Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Heteropolysaccharides from S. cerevisiae show anti-adhesive properties against E. coli associated with Crohn's disease.

The Saccharomyces cerevisiae CNCM I-3856 was previously reported to strongly inhibit adherent-invasive Escherichia coli (AIEC) adhesion to intestinal epithelial cells in vitro and to favor AIEC elimination from the gut in a murine model of Crohn's disease in vivo. In order to identify which cell wall components of yeast are responsible for AIEC elimination, constituent polysaccharides of yeast were isolated and their anti-adhesive ability against AIEC adhesion in vitro was screened. A fraction containing mannan, ß-glucan and α-glucan extracted from yeast cell-walls was shown to inhibit 95% of AIEC adhesion in vitro and was thus identified as the strongest anti-adhesive yeast cell wall component. Furthermore, this mannan-glucan-containing fraction was shown to accelerate AIEC decolonization from gut in vivo. This fraction could be proposed as a treatment to eliminate AIEC bacteria in patients with Crohn's disease, a microbial trigger of intestinal inflammation.

Macrobrachium rosenbergii nodavirus virus-like particles attach to fucosylated glycans in the gills of the giant freshwater prawn.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.

Permethylation and Microfractionation of Sulfated Glycans for MS Analysis.

Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ion mode, respectively. Nevertheless, the negative charge imparted by sulfate can be advantageous for selective detection in the negative ion mode if the sialic acids can first be neutralized. This is most conveniently achieved by a concerted sample preparation workflow in which permethylation is followed by solid phase fractionation to isolate the sulfated glycans prior to MS analysis. Importantly, we demonstrated that conventional NaOH/DMSO slurry permethylation method can retain the sulfates. Instead of extracting permethylated glycans into chloroform for sample clean-up, reverse phase C18 cartridge coupled with self-packed amine-tip or mixed mode weak anion exchange cartridge can be utilized to obtain in good yield the non-sulfated, mono-sulfated, and multiply sulfated permethylated glycans in separate fractions for sulfoglycomic analysis.

Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans.

We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of permethylated sulfated glycans in the negative ion mode. The advantages are that extra sulfates on permethylated di- and multiply sulfated glycans will survive in nanoESI conditions to allow detection of multiply charged intact molecular ions, and more comprehensive MS/MS can be performed in an automated fashion at higher sensitivity, compared with MALDI-MS/MS. Parallel higher energy collision dissociation (HCD) and ion trap collision induced dissociation (CID)-based MS2, coupled with product-dependent MS3 in data dependent acquisition mode proved to be highly productive when applied to resolve and identify the isomeric sulfated glycan structures. In-house glycomic data mining software, GlyPick, was developed and used to automate the downstream process of identification and relative quantification of target sulfated glycotopes based on summed intensity of their diagnostic MS2 ions extracted from thousands of HCD-MS2 and/or CID-MS2 data.

Systems glycomics of adult zebrafish identifies organ-specific sialylation and glycosylation patterns.

The emergence of zebrafish Danio rerio as a versatile model organism provides the unique opportunity to monitor the functions of glycosylation throughout vertebrate embryogenesis, providing insights into human diseases caused by glycosylation defects. Using a combination of chemical modifications, enzymatic digestion and mass spectrometry analyses, we establish here the precise glycomic profiles of eight individual zebrafish organs and demonstrate that the protein glycosylation and glycosphingolipid expression patterns exhibits exquisite specificity. Concomitant expression screening of a wide array of enzymes involved in the synthesis and transfer of sialic acids shows that the presence of organ-specific sialylation motifs correlates with the localized activity of the corresponding glycan biosynthesis pathways. These findings provide a basis for the rational design of zebrafish lines expressing desired glycosylation profiles.

Distinct substrate specificities of human GlcNAc-6-sulfotransferases revealed by mass spectrometry-based sulfoglycomic analysis.

Sulfated glycans are known to be involved in several glycan-mediated cell adhesion and recognition pathways. Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O-sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases. On the contrary, GlcNAc6ST-3 (CHST5) was only expressed in nonmalignant epithelial cells, whereas GlcNAc6ST-1 (CHST2) was expressed equally in both cancerous and nonmalignant epithelial cells. These results suggest that 6-O-sulfated glycans that are synthesized only by GlcNAc6ST-2 may be highly colon cancer-specific, as supported by immunohistochemical staining of cancer cells using the MECA-79 antibody known to be relatively specific to the enzymatic reaction products of GlcNAc6ST-2. By more precise MS-based sulfoglycomic analyses, we sought to further infer the substrate specificities of GlcNAc6STs via a definitive mapping of various sulfo-glycotopes and O-glycan structures expressed in response to overexpression of transfected GlcNAc6STs in the SW480 colon cancer cell line. By detailed MS/MS sequencing, GlcNAc6ST-3 was shown to preferentially add sulfate onto core 2-based O-glycan structures, but it does not act on extended core 1 structures, whereas GlcNAc6ST-1 prefers core 2-based O-glycans to extended core 1 structures. In contrast, GlcNAc6ST-2 could efficiently add sulfate onto both extended core 1- and core 2-based O-glycans, leading to the production of unique sulfated extended core 1 structures such as R-GlcNAc(6-SO3-)ß1-3Galß1-4GlcNAc(6-SO3-)ß1-3Galß1-3GalNAcα, which are good candidates to be targeted as cancer-specific glycans.

Investigating the function of Gdt1p in yeast Golgi glycosylation.

The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca2+/Mn2+ homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca2+ concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn2+ confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca2+. The use of Pmr1p mutants either defective for Ca2+ or Mn2+ transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca2+ requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn2+ inside the Golgi lumen when Pmr1p exclusively transports Ca2+. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn2+ sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn2+/Ca2+transport.

Concerted mass spectrometry-based glycomic approach for precision mapping of sulfo sialylated N-glycans on human peripheral blood mononuclear cells and lymphocytes.

Despite well-recognized biological importance, mass spectrometry (MS)-based glycomic identification of sulfo-, sialylated terminal glyco-epitopes on the N-glycans of various immune cell types remains technically challenging and rarely reported. Previous studies with monoclonal antibody have implicated a regulated expression of 6-sulfo-α2-6-sialyl LacNAc on B cells in peripheral lymph nodes and the circulating peripheral blood lymphocytes but its occurrence on leukemia cells or lymphomas have not been critically addressed. In this study, we have extended our previously developed MS-based sulfoglycomic platform by incorporating additional complementary analytical approaches in order to achieve a high sensitivity mapping and relative quantification of the detected sulfated glycotopes down to the level of defining their sialyl linkages. We showed that discovery mode sulfoglycomics and precise location of sulfate were best achieved by multimode MS analyses of fractionated, permethylated sulfated N-glycans. On the other hand, the relative degree of sulfation on individual N-glycans could be more efficiently inferred from the respective extracted ion chromatograms of native, non-sulfated and sulfated target N-glycans in single LC-MS/MS runs. The GlcNAc-6-O-sulfated α2-6-sialyl LacNAc, which constitutes the higher affinity ligand for the human inhibitory co-receptor of B cells, CD22, was found to be commonly carried on a range of complex type N-glycans from human CD19+ and CD4+ lymphocytes. We further showed that its occurrence on the most abundant α2-6-disialylated biantennary structure from the peripheral blood mononuclear cells of patients diagnosed as B-cell chronic lymphocytic leukemia varied within ±2-fold abundance from the mean value determined for isolated CD19+ lymphocytes and cultured B-CLL cells.

KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin-dependent lymphocyte homing.

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6'-disulfo-3'sLN (Siaα2→3[6S]Galß1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.

Priming mass spectrometry-based sulfoglycomic mapping for identification of terminal sulfated lacdiNAc glycotope.

In an effort to prime our mass spectrometry (MS)-based sulfoglycomic mapping platform technology for facile identification of sulfated lacdiNAc (GalNAcß1-4GlcNAcß1-), we have re-examined the N-glycans of bovine thyroid stimulating hormone. We showed that MALDI-MS mapping of permethylated glycans in negative ion mode can give an accurate representation of the sulfated glycans and, through MS/MS, diagnostic ions can be derived that we can collectively define the presence of a terminal sulfated lacdiNAc moiety at high sensitivity. Based on these ions, which can also be produced by nanoESI-MS(n), we demonstrated that the glycome of an ovarian carcinoma cell line, RMG-1, comprises a high abundance of sulfated lacdiNAc epitopes carried on multiantennary complex type N-glycans alongside fucosylated, sialylated and/or sulfated lacNAc antennae. This represents the first report of a natural glycomic occurrence of sulfated lacdiNAc on a cell line, as opposed to other better-characterized presence on secreted glycoproteins from a handful of sources. It is anticipated that with improved methods of detection such as that developed in this work, we are likely to identify a wider occurrence of sulfated lacdiNAc and be able to more accurately delineate the regulatory mechanism dictating the choice of a cell type in synthesizing sulfated, sialylated, fucosylated and/or non-substituted lacdiNAc.

Prominent expression of sialyl Lewis X-capped core 2-branched O-glycans on high endothelial venule-like vessels in gastric MALT lymphoma.

High endothelial venule (HEV)-like vessels have been observed in gastric B cell lymphoma of mucosa-associated lymphoid tissue type (MALT lymphoma), as well as in its preceding lesion, chronic Helicobacter pylori gastritis. Previously we reported that glycans on HEV-like vessels in the latter lesion served as L-selectin ligands, although their function is unclear. We have investigated sialyl Lewis X (sLeX)-related glycoepitopes and found that MECA-79(-) /HECA-452(+) /NCC-ST-439(+) HEV-like vessels preferentially mark gastric MALT lymphoma compared to chronic H. pylori gastritis. We then constructed CHO cell lines expressing potential MECA-79(-) /HECA-452(+) /NCC-ST-439(+) glycans, as well as other sLeX-type glycans, on CD34 and evaluated L-selectin binding to those cells, using L-selectin-IgM chimera binding and lymphocyte adhesion assays. L-selectin-IgM chimeras bound to CHO cells expressing 6-sulpho-sLeX attached to core 2-branched O-glycans with or without 6-sulpho-sLeX attached to extended core 1 O-glycans, but only marginally to other CHO cell lines. By contrast, CHO cells expressing 6-sulpho-sLeX attached to extended core 1 and/or core 2-branched O-glycans, as well as non-sulphated sLeX attached to core 2-branched O-glycans, showed substantial lymphocyte binding, while binding was negligible on lines expressing 6-sulpho- and non-sulphated sLeX attached to N-glycans and non-sulphated sLeX attached to extended core 1 O-glycans. These results indicate that MECA-79(-) /HECA-452(+) /NCC-ST-439(+) glycans, specifically, 6-sulpho- and non-sulphated sLeXs attached to core 2-branched O-glycans, expressed on HEV-like vessels in gastric MALT lymphoma function as L-selectin ligands and likely contribute to H. pylori-specific T cell recruitment in the progression of gastric MALT lymphoma.

Core2 O-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-IV during intestinal cell differentiation.

Alterations in glycosylation play an important role during intestinal cell differentiation. Here, we compared expression of mucin-type O-glycan synthases from proliferating and differentiated HT-29 and Caco-2 cells. Mucin-type O-glycan structures were analyzed at both stages by mass spectrometry. Core2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GnT-2) was markedly increased in differentiated HT-29 and Caco-2 cells, but the core3 structure was hardly detectable. To determine whether such differential expression of mucin-type O-glycan structures has physiological significance in intestinal cell differentiation, expression of sucrase isomaltase (SI) and dipeptidyl-peptidase IV (DPP-IV), two well known intestinal differentiation markers, was examined. Interestingly, the fully glycosylated mature form of SI was decreased in C2GnT-2 knock-out mice but not in core2 N-acetylglucosaminyltransferase-3 (C2GnT-3) nulls. In addition, expression of SI and DPP-IV was dramatically reduced in C2GnT-1-3 triple knock-out mice. These patterns were confirmed by RNAi analysis; C2GnT-2 knockdown significantly reduced cell surface expression of SI and DPP-IV in Caco-2 cells. Similarly, overexpression of the core3 structure in HT-29 cells attenuated cell surface expression of both enzymes. These findings indicate that core3 O-glycan structure regulates cell surface expression of SI and DPP-IV and that core2 O-glycan is presumably an essential mucin-type O-glycan structure found in both molecules in vivo. Finally, goblet cells in the upper part of the crypt showed impaired maturation in the core2 O-glycan-deficient mice. These studies are the first to clearly identify functional mucin-type O-glycan structures modulating cell surface expression of SI and DPP-IV during the intestinal cell differentiation.

Mass spectrometric analysis of sulfated N- and O-glycans.

Sulfated N- and O-glycans carried on a myriad of cell-surface adhesion molecules and receptors are often not detected by current approaches in mass spectrometry (MS)-based glycomic mapping of cells and tissues. This is in part due to a natural lower abundance, compounded further by their negatively charged nature, which adversely disfavors their ionization and detection amid a sea of often much more abundant, nonsulfated, sialylated glycans. However, this particular limitation can actually be taken advantage of to effect highly selective enrichment and sensitive MS screening in negative ion mode, provided the ubiquitous sialic acids can first be neutralized. It has been demonstrated that permethylation not only fulfills this role adequately but further confers better MS/MS fragmentation characteristics for more efficient structural mapping and sequencing. Protocols and general practical considerations are described here which would enable one to readily prepare permethylated sulfated glycans, fractionate them away from the more abundant nonsulfated ones in simple steps for high-sensitivity MS analysis, and sensibly interpret the initial sulfoglycomic screening data thus obtained.

N-Glycosylation profiling of turtle egg yolk: expression of galabiose structure.

To understand the roles of species-specific carbohydrates, systematic studies of interspecific glycan analyses are imperative. An extensive series of glycomics studies on approximately 180 kinds of bird eggs have demonstrated that 60-70% of the birds, which are closely related in phylogeny, express the alpha-Galp-(1-->4)-Galp structure on their egg glycoproteins. This prompted us to investigate the glycosylation profiles of eggs from an evolutionarily related organism, a sea turtle (reptilian). We performed N-glycosylation profiling of turtle egg yolk by using HPLC mapping in conjunction with mass spectrometric methods and thereby demonstrated that the alpha-Galp-(1-->4)-Galp groups are displayed on approximately 38% of total N-glycans. Our findings suggest that the ability to express the galabiose structure was acquired at an early stage of diversification in amniotes.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.

Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.

Identification of blood group A/A-Leb/y and B/B-Leb/y active glycotopes co-expressed on the O-glycans isolated from two distinct human ovarian cyst fluids.

Although the individual human blood group A and B determinants are well defined, their co-expression pattern on a particular glycan carrier in individuals of blood group AB status has not been delineated. To address this issue, complex O-glycans were isolated from two distinct sources of human ovarian cyst glycoproteins (HOC 89 and Cyst 19) and profiled by advanced MS analyses, in conjunction with defining their binding characteristics against a panel of lectins and monoclonal antibodies. The major O-glycans of HOC 89 were found to correspond to sialyl Tn, mono- and di-sialyl T structures, whereas those of Cyst 19 were apparently more heterogeneous and extended to larger sizes. A minimal structure that carries both A and B determinants on the same molecule was identified, in which the A epitope is attached directly to the core GalNAc, whereas the B epitope is preferentially located on the six arms of a core 2 structure. Both arms can be further extended with internal fucosylation that appears to be restricted to those non-sialylated chains already carrying the terminal ABH determinants, thus giving rise to rather prominent A/B-Le(b/y) glycotopes on larger O-glycans.

Core3 O-glycan synthase suppresses tumor formation and metastasis of prostate carcinoma PC3 and LNCaP cells through down-regulation of alpha2beta1 integrin complex.

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

A single step method for purification of sulfated oligosaccharides.

Purifying and analysing sulfated oligosaccharides by mass spectrometry often constitutes a challenge. We present here a single step method to isolate sulfated compounds from a complex mixture of neutral and acidic oligosaccharide-alditols. The strategy relies on the exclusion of sulfated molecules from strong cation exchange resin. By testing a wide range of reduced mucin type O-glycans isolated from different biological sources, we demonstrate that this method permits, without prior chemical modification, the specific purification of sulfate-containing oligosaccharides present in any quantity from very complex mixtures of molecules.