Evaluation of the context of downstream N- and free N-glycomic alterations induced by swainsonine in HepG2 cells.

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.

Comprehensive Glycomic Approach Reveals Novel Low-Molecular-Weight Blood Group-Specific Glycans in Serum and Cerebrospinal Fluid.

ABO blood antigens on the human red blood cell membrane as well as different cells in various human tissues have been thoroughly studied. Anti-A and -B antibodies of IgM are present in serum/plasma, but blood group-specific glyco-antigens have not been extensively described. In this study, we performed comprehensive and quantitative serum glycomic analyses of various glycoconjugates and free oligosaccharides in all blood groups. Our comprehensive glycomic approach revealed that blood group-specific antigens in serum/plasma are predominantly present on glycosphingolipids on lipoproteins rather than glycoproteins. Expression of the ABO antigens on glycosphingolipids depends not only on blood type but also on secretor status. Blood group-specific glycans in serum/plasma were classified as type I, whereas those on RBCs had different structures including hexose and hexosamine residues. Analysis of free oligosaccharides revealed that low-molecular-weight blood group-specific glycans, commonly containing lacto-N-difucotetraose, were expressed in serum/plasma according to blood group. Furthermore, comprehensive glycomic analysis in human cerebrospinal fluid showed that many kinds of free oligosaccharides were highly expressed, and low-molecular-weight blood group-specific glycans, which existed in plasma from the same individuals, were present. Our findings provide the first evidence for low-molecular-weight blood group-specific glycans in both serum/plasma and cerebrospinal fluid.

Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways.

Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG) program. TAG consists of three units: 'TAG List' creates a glycan list that is used for database searching in TAG Expression; 'TAG Expression' automatically annotates and quantifies glycan signals and draws graphs; and 'TAG Pathway' maps the obtained expression information to biosynthetic pathways. Herein, we discuss the concepts, outline the TAG process, and demonstrate its potential using glycomic expression profile data from Chinese hamster ovary (CHO) cells and mutants lacking a functional Npc1 gene (Npc1 knockout (KO) CHO cells). TAG not only drastically reduced the amount of time and labor needed for glycomic analysis but also detected and quantified more glycans than manual analysis. Although this study was limited to the analysis of N-glycans and free oligosaccharides, the glycomic platform will be expanded to facilitate the analysis of O-glycans and glycans of glycosphingolipids.

Alteration of the Total Cellular Glycome during Late Differentiation of Chondrocytes.

In normal articular cartilage, chondrocytes do not readily proliferate or terminally differentiate, and exhibit a low level of metabolism. Hypertrophy-like changes of chondrocytes have been proposed to play a role in the pathogenesis of osteoarthritis by inducing protease-mediated cartilage degradation and calcification; however, the molecular mechanisms underlying these changes are unclear. Glycans are located on the outermost cell surface. Dynamic cellular differentiation can be monitored and quantitatively characterized by profiling the glycan structures of total cellular glycoproteins. This study aimed to clarify the alterations in glycans upon late differentiation of chondrocytes, during which hypertrophy-like changes occur. Primary mouse chondrocytes were differentiated using an insulin-induced chondro-osteogenic differentiation model. Comprehensive glycomics, including N-glycans, O-glycans, free oligosaccharides, glycosaminoglycan, and glycosphingolipid, were analyzed for the chondrocytes after 0-, 10- and 20-days cultivation. The comparison and clustering of the alteration of glycans upon hypertrophy-like changes of primary chondrocytes were performed. Comprehensive glycomic analyses provided complementary alterations in the levels of various glycans derived from glycoconjugates during hypertrophic differentiation. In addition, expression of genes related to glycan biosynthesis and metabolic processes was significantly correlated with glycan alterations. Our results indicate that total cellular glycan alterations are closely associated with chondrocyte hypertrophy and help to describe the glycophenotype by chondrocytes and their hypertrophic differentiation. our results will assist the identification of diagnostic and differentiation biomarkers in the future.

Sialic Acid Linkage Specific Derivatization of Glycosphingolipid Glycans by Ring-Opening Aminolysis of Lactones.

Sialic acids occur widely as glycoconjugates at the nonreducing ends of glycans. Glycosphingolipids (GSLs) include a large number of sialyl-linked glycan isomers with α2,3-, α2,6-, and α2,8-linked polysialic acids. Thus, it is difficult to distinguish structural isomers with the same mass by mass spectrometry. The sialic acid linkage specific alkylamidation (SALSA) method has been developed for discriminating between α2,3- and α2,6-linked isomers, but sequential amidation of linkage-specific sialic acids is generally complicated and time-consuming. Moreover, analysis of GSL-glycans containing α2,8-linked polysialic acids using solid-phase SALSA has not been reported. Herein, we report a novel SALSA method focused on ring-opening aminolysis (aminolysis-SALSA), which shortens the reaction time and simplifies the experimental procedures. We demonstrate that aminolysis-SALSA can successfully distinguish serum GSL-glycan isomers by mass spectrometry. In addition, ring-opening aminolysis can easily be applied to amine and hydrazine derivatives.

Glycomics of human embryonic stem cells and human induced pluripotent stem cells.

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.

Impact of the Niemann-Pick c1 Gene Mutation on the Total Cellular Glycomics of CHO Cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder, and the majority of cases are caused by mutations in the NPC1 gene. In this study, we clarified how a single gene mutation in the NPC1 gene impacts the cellular glycome by analyzing the total glycomic expression profile of Chinese hamster ovary cell mutants defective in the Npc1 gene (Npc1 KO CHO cells). A number of glycomic alterations were identified, including increased expression of lactosylceramide, GM1, GM2, GD1, various neolacto-series glycosphingolipids, and sialyl-T (O-glycan), which was found to be the major sialylated protein-bound glycan, as well as various N-glycans, which were commonly both fucosylated and sialylated. We also observed significant increases in the total amounts of free oligosaccharides (fOSs), especially in the unique complex- and hybrid-type fOSs. Treatment of Npc1 KO CHO cells with 2-hydroxypropyl-ß-cyclodextrin (HPBCD), which can reduce cholesterol and glycosphingolipid (GSL) storage, did not affect the glycomic alterations observed in the GSL-, N-, and O-glycans of Npc1 KO CHO cells. However, HPBCD treatment corrected the glycomic alterations observed in fOSs to levels observed in wild-type cells.

Glycomics of human embryonic stem cells and human induced pluripotent stem cells.

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method.

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 µl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1-21.4 µM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.

POMGNT1 Is Glycosylated by Mucin-Type O-Glycans.

Protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the N-acetylglucosamine (GlcNAc) ß1→2Man linkage of O-mannosyl glycan. POMGNT1 is not modified by N-glycans because there are no potential N-glycosylation sites; however, it is not clear whether POMGNT1 is modified by O-glycans. To determine whether POMGNT1 is O-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by Sambucus sieboldiana lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of Arachis hypogaea lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 O-glycan. Next, we analyzed the structures of the O-glycans on POMGNT1 by ß-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type O-glycans containing (NeuAc)1(Hex)1(HexNAc)1, (NeuAc)2(Hex)1(HexNAc)1, and (NeuAc)2(Hex)2(HexNAc)2. To examine whether the O-glycans affect the functions and properties of POMGNT1, we compared glycosylated and non-glycosylated forms of recombinant sPOMGNT1 for their activity and surface hydrophobicity using the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS). POMGNT1 activity and surface hydrophobicity were not affected by the presence or absence of O-glycans.

Comprehensive Glycomics of a Multistep Human Brain Tumor Model Reveals Specific Glycosylation Patterns Related to Malignancy.

Cancer cells frequently express glycans at different levels and/or with fundamentally different structures from those expressed by normal cells, and therefore elucidation and manipulation of these glycosylations may provide a beneficial approach to cancer therapy. However, the relationship between altered glycosylation and causal genetic alteration(s) is only partially understood. Here, we employed a unique approach that applies comprehensive glycomic analysis to a previously described multistep tumorigenesis model. Normal human astrocytes were transformed via the serial introduction of hTERT, SV40ER, H-RasV12, and myrAKT, thereby mimicking human brain tumor grades I-IV. More than 160 glycans derived from three major classes of cell surface glycoconjugates (N- and O-glycans on glycoproteins, and glycosphingolipids) were quantitatively explored, and specific glycosylation patterns related to malignancy were systematically identified. The sequential introduction of hTERT, SV40ER, H-RasV12, and myrAKT led to (i) temporal expression of pauci-mannose/mono-antennary type N-glycans and GD3 (hTERT); (ii) switching from ganglio- to globo-series glycosphingolipids and the appearance of Neu5Gc (hTERT and SV40ER); (iii) temporal expression of bisecting GlcNAc residues, α2,6-sialylation, and stage-specific embryonic antigen-4, accompanied by suppression of core 2 O-glycan biosynthesis (hTERT, SV40ER and Ras); and (iv) increased expression of (neo)lacto-series glycosphingolipids and fucosylated N-glycans (hTERT, SV40ER, Ras and AKT). These sequential and transient glycomic alterations may be useful for tumor grade diagnosis and tumor prognosis, and also for the prediction of treatment response.

Quantitative O-Glycomics by Microwave-Assisted ß-Elimination in the Presence of Pyrazolone Analogues.

O-Linked glycosylation of serine/threonine residues is a posttranslational modification of proteins and is essential for protein recognition and lipid functions on cell surfaces and within cells. The characterization of differently structured O-linked glycans (O-glycans) is particularly challenging because there is no known endoglycosidase for such groups. Therefore, chemical digestion approaches have been widely used; however, it is sometimes difficult to suppress unwanted side reactions. Recently, we reported a novel O-glycomics procedure using ß-elimination in the presence of pyrazolone analogues (BEP). In the present study, we describe a microwave (MW)-assisted BEP procedure for rapid and quantitative O-glycomic analysis. Following optimization of the reaction conditions, the MW-assisted BEP reaction substantially improved the recovery of total O-glycans from model glycoproteins (PSM) and the reaction time was reduced from 16 to 2 h. Combined with sequential solid-phase extractions, this MW-assisted BEP procedure enabled O-glycomic analyses of various biological samples.

Persistent verbal and behavioral deficits after resection of the left supplementary motor area in epilepsy surgery.

An 8-year-old boy underwent a resection for focal cortical dysplasia at the left supplementary motor area (SMA) for the treatment of intractable epilepsy. The manifestations of SMA syndrome, such as transient mutism and right hemiparesis, resolved within a few weeks. Verbal disfluency and impaired executive function, accompanied by impulsivity and distractibility, persisted for more than 12months. The verbal and behavioral problems caused serious difficulties in the school life of the patient, until they became less evident at 18months after surgery. Tractography performed 18months after surgery revealed a defect in the subportion of fronto-parietal association fibers within the left superior longitudinal fascicles. Verbal influency can persist with unusually long duration after resection of SMA during childhood. Although not discernible on the routine neuroimaging, white matter damage beneath the SMA region could result in serious disabilities in executive function. These complications should be recognized for the prediction and assessment of deficits in children after surgical intervention involving this region.

[Two-way deficit between phonological and arabic representation of numbers after left parieto-occipital hemorrhage].

A 52-year-old right-handed man developed a deficit of two-way transcoding between phonological and Arabic representations of numbers after cerebral hemorrhage in the left occipital lobe, intraparietal sulcus, and the surrounding of inferior parietal lobule. He showed right hemianopia, mild Wernicke's aphasia, agraphia, and phonological dyslexia. Calculation, comparison between numbers and comprehension of arithmetic signs were almost preserved. Retrieval of the mnemonic rhymes for the multiplication table (kuku) was moderately compromised. He showed good performance in the following tasks: selecting coins corresponding to the number orally designated by the experimenter, reporting orally the amount of arranged coins, selecting coins according to written amount, indicating the amount of arranged coins by writing numbers, and choosing the amount of arranged coins from several alternatives of written numbers. Thus, encoding the phonological and visual representations of numbers from the meanings as well as decoding from these 2 representations into their meanings were preserved. However, he showed grave difficulty in dictation of numbers, in choosing the verbally indicated numbers from among a set of written numbers, and in reading aloud a number. Thus, two-way impairment of association between phonological and visual representations of numbers was confirmed. Regarding other categories of nouns, marked deterioration was detected only in dictation and writing the names of indicated objects. We taught him a strategy of imaging the coins when he transcoded from Arabic representation of numbers to phonological representation and vice versa. This strategy improved his performance to some extent. These results suggest that there is a direct, bi-directional transcoding route between phonological and visual representations of numbers, which is not mediated by semantic representation. This route can be impaired by a lesion located in the left parieto-occipital cortex.

[Normative data on tests for frontal lobe functions: Trail Making Test, Verbal fluency, Wisconsin Card Sorting Test (Keio version)].

Trail Making Test (TMT Part A, B), Verbal fluency test (phonemic, semantic) and Wisconsin Card Sorting Test (Keio version : KWCST) are useful to evaluate frontal lobe functions and are commonly used in clinical settings. However, few normative data for aged Japanese have been reported. We collected normative data on these tests in elderly population, and examined the effects of age and education on the performance of these tests. Seventy-six healthy adults, aged 45 to 74 years, participated in this study. Subjects were classified into three groups by age (45-54, 55-64, 65-74). Fifty-five subjects repeated the same tests after 6 months to examine the test-retest reliability. Performance of the TMT Part A was correlated with age (r= 0.530) and that of Part B was correlated with age (r=0.500) and education (r=-0.340). Performance on both phonemic and semantic fluency was correlated with education (phonemic: r=0.357, semantic: r=0.279). Number of Categories Achieved (CA) of KWCST was correlated with education (r=0.376). The test-retest reliability of all these tests except for semantic verbal fluency and difficulty maintaining set (DMS) of KWCST was good enough for longitudinal studies.