Michael Pierce

The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays

A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of β(1–4)galactosyltransferase, α(1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated.

N-Acetylglucosaminyltransferase V Expression Levels Regulate Cadherin-associated Homotypic Cell-Cell Adhesion and Intracellular Signaling Pathways

A common glycan alteration in transformed cells and human tumors is the highly elevated levels of N-linked β(1,6)glycans caused by increased transcription of N-acetylglucosaminyltransferase V (GnT-V). Here, we define the involvement of GnT-V in modulation of homotypic cell-cell adhesion in human fibrosarcoma HT1080 and mouse NIH3T3 cells. Increased GnT-V expression resulted in a significant decrease in the rates of calcium-dependent cell-cell adhesion. Reduced cell-cell adhesion was blocked by function-blocking antibody against N-cadherin and abrogated by pre-treatment of cells with swainsonine, demonstrating the involvement of N-cadherin in the cell-cell adhesion and that changes in N-linked β(1,6)glycan expression are responsible for the reduction in rates of adhesion, although this reduction could be mediated by the altered N-linked glycosylation of glycoproteins other than N-cadherin. Overexpression of GnT-V had no effect on the levels of cell surface expression of N-cadherin; however, it did cause a marked enhancement of both β(1,6) branching and poly-N-acetyllactosamine expression on N-cadherin. GnT-V overexpression resulted in decreased N-cadherin clustering on the cell surface induced by anti-N-cadherin antibody and affected the outside-in signal transduction pathway of ERK mediated by N-cadherin. Overexpression of GnT-V sensitized stimulation of tyrosine phosphorylation of catenins by growth factors and expression of v-src, which is consistent with its reduction of cell-cell adhesion. In vitro, GnT-V-overexpressing cells showed increased motility concomitant with increased phosphorylation of catenins. Moreover, GnT-V-deficient embryo fibroblasts from GnT-V homozygous null mice (GnT-V-/-) express N-cadherin and showed significantly increased levels of N-cadherin-based cell-cell adhesion compared with those from GnT-V+/- mice. These levels of adhesion were inhibited significantly by transient expression of GnT-V, confirming the hypothesis that levels of GnT-V can regulate cadherin-associated homotypic cell-cell adhesion. Aberrant N-linked β(1,6) branching that occurs during oncogenesis can, therefore, lessen cell-cell adhesion, contributing to increased cellular motility and invasiveness.

Transcriptional Regulation ofN-Acetylglucosaminyltransferase V by the srcOncogene

Transformation of baby hamster kidney fibroblasts by the Rous sarcoma virus causes a significant increase in the GlcNAcβ(1,6)Man-branched oligosaccharides by elevating the activity and mRNA transcript levels encodingN-acetylglucosaminyltransferase V (GlcNAc-T V). Elevated activity and mRNA levels could be inhibited by blocking cell proliferation with herbimycin A, demonstrating that Src kinase activity can regulate GlcNAc-T V expression. 5′ RACE analysis was used to identify a 3-kilobase 5′-untranslated region from GlcNAc-T V mRNA and locate a transcriptional start site in a 25-kilobase pair GlcNAc-T V human genomic clone. A 6-kilobase pair fragment of the 5′ region of the gene contained AP-1 and PEA3/Ets binding elements and, when co-transfected with a src expression plasmid into HepG2 cells, conferred src-stimulated transcriptional enhancement upon a luciferase reporter gene. This stimulation by srccould be antagonized by co-transfection with a dominant-negative mutant of the Raf kinase, suggesting the involvement of Ets transcription factors in the regulation of GlcNAc-T V gene expression. Thesrc-responsive element was localized by 5′ deletion analysis to a 250-base pair region containing two overlapping Ets sites. src stimulation of transcription from this region was inhibited by co-transfection with a dominant-negative mutant of Ets-2, demonstrating that the effects of the src kinase on GlcNAc-T V expression are dependent on Ets.

Innate BALB/c Enteric Epithelial Responses to Trichinella spiralis: Inducible Expression of a Novel Goblet Cell Lectin, Intelectin-2, and Its Natural Deletion in C57BL/10 Mice

Infection of mice with the nematode parasite Trichinella spiralis induces changes in the proteome of the jejunal epithelium, including substantial up-regulation of a novel variant of interlectin. In this study we sequence this novel lectin, termed intelectin-2, and compare expression levels during T. spiralis infection of resistant (BALB/c) with susceptible (C57BL/10) mouse strains. Intelectin-2 was cloned and sequenced from BALB/c mRNA extracted on day 14 of infection, and was found to have 91% amino acid identity with intelectin (within our study termed intelectin-1). Intelectin-2 transcripts were up-regulated early (day 3) during infection with T. spiralis in BALB/c mice, suggesting an innate response, and levels remained high through to day 14 (time of parasite rejection). Immunohistochemistry of jejunal sections with a rabbit polyclonal Ab to Xenopus laevis 35-kDa cortical granule lectin (XL35; 68% identity with intelectin-2) followed a similar pattern, with intense labeling of goblet and Paneth cells at day 14. However, intelectin-2 transcripts and protein were absent, and immunohistochemistry negative when C57BL/10 mice were infected with T. spiralis. Genomic PCR and Southern blotting confirmed that the intelectin-2 gene is absent from the C57BL/10 genome. The presence of intelectin-2 in resistant BALB/c mice, its absence from the susceptible C57BL/10 strain and the kinetics of its up-regulation during T. spiralis infection suggest that this novel lectin may serve a protective role in the innate immune response to parasite infection.

Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis.

Epithelial ovarian cancer is diagnosed less than 25% of the time when the cancer is confined to the ovary, leading to 5‐year survival rates of less than 30%. Therefore, there is an urgent need for early diagnostics for ovarian cancer. Our study using glycotranscriptome comparative analysis of endometrioid ovarian cancer tissue and normal ovarian tissue led to the identification of distinct differences in the transcripts of a restricted set of glycosyltransferases involved in N‐linked glycosylation. Utilizing lectins that bind to glycan structures predicted to show changes, we observed differences in lectin‐bound glycoproteins consistent with some of the transcript differences. In this study, we have extended our observations by the use of selected lectins to perform a targeted glycoproteomic analysis of ovarian cancer and normal ovarian tissues. Our results have identified several glycoproteins that display tumor‐specific glycosylation changes. We have verified these glycosylation changes on glycoproteins from tissue using immunoprecipitation followed by lectin blot detection. The glycoproteins that were verified were then analyzed further using existing microarray data obtained from benign ovarian adenomas, borderline ovarian adenocarcinomas, and malignant ovarian adenocarcinomas. The verified glycoproteins found to be expressed above control levels in the microarray data sets were then screened for tumor‐specific glycan modifications in serum from ovarian cancer patients. Results obtained from two of these glycoprotein markers, periostin and thrombospondin, have confirmed that tumor‐specific glycan changes can be used to distinguish ovarian cancer patient serum from normal serum.

The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products.

Malignant transformation is associated with changes in the glycosylation of cell surface proteins. For example, the N-linked oligosaccharides containing the [GlcNAcβ(1,6)Man] branch are increased after transformation of many cell types by a number of tumor viruses and oncogenes which induce the expression of N-acetylglucosaminyl-transferase V (GlcNAc-T V), the enzyme that adds this branch. A large percentage of human breast carcinomas have increased N-linked β(1,6) branches on glycoproteins, while up to 30% of breast carcinomas have amplified the oncogene her-2/neu (erb-B2). We tested the hypothesis that expression of her-2/neu stimulates GlcNAc-T V gene expression and increases the β(1,6) branching of N-linked oligosaccharides. We found that neu-transformed NIH3T3 cells have a threefold increase in GlcNAc-T V enzyme activity and increased β(1,6) branching on a specific set of glycoproteins. Promoter/reporter experiments showed that her-2/neu stimulates transcription from the human GlcNAc-T V promoter and that the her-2/neu response element was located about 400 bp 5′ of the transcription initiation site and includes three Ets transcription factor binding sequences. Co-transfections with dominant-negative Raf and Ets expression plasmids demonstrated that the transcriptional activation of the GlcNAc-T V promoter by neu is mediated by the Ras-Raf-Ets signal transduction pathway.

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.

Targeted Glycoproteomic Identification of Biomarkers for Human Breast Carcinoma

Glycosylation is a dynamic post-translational modification that changes during the development and progression of various malignancies. During the oncogenesis of breast carcinoma, the glycosyltransferase known as N-acetylglucosaminyltransferase Va (GnT-Va) transcript levels and activity are increased due to activated oncogenic signaling pathways. Elevated GnT-V levels leads to increased beta(1,6)-branched N-linked glycan structures on glycoproteins that can be measured using a specific carbohydrate binding protein or lectin known as L-PHA. L-PHA does not bind to nondiseased breast epithelial cells, but during the progression to invasive carcinoma, cells show a progressive increase in L-PHA binding. We have developed a procedure for intact protein L-PHA-affinity enrichment, followed by nanospray ionization mass spectrometry (NSI-MS/MS), to identify potential biomarkers for breast carcinoma. We identified L-PHA reactive glycoproteins from matched normal (nondiseased) and malignant tissue isolated from patients with invasive ductal breast carcinoma. Comparison analysis of the data identified 34 proteins that were enriched by L-PHA fractionation in tumor relative to normal tissue for at least 2 cases of ductal invasive breast carcinoma. Of these 34 L-PHA tumor enriched proteins, 12 are common to all 4 matched cases analyzed. These results indicate that lectin enrichment strategies targeting a particular glycan change associated with malignancy can be an effective method of identifying potential biomarkers for breast carcinoma.

Focused glycomic analysis of the N-linked glycan biosynthetic pathway in ovarian cancer

Epithelial ovarian cancer is the deadliest female reproductive tract malignancy in Western countries. Less than 25% of cases are diagnosed when the cancer is confined, however, pointing to the critical need for early diagnostics for ovarian cancer. Identifying the changes that occur in the glycome of ovarian cancer cells may provide an avenue to develop a new generation of potential biomarkers for early detection of this disease. We performed a glycotranscriptomic analysis of endometrioid ovarian carcinoma using human tissue, as well as a newly developed mouse model that mimics this disease. Our results show that the N‐linked glycans expressed in both nondiseased mouse and human ovarian tissues are similar; moreover, malignant changes in the expression of N‐linked glycans in both mouse and human endometrioid ovarian carcinoma are qualitatively similar. Lectin reactivity was used as a means for rapid validation of glycan structural changes in the carcinomas that were predicted by the glycotranscriptome analysis. Among several changes in glycan expression noted, the increase of bisected N‐linked glycans and the transcripts of the enzyme responsible for its biosynthesis, GnT‐III, was the most significant. This study provides evidence that glycotranscriptome analysis can be an important tool in identifying potential cancer biomarkers.

Receptor Tyrosine Phosphatase β (RPTPβ) Activity and Signaling Are Attenuated by Glycosylation and Subsequent Cell Surface Galectin-1 Binding

O-Mannosyl-linked glycosylation is abundant within the central nervous system, yet very few glycoproteins with this glycan modification have been identified. Congenital diseases with significant neurological defects arise from inactivating mutations found within the glycosyltransferases that act early in the O-mannosyl glycosylation pathway. The N-acetylglucosaminyltransferase known as GnT-Vb or -IX is highly expressed in brain and branches O-mannosyl-linked glycans. Our results using SH-SY5Y neuroblastoma cells indicate that GnT-Vb activity promotes the addition of the O-mannosyl-linked HNK-1 modification found on the developmentally regulated and neuron-specific receptor protein-tyrosine phosphatase β (RPTPβ). These changes in glycosylation accompany decreased cell-cell adhesion and increased rates of migration on laminin. In addition, we show that expression of GnT-Vb promotes its dimerization and inhibits RPTPβ intrinsic phosphatase activity, resulting in higher levels of phosphorylated β-catenin, suggesting a mechanism by which GnT-Vb glycosylation couples to changes in cell adhesion. GnT-Vb-mediated glycosylation of RPTPβ promotes galectin-1 binding and RPTPβ levels of retention on the cell surface. N-Acetyllactosamine, but not sucrose, treatment of cells results in decreased RPTP retention, showing that galectin-1 binding contributes to the increased retention after GnT-Vb expression. These results place GnT-Vb as a regulator of RPTPβ signaling that influences cell-cell and cell-matrix interactions in the developing nervous system.