well as biotin-ACA/and biotin-MPA/streptavidin-immobilized magnetic beads were isolated, labeled with iTRAQ, and identified using quantitative mass spectrometry. It was found that the ACA- and MPA-recognizable clusterin was more enriched in patients' sera from prostate cancer compared with those from benign prostatic diseases. Following this discovery, we constructed a Luminex-based assay to quantify O-glycosylated clusterin, in which total serum clusterin was first captured on anti-clusterin antibody-immobilized beads, and then clusterin-associated O-glycans were determined by the pair of biotin-MPA and streptavidin-phycoerythrin. When PSA values registered less than 10 ng/ml, the corresponding serum level of MPA-recognized clusterin determined by this assay was beneficial for distinguishing the patients with prostate cancer from the patients with benign prostatic disease. CONCLUSION: For PSA values that measure less than 10 ng/ml, the serum O-glycosylated clusterin level can be a complementary indicator for the malignancy of prostate cancer.

C-terminal peptide (hCTP) of human chorionic gonadotropin enhances in vivo biological activity of recombinant Japanese eel follicle-stimulating hormone and luteinizing hormone produced in FreeStyle 293-F cell lines.

Gonadotropins (Gths), follicle-stimulating hormone (Fsh), and luteinizing hormone (Lh) play central roles in the reproductive biology of vertebrates. In this study, recombinant single-chain Japanese eel Gths (rGth: rFsh and rLh), and recombinant chimeric Gths (rGth-hCTPs: rFsh-hCTP and rLh-hCTP; rGth-eCTPs: rFsh-eCTP and rLh-eCTP) with an extra O-glycosylation site (either a C-terminal peptide of human (hCTP) or equine (eCTP) chorionic gonadotropin), which are known to prolong the half-life of glycoprotein were produced in HEK293 cells and highly purified. Lectin blot analyses demonstrated that all these recombinant Gths contained N-glycans of the high mannose and complex types. In contrast, only rGth-hCTPs and rGth-eCTPs possessed highly sialylated O-linked oligosaccharides. Further analyses of glycans by liquid chromatography-mass spectrometry suggested that the species, amount, and degree of sialylation of N-glycans were comparable among recombinant Fshs and recombinant Lhs, while the amount of O-glycans with sialic acids in rGth-hCTPs was higher than that in the corresponding rGth-eCTPs. The serum levels of recombinant Gths in male eels significantly increased 12-24 h after a single injection of the Gths. The levels of rGth-hCTPs tended to be higher than those of the corresponding rGths and rGth-eCTPs throughout the experimental period, coinciding with the serum fluctuations of 11-ketotestosterone (11KT). The long-term treatment of male eels with these recombinant Gths also revealed the superiority of rGth-hCTPs in assisted reproduction; thus, the serum levels of 11KT and gonadosomatic indices in eels treated with rGth-hCTPs were higher than those in eels treated with the corresponding rGths and rGth-eCTPs. The induction of the entire process of spermatogenesis was only histologically observed in rGth-hCTPs-treated eels. These findings strongly suggest that hCTP enhances the in vivo biological activity of recombinant Japanese eel Gths due to the high abundance of O-linked glycans with sialylated antennae.

A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase.

O-Linked glycan liberation from proteins through reductive beta-elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan analysis in glycan biomarker research and microheterogeneity evaluation of biopharmaceutical glycosylation. Here, we introduce an alternative method by using hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and optimize the reaction conditions. The developed method afforded comparable results to those of hydrazinolysis, but with less degraded products. In addition, we examined the compatibility of the optimized O-linked glycan liberation with denaturant and detergents. The optimized method also released glycans containing NeuGc without degradation or deacylation. To demonstrate the feasibility of the developed method, we analyzed O-linked glycans of porcine submaxillary mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. The method for O-linked glycan liberation and fluorescent labeling presented here was easy and rapid, and will be practically useful for O-linked glycan analyses.

Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins.

The principal aim of this study was to demonstrate the optimization and fine-tuning of quantitative and nonselective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans were examined: ammonia-based ß-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity was observed between the ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins was approximately 20 to 30 times more efficient with hydrazine compared with ammonia-based ß-elimination reagent. In addition, the ammonia-based reagent demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure was developed for characterization of O-glycans. The method performance parameters were evaluated. It was shown that this procedure is superior for quantitative nonselective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from hydrophilic interaction chromatography (HILIC) fractions was also demonstrated using linear ion trap Fourier transform mass spectrometry (LTQ FT MS) with mass accuracy below 1ppm.

Eliminative Oximation of O-Glycans from Mucins.

The large variety and high concentration of O-glycans are characteristic properties of mucins and play a crucial role in their unique functions. Analyzing the O-glycans of mucins is essential for investigating the functions of mucins. Eliminative oximation is an aqueous reaction that can be used to obtain O-glycan oximes from mucins. Using diazabicyclo undec-7ene (DBU) as a base, an organic superbase that can be removed with an organic solvent during solid-phase extraction, and adding hydroxylamine to the reaction mixture in advance, the O-glycans released from the mucin are immediately converted to the corresponding glycan oximes. The glycan oxime can then be fluorescently labeled with a fluorescent labeling reagent and 2-picoline borane via reductive amination. O-glycans that have been fluorescently labeled can be analyzed using conventional HPLC techniques.

Preparation of Soluble Mucin Solutions from the Salivary Glands.

Studying salivary gland mucins is important for elucidating the pathogenesis of salivary gland diseases, including tumors and xerostomia, and developing diagnostic methods for them. Classic methods for isolating mucins from salivary glands require sacrificing several animals to obtain sufficient quantities of mucin and are time-consuming. Supported molecular matrix electrophoresis (SMME) was used to characterize mucins and their glycans. With this method, mucins can be analyzed within 2 days using less than 100 mg of tissue and without using expensive equipment, such as an ultracentrifuge. This chapter describes a method for preparing mucin solutions for SMME analysis of salivary gland mucins.

Comprehensive Cellular Glycan Profiling of Glycoproteins and Glycosphingolipids by Glycoblotting and BEP Methods.

The glycocalyx is a layer of glycans that covers the surface of every cell. Glycans are covalently attached to proteins and lipids, and are classified into subclasses such as N-linked glycans, glycosaminoglycans, glycosphingolipid-glycans, free oligosaccharides, and O-linked glycans according to their biosynthetic pathways. These complex glycans affect various biological and pathological processes, such as cell growth, differentiation, and adhesion. During infection, bacteria and viruses often use glycans to recognize and attack host cells. In this chapter, we describe detailed protocols to prepare glycans, and perform comprehensive cellular glycomic analysis using glycoblotting and ß-elimination with pyrazolone methods.

Alteration of mucins in the submandibular gland during aging in mice.

OBJECTIVE: Mucins are large glycosylated glycoproteins that are produced in the salivary glands, and their changes may contribute to the development of xerostomia due to aging and the accompanying deterioration of oral hygiene. This study aimed to characterize the changes in the mucins produced in submandibular gland (SMG) during the aging process. METHODS: SMG mucins derived from mice of each age were separated using supported molecular matrix electrophoresis (SMME). Subsequently, the membranes were stained with Alcian blue (AB) or blotted with MAL-II lectin. The SMME membranes stained with AB were subjected to densitometric analysis and glycan analysis. The detailed structures of O-glycan were investigated by tandem mass spectrometry (MS/MS). RESULTS: The SMG of mice secreted three mucins with different glycan profiles: age-specific mucin, youth-specific mucin, and a mucin expressed throughout life, and the expression patterns of these mucins change during aging. Additionally, age-specific mucin began to be detected at about 12 months of age. A mucin expressed throughout life and age-specific mucin had the same mass of major glycans but different structures. Furthermore, the proportion of mucin glycan species expressed throughout life changed during the aging process, and aging tended to decrease the proportion of fucosylated glycans and increase the proportion of sialoglycans. CONCLUSION: There are three secretory mucins with different glycan profiles in the SMG of mice, and their expression patterns change according to the period of the aging process. The proportion of glycan species of mucin expressed throughout life also changes during the aging process.

High expression of tissue O-linked glycans is associated with a malignant phenotype of cholangiocarcinoma.

OBJECTIVE: This study aimed to investigate the expression of O-linked glycoprotein glycans in tissue of patients with cholangiocarcinoma compared with adjacent normal tissue. METHODS: Sixty patients with cholangiocarcinoma were included in the study. Permethylated O-linked glycans from intrahepatic cholangiocarcinoma tissue and adjacent normal tissue were analyzed using nano-spray ionization-linear ion trap mass spectrometry. Histochemistry of peanut agglutinin lectin was used for detection and localization of galactose (Gal) 1, N-acetyl-galactosamine (GalNAc) 1. RESULTS: O-linked glycans from patients with cholangiocarcinoma were composed of di- to hexa-saccharides with a terminal galactose and sialic acids (N-acetylneuraminic acid [NeuAc]). A total of eight O-linked glycan structures were detected. Gal1GalNAc1 and Gal2 N-acetyl-glucosamine 1 GalNAc1 expression was significantly higher in tissue from patients with cholangiocarcinoma compared with adjacent normal tissue, while NeuAc1Gal1GalNAc1 expression was significantly lower. High Gal1GalNAc1 expression was significantly associated with the late stage of cholangiocarcinoma (stages II-IV), lymphatic invasion, and vascular invasion. CONCLUSION: Our study shows expression of O-linked glycans in progression of cholangiocarcinoma and highlights the association of Gal1GalNAc1 with lymphatic and vascular invasion of cholangiocarcinoma.

A rapid separation and characterization of mucins from mouse submandibular glands by supported molecular matrix electrophoresis.

Mucins are heavily glycosylated proteins with high molecular mass, and are involved in various diseases including infection, inflammation, and cancer. As easy separation method, such as gel electrophoresis, however, does not exist for mucins, due to their large molecular sizes and heterogeneities. In 2009, we published a supported molecular matrix electrophoresis (SMME) method that can be used to characterize mucins. For SMME analysis, mucins have been enriched by ultrafiltration of trypsin digests using a 100 KDa cutoff filter. However, this enrichment results in a loss of protein identification capability using proteomic approaches. In this study, we describe a simple enrichment of mucins without trypsinization for SMME analysis. The enrichment was developed using a porcine submandibular gland and then was applied to study and compare mouse submandibular glands between young and aged mice. From mouse submandibular glands, hyaluronic acid and some mucins were observed by SMME. One of the mucins was identified as MUC10 by proteomic analysis of the band on the SMME membrane and immunostaining using anti-MUC10 antibody. A major O-glycan of MUC10 was determined to be NeuAcα2-3Galß1-3GalNAc. Furthermore, our experiments revealed that the concentrations of these molecules were lower in aged mice than in young mice, and that an unknown mucin-like molecule was detected only from the aged mouse submandibular gland.

A novel core 1 O-linked glycan-specific binding lectin from the fruiting body of Hericium erinaceus.

Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galß(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays.

Identification of N- and O-linked glycans recognized by AAL in saliva of patients with atrophic gastritis and gastric cancer.

BACKGROUND AND AIM: Gastric cancer (GC) is a common and fatal malignancy with a worldwide occurrence. There still lacks effective biomarkers for precisely evaluating GC. Saliva is a biological fluid with enormous diagnostic potentials which emerged many advantages. We aimed to discover the novel biomarkers for accurately distinguishing early GC based on saliva glycopatterns. METHODS: We used Aleuria Aurantia Lectin (AAL)-magnetic particle conjugates to isolate fucosylated glycoproteins in the pooled saliva of healthy volunteers (HV, n= 51) and patients with atrophic gastritis (AG, n= 51) or GC (n= 51), following to release the N- and O-linked glycans from the isolated proteins with PNGase F and NaClO, and further identified the released glycans by MALDI-TOF/TOF-MS, respectively. RESULTS: A total of 9/9, 8/11, and 9/9 fucosylated N-/O-linked glycans were annotated in the isolated salivary proteins from HV, AG, and GC, respectively. Among these, six fucosylated N-linked glycansand four O-linked glycans exhibited significantly increased expression levels in GC, while five fucosylated N-linked glycans and ten fucosylated O-linked glycans exhibited significantly decreased expression levels in GC. The proportion of fucosylated N-linked glycans was decreased in GC (41.66%) compared with AG (43.63%) and HV (52.57%), as well as the fucosylated O-linked glycans was apparently decreased in GC (19.58%) compared with AG (25.43%) and HV (55.54%). CONCLUSIONS: This study could provide pivotal information to distinguish among HV, AG, and GC, and facilitate the discovery of biomarkers for GC diagnosis based on precise alterations of N- and O-linked glycans in saliva.

Hericium erinaceus isolectins recognize mucin-type O-glycans as tumor-associated carbohydrate antigens on the surface of K562 human leukemia cells.

In Hericium erinaceus mushroom fruiting body, two different lectin groups, HEL1 and HEL2, were identified by using peptide mass fingerprinting based on customized protein sequence databases derived from RNA-Seq data. The HEL2 group included four isoforms designated HEL2a-d. Codon-optimized genes encoding HEL1, HEL2a, and HEL2b were expressed in Escherichia coli to produce fully active soluble proteins designated rHEL1, rHEL2a, and rHEL2b. Interestingly, these lectins showed different molecular weights: approximately 15 kDa for rHEL1 and approximately 120 kDa for rHEL2a and rHEL2b under non-denaturing conditions. rHEL2a and rHEL2b exhibited agglutination activities, but rHEL1 did not show any agglutination activity toward animal erythrocytes. The hemagglutination activity of rHEL2 lectins was strongly inhibited by glycoproteins containing mucin-type O-glycans. Glycan array analysis and isothermal titration calorimetry revealed that rHEL2 isolectins interacted strongly with O-glycans harboring the core 1 O-glycan motif, Galß(1,3)GalNAc. Moreover, the glycan binding specificities of rHEL2 isolectins were comparable to that of peanut agglutinin in their ability to recognize O-glycans attached to leukosialin as tumor-associated carbohydrate antigens on the surface of K562 human leukemia cells. These results indicate that rHEL2 isolectins could be used as a powerful tool for analyzing mucin-type O-glycans expressed on the surface of cancer cells.

Biosynthetic Machinery Involved in Aberrant Glycosylation: Promising Targets for Developing of Drugs Against Cancer.

Cancer cells depend on altered metabolism and nutrient uptake to generate and keep the malignant phenotype. The hexosamine biosynthetic pathway is a branch of glucose metabolism that produces UDP-GlcNAc and its derivatives, UDP-GalNAc and CMP-Neu5Ac and donor substrates used in the production of glycoproteins and glycolipids. Growing evidence demonstrates that alteration of the pool of activated substrates might lead to different glycosylation and cell signaling. It is already well established that aberrant glycosylation can modulate tumor growth and malignant transformation in different cancer types. Therefore, biosynthetic machinery involved in the assembly of aberrant glycans are becoming prominent targets for anti-tumor drugs. This review describes three classes of glycosylation, O-GlcNAcylation, N-linked, and mucin type O-linked glycosylation, involved in tumor progression, their biosynthesis and highlights the available inhibitors as potential anti-tumor drugs.

Glycosylation Analysis for Congenital Disorders of Glycosylation.

Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the first description of a CDG in 1980, approximately 100 disorders have been identified. Most of these are defects in protein glycosylation, although an increasing number are defects of glycolipid or proteoglycan biosynthesis. A group of biochemical markers has been used to characterize protein glycosylation abnormalities in CDG. This unit describes three protocols that can be used to measure plasma or serum carbohydrate deficient transferrin (CDT) profile, N-glycan profile, and O-glycan profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). The quantification of particular biomarkers, such as T antigens or sialylated T antigens, could also be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These techniques can be used to identify a majority of patients with defects in protein glycosylation, although different techniques, such as flow cytometry with immunostaining, are necessary to detect defects in glycolipid or proteoglycan biosynthesis which is not included in this unit.

Sialylation of Thomsen-Friedenreich antigen is a noninvasive blood-based biomarker for GNE myopathy.

AIM: The exact pathomechanism of GNE myopathy remains elusive, but likely involves aberrant sialylation. We explored sialylation status of blood-based glycans as potential disease markers. METHODS: We employed immunoblotting, lectin histochemistry and mass spectrometry. RESULTS: GNE myopathy muscle showed hyposialylation of predominantly O-linked glycans. The O-linked glycome of patients' plasma compared with controls showed increased amounts of desialylated Thomsen-Friedenreich (T)-antigen, and/or decreased amounts of its sialylated form, ST-antigen. Importantly, all patients had increased T/ST ratios compared with controls. These ratios were normalized in a patient treated with intravenous immunoglobulins as a source of sialic acid. DISCUSSION:  GNE myopathy clinical trial data will reveal whether T/ST ratios correlate to muscle function.  CONCLUSION: Plasma T/ST ratios are a robust blood-based biomarker for GNE myopathy, and may also help explain the pathology and course of the disease.