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Dauber A.,Boston Childrens Hospital | Dauber A.,Cambridge Broad Institute | Ercan A.,Immunology and Allergy | Lee J.,5 Francis Street | And 17 more authors.
Human Molecular Genetics

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migrationcausedbymutationsin the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.© The Author 2014. Published by Oxford University Press. All rights reserved.All rights reserved. Source

Ashline D.J.,Glycan Connections LLC. | Hanneman A.J.S.,Glycan Connections LLC. | Zhang H.,University of New Hampshire | Reinhold V.N.,Glycan Connections LLC. | Reinhold V.N.,University of New Hampshire
Journal of the American Society for Mass Spectrometry

Documenting mass spectral data is a fundamental aspect of accepted protocols. In this report, we contrast MSn sequential disassembly spectra obtained from natural and synthetic glycan epitopes. The epitopes considered are clusters found on conjugate termini of lipids and N- and O-glycans of proteins. The latter are most frequently pendant through a CID-labile HexNAc glycosidic linkage. The synthetic samples were supplied by collaborating colleagues and commercial sources and usually possessed a readily released reducing-end linker, a by-product of synthesis. All samples were comparably methylated, extracted, and MSn disassembled to compare their linkage and branching spectral details. Both sample types provide B-ion type fragments early in a disassembly pathway and their compositions are a suggestion of structure. Further steps of disassembly are necessary to confirm the details of linkage and branching. Included in this study were various Lewis and H antigens, 3- and 6-linked sialyl-lactosamine, NeuAc-2,8-NeuAc dimer, and Galα1,3Gal. Sample infusion provided high quality spectral data whereas disassembly to small fragments generates reproducible high signal/noise spectra for spectral matching. All samples were analyzed as sodium adducted positive ions. This study includes comparability statistics and evaluations on several mass spectrometers. [Figure not available: see fulltext.] © 2013 American Society for Mass Spectrometry. Source

Zhou H.,University of New Hampshire | Zhou H.,Harvard University | Hanneman A.J.,University of New Hampshire | Hanneman A.J.,Glycan Connections LLC. | And 3 more authors.
Journal of Proteome Research

This report describes the structural details of a unique N-linked valence epitope on the major protein within the extrapallial (EP) fluid of the mollusk, Mytilus edulis. Fluids from this area are considered to be responsible for shell expansion by a self-assembly process that provides an organic framework for the growth of CaCO3 crystals. Previous reports from our laboratories have described the purification and amino acid sequence of this EP protein, which was found to be a glycoprotein (EPG) of approximately 28 KDa with 14.3% carbohydrate on a single N-linked consensus site. Described herein is the de novo sequence of the major glycan and its glycomers. The sequence was determined by ion trap sequential mass spectrometry (ITMSn) resolving structure by tracking precursor-product relationships through successive rounds of collision induced disassociation (CID), thereby spatially resolving linkage and branching details within the confines of the ion trap. Three major glycomers were detected, each possessing a 6-linked fucosylated N-linked core. Two glycans possessed four and five identical antennae, while the third possessed four antennas, but with an additional methylfucose 2-linked to the glucuronic acid moiety, forming a pentasaccharide. The tetrasaccharide structure was: 4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc, while the pentasaccharide was shown to be as follows: mono-O-methyl-Fuc(1-2)-4-O-methyl-GlcA(1-4)[GlcNAc(1-3)] Fuc(1-4)GlcNAc. Samples were differentially deuteriomethylated (CD 3/CH3) to localize indigenous methylation, further analyzed by high resolution mass spectrometry (HRMS) to confirm monomer compositions, and finally gas chromatography mass spectrometry (GC-MS) to assign structural and stereoisomers. The interfacial shell surface location of this major extrapallial glycoprotein, its calcium and heavy metal binding properties and unique structure suggests a probable role in shell formation and possibly metal ion detoxification. A closely related terminal tetrasaccharide structure has been reported in spermatozoan glycolipids of freshwater bivalves. © 2013 American Chemical Society. Source

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