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Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glycophorins C and D are highly glycosylated integral sialoglycoproteins of human red blood cell membranes carrying the Gerbich blood group antigens. The O- and N-glycosidic chains of the major erythrocyte glycoprotein (Lisowska E. 2001, Antigenic properties of human glycophorins - an update. Adv Exp Med Biol, 491:155-169; Tomita M and Marchesi VT. 1975, Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci USA, 72:2964-2968.) are well characterized but the structure of GPC N-glycans has remained unknown. This problem became important since it was reported that GPC N-glycans play an essential role in the interaction with Plasmodium falciparum EBA-140 merozoite ligand. The elucidation of these structures seems essential for full characterization of the GPC binding site for the EBA-140 ligand. We have employed detailed structural analysis using sequential mass spectrometry to show that many GPC N-glycans contain H2 antigen structures and several contain polylactosamine structures capped with fucose. The results obtained indicate structural heterogeneity of the GPC N-glycans and show the existence of structural elements not found in glycophorin A N-glycans. Our results also open a possibility of new interpretation of the data concerning the binding of P. falciparum EBA-140 ligand to GPC. We hypothesize that preferable terminal fucosylation of N-glycosidic chains containing repeating lactosamine units of the GPC Gerbich variant could be an explanation for why the EBA-140 ligand does not react with GPC Gerbich and an indication that the EBA-140 interaction with GPC is distinctly dependent on the GPC N-glycan structure., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The conserved oligomeric Golgi complex (COG) is a hetero-octomeric peripheral membrane protein required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi. Mutations in subunits 1, 4, 5, 6, 7 and 8 are the basis for a rare inheritable human disease termed congenital disorders of glycosylation type-II. Defects to COG complex function result in aberrant glycosylation, protein trafficking and Golgi structure. The cellular function of the COG complex and its role in protein glycosylation are not completely understood. In this study, we report the first detailed structural analysis of N-glycans from a COG complex-deficient organism. We employed sequential ion trap mass spectrometry of permethylated N-glycans to demonstrate that the COG complex is essential for the formation of fucose-rich N-glycans, specifically antennae fucosylated structures in Caenorhabditis elegans. Our results support the supposition that disruption to the COG complex interferes with normal protein glycosylation in the medial and/or trans-Golgi.

LT-IIb, a type II heat-labile enterotoxin of Escherichia coli, is a potent immunologic adjuvant with high affinity binding for ganglioside GD1a. Earlier study suggested that LT-IIb bound preferentially to the terminal sugar sequence NeuAcalpha2-3Galbeta1-3GalNAc. However, studies in our laboratory suggested a less restrictive binding epitope. LT-IIb(T13I), an LT-IIb variant, engineered by a single isoleucine-threonine substitution, retains biological activity, but with less robust inflammatory effects. We theorized that LT-IIb has a less restrictive binding epitope than previously proposed and that immunologic differences between LT-IIb and LT-IIb (T13I) correlate with subtle ganglioside binding differences. Ganglioside binding epitopes, determined by affinity overlay immunoblotting and enzymatic degradation of ganglioside components of RAW264.7 macrophages, indicated that LT-IIb bound to a broader array of gangliosides than previously recognized. Each possessed NeuAcalpha2-3Galbeta1-3GalNAc, although not necessarily as a terminal sequence. Rather, each had a requisite terminal or penultimate single sialic acid and binding was independent of ceramide composition. RAW264.7 enterotoxin-binding and non-binding ganglioside epitopes were definitively identified as GD1a and GM1a, respectively, by enzymatic degradation and mass spectroscopy. Affinity overlay immunoblots, constructed to the diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIb bound NeuAc- and NeuGc-gangliosides with nearly equal affinity. However, LT-IIb(T13I) exhibited enhanced affinity for NeuGc-gangliosides and more restrictive binding. These studies further elucidate the binding epitope for LT-IIb and suggest that the diminished inflammatory activity of LT-IIb(T13I) is mediated by a subtle shift in ganglioside binding. These studies underscore the high degree of specificity required for ganglioside-protein interactions.

In an effort to understand the role of molecular glycosylation in cancer a murine model has been used to characterize and fingerprint malignancies in established cell lines that manifest all the hallmarks of metastatic disease: spontaneous development, local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Using astrocyte cell controls, we compared N-linked glycosylation from a nonmetastatic brain tumor cell line and two different metastatic brain tumor cells. Selected ions in each profile were disassembled by ion trap mass spectrometry (MS(n)) which exhibited multiple structural differences between each tissue. These unique structures were identified within isomeric compositions as pendant nonreducing termini of di- and trisaccharide fragments, probably transparent to a tandem MS approach but distinctively not to sequential ion trap MS(n) detection.

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

Rare polyagglutinable erythrocytes of NOR phenotype were found to contain two unique glycosphingolipids (designated NOR1 and NOR2). These components (not detected in normal erythrocytes) were reactive with Griffonia simplicifolia isolectin IB4 (GSL-IB4) and commonly present human anti-NOR antibodies. The NOR1 component has been reported to be a globoside containing a single galactose residue linked alpha1,4 to the terminal N-acetylgalactosamine. Here, we report the structural studies on a second glycolipid, NOR2, and a third novel component migrating in high-performance thin-layer chromatography (HPTLC) between NOR1 and NOR2. The structures were determined by a combination of ion trap sequential mass spectrometry (MALDI-QIT-TOF) and step-wise treatment with glycosidases, followed by identification of products on HPTLC plates with lectins and mouse monoclonal anti-NOR antibody. The NOR2 component was found to be a disaccharide extension of NOR1 with the following structure: Galalpha1-4GalNAcbeta1-3Galalpha1-4GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-Cer. Treatment of NOR2 with alpha-galactosidase gave a glycolipid migrating between NOR1 and NOR2, which did not react with either GSL-IB4 or anti-NOR antibodies but did react with GalNAc-specific soybean agglutinin. This intermediate glycolipid (now designated NOR(int)) was identified as a relatively abundant component of a neutral glycolipid fraction from NOR erythrocytes, suggesting its presence as a precursor to NOR2. The structure of NOR(int) was also confirmed by sequential mass spectrometry studies. These results indicate that polyagglutination in NOR subjects is due to unique erythrocyte glycolipids that are synthesized by sequential addition of Galalpha1,4 and GalNAcbeta1,3 to globoside.

Analysis of protein glycosylation within the nematode Caenorhabditis elegans has revealed an abundant and unreported set of core chitobiose modifications (CCMs) to N-linked glycans. With hydrazine release, an array of glycomers and isobars were detected with hexose extensions on the 3- and 3,6-positions of the penultimate and reducing terminus, respectively. A full complement of structures includes a range of glycomers possessing a Galbeta(1-4)Fuc disaccharide at the 3- and 6-positions of the protein-linked GlcNAc. Importantly, enzymatic (PNGase F/A) release failed to liberate many of these extended structures from reduced and alkylated peptides and, as a consequence, such profiles were markedly deficient in a representation of the worm glycome. Moreover, the 3-linked Galbeta(1-4)Fuc moiety was notably resistant to a range of commercial galactosidases. For identification, the fragments were spectrum-matched with synthetic products and library standards using sequential mass spectrometry (MS(n)). A disaccharide observed at the 3-position of penultimate GlcNAc, indicating a Hex-Fuc branch on some structures, was not further characterized because of low ion abundance in MS(n). Additionally, a Hex-Hex-Fuc trisaccharide on the 6-position of proximal GlcNAc was also distinguished on select glycomers. Similar branch extensions on 6-linked core fucosyl residues have recently been reported among other invertebrates. Natural methylation and numerous isobars complement the glycome, which totals well over 100 individual structures. Complex glycans were detected at lower abundance, indicating glucosaminyltransferase-I (GnT-I) and GnT-II activity. A range of phosphorylcholine (PC)-substituted complex glycans were also confirmed following a signature two-stage loss of PC during MS(n) analysis, although the precursor ion was not observed in the mass profiles. In a similar manner, numerous other minor glycans may be present but unobserved in hydrazine-release profiles dominated by fucosylated structures. All CCM structures, including multiple isomers, were determined without chromatography by gas-phase disassembly (MS(n)) in Paul and linear ion trap (IT) instruments.

Sialoglycosphingolipids (gangliosides) are membrane components of eukaryotic cells that modulate cell signal transduction events. Discrepancies exist in the published descriptions of the gangliosides present in the human peripheral monocyte/macrophage. Macrophages were isolated from healthy human volunteers by two different methods. Their ganglioside fractions were isolated and examined by 2D thin-layer mobility, enzymatic susceptibility, and mass spectral-collision induced dissociation-mass spectral analyses. Thin-layer ganglioside chromatographic patterns displayed four major doublets and were similar for monocytes/macrophages isolated by either apheresis/elutriation or density gradient centrifugation. All gangliosides were resistant to beta-galactosidase but sensitive to Clostridium perfringens sialidase, indicating the absence of terminal galactose residues and sialidase-resistant sialic acid moieties. Mass spectra indicated only three major sets of glycolipid components with mass heterogeneity in the ceramide portion of each set. In all the gangliosides, the ceramide moiety contained only C18 sphingosine with the heterogeneity produced by the presence of C16 or C24 fatty acid. One doublet was resistant to Newcastle disease virus sialidase, indicating the presence of an alpha(2-6)-linked sialic acid residue with the same mass as another doublet. All data was consistent with the following structures as the major gangliosides of human peripheral monocyte/macrophages: II(3)NeuAcLacCer (sialolactosyl ceramide, GM3), IV(3)- and IV(6)NeuAcnLcOse(4)Cer (sialoparagloboside, nLM1), and IV(3)NeuAcnLcOse(6)Cer (a sialohexosylceramide).

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.

Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events. Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component. Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods. All detectable components of the polysialo fraction were determined to be disialogangliosides. Treatment of the polysialo fraction with Clostridium perfringens sialidase produced mostly the sialidase-resistant monosialoganglioside, GM1a, and a minor amount of asialoGM1. Periodate oxidation and mass spectrometry analyses demonstrated the lack of tandem disialo moieties which indicated the absence of GD1b or GD1c (GD1) entities. The combined data showed the major disialogangliosides consisted of GD1a entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer, IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, and IV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted of GD1alpha entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGc-GgOse4Cer, and also positional isomer(s) of GD1alpha(NeuAc, NeuGc). These isomeric components were identified by collision analysis and tandem mass spectrometry. Consistent with previous analyses, the ceramide portion of all polysialo (disialo) gangliosides contained solely C18 sphingosine with C16 and C24 fatty acid moieties. These results, combined with the previous characterization of macrophage monosialogangliosides, indicate normal murine macrophage ganglioside biosynthesis proceeds along the "a" ganglioside pathway, e.g., GM3-->GM2-->GM1a-->GD1a, and the proposed asialoganglioside or "alpha" pathway, asialoGM1-->GM1b-->GD1alpha. The presence of totally sialidase-sensitive gangliosides appears to be characteristic of functional murine peritoneal macrophages while they are reduced in genetically impaired cells.