Gerardo Alvarez-Manilla

IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells.

Robust quantification is an essential component of comparative -omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, has not been described. Here, we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-(15)N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we demonstrate that culturing mouse embryonic stems cells for 72 h in the presence of amide-(15)N-Gln media results in nearly complete incorporation of (15)N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is a simple to implement, yet powerful quantitative tool for the glycomics toolbox.

A novel β(1,6)-N-acetylglucosaminyltransferase V (GnT-VB)1

UDP‐N‐acetylglucosamine:α(1,6)‐D‐mannoside β(1,6)‐N‐acetylglucosaminyltransferase (GnT‐V, Mgat5) functions in the biosynthesis of N‐linked glycans and is transcriptionally upregulated by oncogene signaling. We report here the cloning and characterization of a human cDNA encoding a distinct enzyme with related substrate specificity, termed GnT‐VB, which is predicted to have 53% similarity to the original amino acid sequence of GnT‐V(A). Transient expression of GnT‐VB cDNA in COS7 cells yielded significant increases of activity toward GnT‐VA acceptors, including synthetic saccharides and N‐linked glycopeptides, with some differences in specificity. Unlike GnT‐VA, GnT‐VB required divalent cation for full activity. EST databases showed expression of a 6 bp (+) splice isoform of GnT‐VB; when expressed, this enzyme showed significantly reduced activity. CHO Lec4 cells, which do not express GnT‐VA or B activity, lack synthesis of the N‐linked β(1,6) branch, and do not bind L‐phytohemagglutinin (L‐PHA), were transfected with GnT‐VB or GnT‐VA; both then bound significant amounts of L‐PHA, demonstrating that both enzymes synthesized N‐linked β(1,6) branched glycans in vivo. Real‐time polymerase chain reaction results showed that GnT‐VB mRNA was highly expressed in brain and testis, with lesser levels in other tissues, while human GnT‐VA showed a more general expression, but with low levels in brain and no expression in skeletal muscle.

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing α-linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Galβ1-4[Fucα1-3]GlcNAcβ1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAcβ1-4[Fucα1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Galβ1-3[Fucα1-4]GlcNAc-R. Although 2′-fucosyllactose Fucα1-2Galβ1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fucα1-2Galβ1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Galβ1-3GlcNAcβ1-3Galβ1-3Glc; LNnT, Galβ1-4GlcNAcβ1-3Galβ1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5′-diphosphate-β-L-fucose

Glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides

Numerous studies have recently focused on the identification of specific glycan biomarkers, given the important roles that protein linked glycans play, for example, during development and disease progression. The identification of protein glycobiomarkers, which are part of a very complex proteome, has involved the use of fractionation techniques such as lectin affinity chromatography. In this study, the glycoproteomic characterization of pluripotent murine embryonic stem cells (ES) and from ES cells that were differentiated into embroid bodies (EB) was performed using immobilized Concanavalin A (ConA). This procedure allowed the isolation of glycopeptides that express biantennary and hybrid N-linked structures (ConA2 fraction) as well as high mannose glycans (ConA3 fraction) that were abundant in both ES and EB stages. A total of 293 unique N-linked glycopeptide sequences (from 180 glycoproteins) were identified in the combined data sets from ES and EB cells. Of these glycopeptides, a total of 119 sequences were identified exclusively in only one of the lectin-bound fractions (24 in the ES-ConA2, 15 in the ES-ConA3, 16 in the EB-ConA2, and 64 in the EB-ConA3). Results from this study allowed the identification of individual N-glycosylation sites of proteins that express specific glycan types. The absence of some of these lectin-bound glycopeptides in a cell stage suggested that they were derived from proteins that were either expressed exclusively on a defined developmental stage or were expressed in both cell stages but carried the lectin-bound oligosaccharides in only one of them. Therefore, these lectin-bound glycopeptides can be considered as stage-specific glycobiomarkers.

Comparison of the substrate specificities and catalytic properties of the sister N-acetylglucosaminyltransferases, GnT-V and GnT-Vb (IX)

N-Acetylglucosaminlyltransferase-V (GnT-V) synthesizes GlcNAcbeta1,6Man branched N-glycans both in vitro and in vivo. A paralog, GnT-Vb (or GnT-IX), has also been shown to synthesize both GlcNAcbeta1,6Man branched N- and O-glycans. GnT-V is expressed in most human and rodent tissues while GnT-Vb expression is limited mainly to neural tissue and testes. It is of interest, therefore, to compare the catalytic properties and reaction kinetics of these sister enzymes. The results demonstrate that while GnT-V was fully active without exogenous cation and in the presence of EDTA, the activity of GnT-Vb was stimulated over 4-fold in the presence of 10 mM Mn(++). The pH optimum for GnT-V was in the range of 6.5-7.0, while that of GnT-Vb was 8.0. common for glycosyltransferases active in brain. Both enzymes transferred GlcNAcbeta1,6 to the Man residue of the GlcNAcbeta1,2Man moiety of glycan substrates, and both enzymes acted effectively on a synthetic GlcNAcbeta1,2Manalpha1,2Glc-O-octyl trisaccharide acceptor. Moreover, although both enzymes utilized an N-linked asialo-agalacto-biantennary glycan as an acceptor, GnT-Vb displayed an almost 2.5-fold higher apparent K(m) value compared to GnT-V. Conversely, GnT-Vb very efficiently glycosylated a synthetic glycopeptide, Ac-H(2)N-Val-Glu-Pro-(GlcNAcbeta1,2-Man-O-)Thr-Ala-Val-CO-Ac, while GnT-V showed relatively poor activity toward this O-Man-linked glycopeptide acceptor, with a K(m) value of 20-fold higher than that of GnT-Vb. When the N-linked asialo-agalacto-biantennary glycan acceptor was utilized with GnT-Vb, the expected triantennary beta1,6-branched product was observed up to 8 h incubation. An additional product with two beta1,6-linked GlcNAc resides, however, was observed after prolonged (>8 h) incubation, consistent with an earlier report. This unusual tetraantennary product was observed with GnT-Vb only after substantial accumulation of the first triantennary product and not during the early stages of incubation.

Loss of expression of N‐acetylglucosaminyltransferase Va results in altered gene expression of glycosyltransferases and galectins

We isolated mouse embryo fibroblasts (MEFs) from N‐acetylglucosaminyltransferase Va (GnT‐Va) knockout mice and studied the effects of loss of expression of GnT‐Va on asparagine‐linked glycans (N‐glycan) synthesis and the gene expression of groups of glycosyltransferases and galectins. Loss of GnT‐Va expression caused aberrant expression of several N‐glycan structures, including N‐linked β(1,6) branching, poly‐N‐lactosamine, bisecting N‐acetylglucosamine (GlcNAc) and sialic acid. Using quantitative reverse transcriptase‐PCR (qRT‐PCR), altered gene expression of several groups of glycosyltransferases and galectins was observed in GnT‐Va null MEFs, supporting the observed changes in N‐glycan structures. These results suggest that genetic disruption of GnT‐Va ultimately resulted in altered MEFs gene expression and decreased tumor progression associated with loss of GnT‐Va observed may result in part from a combination of effects from these altered gene expressions.

N-acetylglucosaminyltranferase VB expression enhances β1 integrin- dependent PC12 neurite outgrowth on laminin and collagen

N‐acetylglucosaminyltransferase VB (GnT‐VB, ‐IX) is a newly discovered glycosyltransferase expressed exclusively in high levels in neuronal tissue during early development. Its homolog, GnT‐V, is expressed in many tissues and modulates cell–cell and cell–matrix adhesion. The ability of GnT‐VB to regulate cell–matrix interactions was initially investigated using the rat pheochromocytoma PC12 neurite outgrowth model. PC12 cells stably transfected with GnT‐VB consistently showed an enhanced rate of nerve growth factor (NGF)‐induced neurite outgrowth on collagen and laminin substrates. Levels of TrkA receptor phosphorylation and downstream ERK activation induced by NGF were not influenced by GnT‐VB expression. No significant difference was observed in the rate of neurite outgrowth when cells were cultured on non‐coated culture dishes, indicating that integrin–ECM interaction is required for the stimulatory effects. Neurite outgrowth induced by manganese‐dependent activation of β1 integrin on collagen and laminin substrates, however, showed a significant increase in neurite length for the PC12/GnT‐VB cells, compared with control cells, suggesting that the enhancement is most likely mediated by alteration of β1 integrin–ECM interaction by GnT‐VB. These results demonstrate that GnT‐VB expression can modulate the rate of neurite outgrowth by affecting β1 integrin–ECM interaction.

A Possible Effect of Concentrated Oolong Tea Causing Transient Ischemic Attack-Like Symptoms.

Aims Tea (green, oolong, and black) is the second most widely consumed beverage worldwide, second only to water. Aside from a few reported adverse effects, tea, particularly green tea, appears to be beneficial for human health. In the case described herein, a male experienced several transient ischemic attack-like symptoms immediately following the consumption of a cup of high quality oolong tea. A thorough medical evaluation uncovered no evidence of such an attack and leads to the suggestion of a heretofore unreported response to oolong tea. Presentation of Case A 72-year old male with hypertension and atrial fibrillation, who takes valsartan/hydrochlorothiazide to control hypertension and warfarin to reduce the risk of thrombosis and thromboembolism, presented at the emergency room of a local hospital describing several transient ischemic attack-like symptoms immediately after consuming a cup of oolong tea. His symptoms included presyncope, disequilibrium, bilateral hand parathesias, mild dysphasia, and visual problems (but apparently not presbyopia or amaurosis fugax), all of which had disappeared in approximately two hours after drinking the tea. (Mild presyncope was previously noted by the patient when ingesting a strong green tea.) No unusual features emerged from his physical examination, and his blood work was unremarkable except for elevation of his partial thromboplastin time (39 sec) and prothrombin time (22.5 sec), giving an international reference of 2.0, all consistent with the effects of warfarin. A battery of tests by the emergency room physician, a cardiologist, and a neurologist, e.g. electrocardiogram, brain computerized tomography, 2-dimensional transthoracic echocardiogram, brain magnetic resonance imaging, with and without 20 ml Gadolinium, and a magnetic resonance angiogram, confirmed the earlier diagnosis of atrial fibrillation but disclosed no additional malfunction in his heart. His brain showed no evidence of a prior hemorrhage, and his carotid arteries were clear. Methodology and Results Analysis of the oolong tea by high performance liquid chromatography and mass spectrometry identified the major catechins and two methylxanthines, caffeine and theophylline, as well as other constituents, but there was no evidence of any extraneous chemicals that could lead to the symptoms. Conclusion In view of the rapid onset of symptoms after the consumption of oolong tea, bilateral as opposed to unilateral parathesis, and the absence of any evidence of a hemorrhage or the presence of impurities in the tea, we suggest that the transient ischemic attack-like symptoms could possibly be attributable to one or more components of the oolong tea and was not an atypical magnetic resonance imaging-negative transient ischemic attack.