Kazuhiro Aoki

E-selectin receptors on human leukocytes

Selectins on activated vascular endothelium mediate inflammation by binding to complementary carbohydrates on circulating neutrophils. The human neutrophil receptor for E-selectin has not been established. We report here that sialylated glycosphingolipids with 5 N-acetyllactosamine (LacNAc, Galbeta1-4GlcNAcbeta1-3) repeats and 2 to 3 fucose residues are major functional E-selectin receptors on human neutrophils. Glycolipids were extracted from 10(10) normal peripheral blood human neutrophils. Individual glycolipid species were resolved by chromatography, adsorbed as model membrane monolayers and selectin-mediated cell tethering and rolling under fluid shear was quantified as a function of glycolipid density. E-selectin-expressing cells tethered and rolled on selected glycolipids, whereas P-selectin-expressing cells failed to interact. Quantitatively minor terminally sialylated glycosphingolipids with 5 to 6 LacNAc repeats and 2 to 3 fucose residues were highly potent E-selectin receptors, constituting more than 60% of the E-selectin-binding activity in the extract. These glycolipids are expressed on human blood neutrophils at densities exceeding those required to support E-selectin-mediated tethering and rolling. Blocking glycosphingolipid biosynthesis in cultured human neutrophils diminished E-selectin, but not P-selectin, adhesion. The data support the conclusion that on human neutrophils the glycosphingolipid NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3[Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3](2)[Galbeta1-4GlcNAcbeta1-3](2)Galbeta1-4GlcbetaCer (and closely related structures) are functional E-selectin receptors.

Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, α-dystroglycan (α-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown α-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle α-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on α-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from α-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.

Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65

The Golgi receives the entire output of newly synthesized cargo from the endoplasmic reticulum (ER), processes it in the stack largely through modification of bound oligosaccharides, and sorts it in the trans-Golgi network (TGN). GRASP65 and GRASP55, two proteins localized to the Golgi stack and early secretory pathway, mediate processes including Golgi stacking, Golgi ribbon linking, and unconventional secretion. Previously we have shown that GRASP depletion in cells disrupts Golgi stack formation. Here we report that knockdown of the GRASP proteins, alone or combined, accelerates protein trafficking through the Golgi membranes but also has striking negative effects on protein glycosylation and sorting. These effects are not caused by Golgi ribbon unlinking, unconventional secretion, or ER stress. We propose that GRASP55/65 are negative regulators of exocytic transport and that this slowdown helps to ensure more complete protein glycosylation in the Golgi stack and proper sorting at the TGN.

Porcine Sperm Bind to Specific 6-Sialylated Biantennary Glycans to Form the Oviduct Reservoir

ABSTRACT After mating, many female mammals store a subpopulation of sperm in the lower portion of the oviduct, forming a reservoir. The reservoir lengthens sperm lifespan, regulates sperm capacitation, controls polyspermy, and selects normal sperm. It is believed that sperm bind to glycans on the oviduct epithelium to form the reservoir, but the specific adhesion molecules that retain sperm are unclear. Herein, using a glycan array to test 377 glycans for their ability to bind porcine sperm, we found two glycan motifs in common among all glycans with sperm-binding ability: the Lewis X trisaccharide and biantennary structures containing a mannose core with 6-sialylated lactosamine at one or more termini. Binding to both motifs was specific; isomers of each motif did not bind sperm. Further work focused on sialylated lactosamine. Sialylated lactosamine was found abundantly on the apical side of epithelial cells collected from the oviduct isthmus, among N-linked and O-linked glycans. Sialylated lactosamine bound to the head of sperm, the region that interacts with the oviduct epithelium. After capacitation, sperm lost affinity for sialylated lactosamine. Receptor modification may contribute to release from the reservoir so that sperm can move to the site of fertilization. Sialylated lactosamine was required for sperm to bind oviduct cells. Simbucus nigra agglutinin or an antibody specific to sialylated lactosamine with a preference for Neu5Acalpha2-6Gal rather than Neu5Acalpha2-3Gal reduced sperm binding to oviduct isthmic cells, as did occupying putative receptors on sperm with sialylated biantennary glycans. These results demonstrate that sperm binding to oviduct 6-sialylated biantennary glycans is necessary for normal adhesion to the oviduct.

Selective Exo‐Enzymatic Labeling of N‐Glycans on the Surface of Living Cells by Recombinant ST6Gal I

A game of tag: N-Glycans on the surface of living cells were selectively tagged by exogenously administering recombinant ST6Gal I sialyltransferase and azide-modified CMP-Neu5Ac. This modification was followed by a strain-promoted cycloaddition using a biotin-modified dibenzylcyclooctynol (red star=biotin). The methodology will make it possible to dissect the mechanisms that underlie altered glycoconjugate recycling and storage in disease.

Targeted release and fractionation reveal glucuronylated and sulphated N- and O-glycans in larvae of dipteran insects

Mosquitoes are important vectors of parasitic and viral diseases with Anopheles gambiae transmitting malaria and Aedes aegypti spreading yellow and Dengue fevers. Using two different approaches (solid-phase extraction and reversed-phase or hydrophilic interaction HPLC fractionation followed by MALDI-TOF MS or permethylation followed by NSI-MS), we examined the N-glycans of both A. gambiae and A. aegypti larvae and demonstrate the presence of a range of paucimannosidic glycans as well as bi- and tri-antennary glycans, some of which are modified with fucose or with sulphate or glucuronic acid residues; the latter anionic modifications were also found on N-glycans of larvae from another dipteran species (Drosophila melanogaster). The sulphate groups are attached primarily to core α-mannose residues (especially the α1,6-linked mannose), whereas the glucuronic acid residues are linked to non-reducing β1,3-galactose. Also, O-glycans were found to possess glucuronic acid and sulphate as well as phosphoethanolamine modifications. The presence of sulphated and glucuronylated N-glycans is a novel feature in dipteran glycomes; these structures have the potential to act as additional anionic glycan ligands involved in parasite interactions with the vector host.

Discrimination between Adenocarcinoma and Normal Pancreatic Ductal Fluid by Proteomic and Glycomic Analysis

Sensitive and specific biomarkers for pancreatic cancer are currently unavailable. The high mortality associated with adenocarcinoma of the pancreatic epithelium justifies the broadest possible search for new biomarkers that can facilitate early detection or monitor treatment efficacy. Protein glycosylation is altered in many cancers, leading many to propose that glycoproteomic changes may provide suitable biomarkers. In order to assess this possibility for pancreatic cancer, we have performed an in-depth LC-MS/MS analysis of the proteome and MS(n)-based characterization of the N-linked glycome of a small set of pancreatic ductal fluid obtained from normal, pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and pancreatic adenocarcinoma patients. Our results identify a set of seven proteins that were consistently increased in cancer ductal fluid compared to normal (AMYP, PRSS1, GP2-1, CCDC132, REG1A, REG1B, and REG3A) and one protein that was consistently decreased (LIPR2). These proteins are all directly or indirectly associated with the secretory pathway in normal pancreatic cells. Validation of these changes in abundance by Western blotting revealed increased REG protein glycoform diversity in cancer. Characterization of the total N-linked glycome of normal, IPMN, and adenocarcinoma ductal fluid clustered samples into three discrete groups based on the prevalence of six dominant glycans. Within each group, the profiles of less prevalent glycans were able to distinguish normal from cancer on this small set of samples. Our results emphasize that individual variation in protein glycosylation must be considered when assessing the value of a glycoproteomic marker, but also indicate that glycosylation diversity across human subjects can be reduced to simpler clusters of individuals whose N-linked glycans share structural features.

Neural-specific α3-fucosylation of N-linked glycans in the Drosophila embryo requires Fucosyltransferase A and influences developmental signaling associated with O-glycosylation

Addition of fucose (Fuc) to glycoprotein N-linked glycans or in O-linkage directly to Ser/Thr residues modulates specific cell-cell interactions and cell signaling events. Vertebrates and invertebrates add Fuc in α6-linkage to the reducing terminal N-acetylglucosamine residue of N-glycans. In Drosophila and other invertebrates, Fuc can also be added in α3-linkage to the same residue. These difucosylated N-glycans are recognized by anti-horseradish peroxidase (anti-HRP) antisera, providing a well-established marker for insect neural tissue. To understand the mechanisms and consequences of tissue-specific glycan expression, we identified a single α3-fucosyltransferase (FucTA) that produces the anti-HRP epitope in Drosophila embryos. FucTA transcripts are temporally and spatially restricted to cells that express the anti-HRP epitope and are missing in a mutant that lacks neural α3-fucosylation. Transgenic expression of FucTA, but not of any other candidate α3-fucosyltransferase, rescues the anti-HRP epitope in the embryonic nervous system of this mutant. Mass spectrometric characterization of the N-glycans of Drosophila embryos overexpressing FucTA confirms that this enzyme is indeed responsible for the biosynthesis of difucosylated glycans in vivo. Whereas ectopic expression of FucTA in the larval wing disc produces mild wing notching, the heterochronic, pan-neural expression of FucTA in early differentiating neurons generates neurogenic and cell migration phenotypes; this latter effect is associated with reduced GDP-Fuc levels in the embryo and indicates that the diversion of fucosylation resources towards fucosylation of N-glycans has an impact on developmental signaling associated with O-fucosylation.

THE GLYCOMICS OF GLYCAN GLUCURONYLATION IN DROSOPHILA MELANOGASTER

As glycan characterization methods increase in sensitivity, new opportunities arise to undertake glycomic analyses on limiting amounts of material. Developing systems present special challenges since the amount of available tissue can restrict deep glycan characterization. We have optimized mass spectrometric methods with the goal of obtaining full glycan profiles from small amounts of tissue derived from organisms of particular interest. A major target of our efforts has been the Drosophila embryo, allowing us to leverage the tools already developed in this organism to meld glycomics, genomics, and molecular genetics. Our analysis of the N-linked, O-linked (non-GAG), and glycosphingolipid (GSL) glycans of the Drosophila embryo have identified expected and unexpected glycan structures. We have verified previous findings regarding the predominance of high-Man and pauci-Man N-linked glycans, but have also detected minor families of sialylated and glucuronylated N-linked structures. Glucuronic acid (GlcA) also presents itself as an abundant modification of O-linked and GSL glycans. We describe critical advancements in our methodology and present the broad range of contexts in which GlcA is found in the Drosophila embryo.

GlyTouCan: an accessible glycan structure repository

Rapid and continued growth in the generation of glycomic data has revealed the need for enhanced development of basic infrastructure for presenting and interpreting these datasets in a manner that engages the broader biomedical research community. Early in their growth, the genomic and proteomic fields implemented mechanisms for assigning unique gene and protein identifiers that were essential for organizing data presentation and for enhancing bioinformatic approaches to extracting knowledge. Similar unique identifiers are currently absent from glycomic data. In order to facilitate continued growth and expanded accessibility of glycomic data, the authors strongly encourage the glycomics community to coordinate the submission of their glycan structures to the GlyTouCan Repository and to make use of GlyTouCan identifiers in their communications and publications. The authors also deeply encourage journals to recommend a submission workflow in which submitted publications utilize GlyTouCan identifiers as a standard reference for explicitly describing glycan structures cited in manuscripts.