Anomalous N-Glycan Structures with an Internal Fucose Branched to GlcA and GlcN Residues Isolated from a Mollusk Shell-Forming Fluid

Abstract

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 the de novo sequence of the major glycan and its glycomers. The sequence was determined by ion trap mass spectrometer (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: mono-O-methyl-Fuc(1-2)-4-O-methyl-GlcA(1-4)[GlcNAc(1-3)]Fuc(1-4)GlcNAc. Samples were differentially deuteriomethylated (CD3/CH3) to localize indigenous methylation, further analyzed by HRMS to confirm monomer compositions, and finally GC-MS to assign structural and stereoisomers. The interfacial shell surface location of this major extrapallial glycoprotein, its calcium 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.

Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

“…To locate the position of the fucosylation, the permethylated N-glycans from both forms were subjected to ESI-MS n fragmentation [15,16]. When the fucosylated composition (Hex3HexNAc2Fuc1) from the tetramer was analyzed, the fucose residue was found to be linked to the reducing end GlcNAc of the oligosaccharide Man3GlcNAc2Fuc1 (Supplemental Fig.1).…”

“…When required, N-glycans were further reduced to the corresponding alditols by the addition of 200 μL of NaBH 4 (10 mg/mL in 0.01M NaOH). The reduced N-glycans were purified using hand-packed cellulose cartridges [15] and permethylated [31] for MS n analysis.…”

Susceptibility of Vibrio aestuarianus 01/032 to the antibacterial activity of Mytilus haemolymph: identification of a serum opsonin involved in mannose‐sensitive interactions

“…EP is an acidic glycoprotein with a high histidine content that can bind Ca 2+ and heavy metals (Renwrantz et al ., ; Hattan et al ., ; Schneeweiss et al ., ; Yin et al ., ; Abebe et al ., ). Recently, by MS‐based approach, a complex and anomalous N‐glycan structure was determined in M. edulis EP (Zhou et al ., ). Such unique structure and calcium and heavy metal‐binding properties suggest a possible role of EP in multiple biological functions including shell formation, metal ion transportation and detoxification (Zhou et al ., ).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…Recent examples of such compounds discovered using MALDI techniques include the high‐mannose glycans from Chlamydomonas reinhardtii which contain one and two xylose residues and up to three methyl mannose units (Mathieu‐Rivet et al, ). Also, Zhou et al () have identified tetra‐antennary glycans with internal fucose residues containing 3‐linked GlcNAc and 4‐linked 4‐methylglycuronic acid in place of each galactose residue ( 75 , 76 , 77 ). In these structures and structures 78–81 , the logical symbols proposed by Harvey et al () rather than the internally inconsistent (as the authors admit) (Varki et al, ), but more commonly used symbols originally proposed by the Consortium for Functional Glycomics.…”