title: 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 de novo sequence of the major glycan and its glycomers. The sequence was determined by ion trap sequential mass spectrometry (ITMS(n)) 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 (CD3/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.
authors.affiliation: Glycomics Center, University of New Hampshire , 35 Colovos Road, Durham, New Hampshire 03824, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Hanneman
authors.forename: N Dennis
authors.initials: ND
authors.lastname: Chasteen
authors.forename: Vernon N
authors.initials: VN
authors.lastname: Reinhold
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D060105:Animal Shells / Q000378:metabolism*; D000818:Animals; D001826:Body Fluids / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005643:Fucose / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D008757:Methylglucosides / Q000378:metabolism; D049880:Mytilus edulis / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D006023:Glycoproteins; D008757:Methylglucosides; D011134:Polysaccharides; D005643:Fucose; D000117:Acetylglucosamine
references.citation: Anal Chem. 2005 Oct 1;77(19):6250-62
references.pmid: 16194086
references.citation: J Biol Chem. 2007 Aug 10;282(32):23253-63
references.pmid: 17558025
references.citation: Comp Biochem Physiol B Biochem Mol Biol. 2000 Sep;127(1):105-12
references.pmid: 11126745
references.citation: Biochemistry. 2005 Aug 9;44(31):10720-31
references.pmid: 16060681
references.citation: Chem Phys Lipids. 1986 Dec 15;42(1-3):27-48
references.pmid: 3549018
references.citation: Prog Mol Subcell Biol. 2011;52:331-52
references.pmid: 21877272
references.citation: Nat Mater. 2010 Dec;9(12):960-1
references.pmid: 21102512
references.citation: Glycobiology. 2006 Sep;16(9):874-90
references.pmid: 16769777
references.citation: Anal Biochem. 2008 May 1;376(1):44-60
references.pmid: 18294950
references.citation: Aquat Toxicol. 2001 Apr;52(2):133-42
references.pmid: 11164535
references.citation: Mar Environ Res. 2011 Feb;71(1):17-21
references.pmid: 20950850
references.citation: Int J Mass Spectrom. 2011 Jun 1;303(2-3):109-117
references.pmid: 21686090
references.citation: Methods Mol Biol. 2009;534:53-64
references.pmid: 19277536
references.citation: Comp Biochem Physiol B Biochem Mol Biol. 2007 May;147(1):74-81
references.pmid: 17276716
references.citation: Curr Top Dev Biol. 2008;80:209-76
references.pmid: 17950376
references.citation: Arch Biochem Biophys. 1999 Jun 1;366(1):8-14
references.pmid: 10334857
references.citation: J Am Chem Soc. 2003 Dec 31;125(52):16213-9
references.pmid: 14692762
references.citation: Environ Toxicol Chem. 2007 May;26(5):872-7
references.pmid: 17521131
references.citation: Biochem Biophys Res Commun. 2005 Jun 3;331(2):562-70
references.pmid: 15850797
references.citation: J Biol Chem. 1983 Feb 25;258(4):2239-45
references.pmid: 6822554
references.citation: Mol Cell Proteomics. 2013 Apr;12(4):866-73
references.pmid: 23438731
references.citation: J Proteome Res. 2010 Sep 3;9(9):4823-30
references.pmid: 20690605
references.citation: Mar Environ Res. 2000 Jul-Dec;50(1-5):99-102
references.pmid: 11460758
references.citation: Glycobiology. 2003 Jul;13(7):41R-53R
references.pmid: 12651883
references.citation: Anal Biochem. 1979 Dec;100(2):324-34
references.pmid: 230749
references.citation: Anal Chem. 2007 May 15;79(10):3830-42
references.pmid: 17397137
references.citation: Biol Bull. 1998 Aug;195(1):43-51
references.pmid: 9739548
references.citation: Rapid Commun Mass Spectrom. 2002;16(12):1199-204
references.pmid: 12112272
references.citation: Environ Toxicol Chem. 2001 Jul;20(7):1596-604
references.pmid: 11434304
references.citation: J Am Soc Mass Spectrom. 2005 May;16(5):622-30
references.pmid: 15862764
references.citation: J Biol Chem. 1981 Nov 10;256(21):10979-85
references.pmid: 7287744
references.citation: Nat Chem Biol. 2010 Oct;6(10):713-23
references.pmid: 20852609
references.citation: J Mass Spectrom. 2005 May;40(5):642-53
references.pmid: 15751107
references.citation: J Mass Spectrom. 2010 May;45(5):528-35
references.pmid: 20446311
references.citation: Comp Biochem Physiol B Biochem Mol Biol. 2004 Jul;138(3):221-8
references.pmid: 15253870
references.citation: J Am Soc Mass Spectrom. 2005 May;16(5):631-46
references.pmid: 15862765
references.citation: J Mass Spectrom. 2009 Jan;44(1):50-60
references.pmid: 18788072
references.citation: Rapid Commun Mass Spectrom. 2010 Mar 15;24(5):571-85
references.pmid: 20131323
references.citation: Comp Biochem Physiol B Biochem Mol Biol. 2000 Mar;125(3):293-306
references.pmid: 10818264
references.citation: Glycobiology. 1991 Mar;1(2):163-71
references.pmid: 1823159
references.citation: Eur J Biochem. 1995 Sep 15;232(3):873-80
references.pmid: 7588729
references.citation: Carbohydr Res. 1986 Nov 1;155:45-56
references.pmid: 3791301
references.citation: J Biol Chem. 2001 Feb 9;276(6):4461-8
references.pmid: 11084027
references.citation: Comp Biochem Physiol A Mol Integr Physiol. 2005 Jul;141(3):319-26
references.pmid: 16006160
references.citation: Eur J Biochem. 2004 Apr;271(7):1348-56
references.pmid: 15030485
references.citation: J Biol Chem. 2011 Feb 11;286(6):4371-81
references.pmid: 21115491
references.citation: Cell. 2010 Nov 24;143(5):672-6
references.pmid: 21111227
references.citation: J Am Soc Mass Spectrom. 2005 May;16(5):647-59
references.pmid: 15862766
references.citation: Carbohydr Res. 2001 Jun 15;332(4):381-8
references.pmid: 11438095