Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase: Novel fragments produced by trifluoroacetic acid

Mark A. Deeg, Dawn R. Humphrey, Shun Hua Yang, Tadd R. Ferguson, Vernon N. Reinhold, Terrone L. Rosenberry

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted α-δ were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan α showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan β was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans α and β were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan α contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan β contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.

Original languageEnglish (US)
Pages (from-to)18573-18580
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number26
StatePublished - Sep 15 1992
Externally publishedYes

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Trifluoroacetic Acid
Acetylcholinesterase
Anchors
Polysaccharides
Glucosamine
Phospholipids
Erythrocytes
Inositol
High performance liquid chromatography
Anions
Hexoses
Mass spectrometry
High Pressure Liquid Chromatography
Mannose
Glycosylphosphatidylinositol Diacylglycerol-Lyase
Stoichiometry
Mass Spectrometry
Ethanolamines
Hydrolysis
Ions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Deeg, M. A., Humphrey, D. R., Yang, S. H., Ferguson, T. R., Reinhold, V. N., & Rosenberry, T. L. (1992). Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase: Novel fragments produced by trifluoroacetic acid. Journal of Biological Chemistry, 267(26), 18573-18580.

Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase : Novel fragments produced by trifluoroacetic acid. / Deeg, Mark A.; Humphrey, Dawn R.; Yang, Shun Hua; Ferguson, Tadd R.; Reinhold, Vernon N.; Rosenberry, Terrone L.

In: Journal of Biological Chemistry, Vol. 267, No. 26, 15.09.1992, p. 18573-18580.

Research output: Contribution to journalArticle

Deeg, MA, Humphrey, DR, Yang, SH, Ferguson, TR, Reinhold, VN & Rosenberry, TL 1992, 'Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase: Novel fragments produced by trifluoroacetic acid', Journal of Biological Chemistry, vol. 267, no. 26, pp. 18573-18580.
Deeg, Mark A. ; Humphrey, Dawn R. ; Yang, Shun Hua ; Ferguson, Tadd R. ; Reinhold, Vernon N. ; Rosenberry, Terrone L. / Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase : Novel fragments produced by trifluoroacetic acid. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 26. pp. 18573-18580.
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abstract = "Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted α-δ were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan α showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan β was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans α and β were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan α contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan β contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50{\%} HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.",
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