Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase

Analysis by mass spectrometry and by protein and DNA sequencing

Robert Haas, Brent C. Jackson, Bruce Reinhold, John D. Foster, Terrone L. Rosenberry

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-PO4-Hex-Hex-Hex(PO4-ethanolamine) (HexNAc)-HexN(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a...CTC... motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.

Original languageEnglish (US)
Pages (from-to)817-825
Number of pages9
JournalBiochemical Journal
Volume314
Issue number3
StatePublished - Mar 15 1996
Externally publishedYes

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Protein Sequence Analysis
Acetylcholinesterase
Protein C
Anchors
DNA Sequence Analysis
Mass spectrometry
Mass Spectrometry
Phospholipids
Erythrocytes
DNA
Proteins
Peptides
Ethanolamine
Monomers
Alkylation
Disulfides
Polysaccharides
Cysteine
Ions
Phosphoinositide Phospholipase C

ASJC Scopus subject areas

  • Biochemistry

Cite this

Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase : Analysis by mass spectrometry and by protein and DNA sequencing. / Haas, Robert; Jackson, Brent C.; Reinhold, Bruce; Foster, John D.; Rosenberry, Terrone L.

In: Biochemical Journal, Vol. 314, No. 3, 15.03.1996, p. 817-825.

Research output: Contribution to journalArticle

Haas, Robert ; Jackson, Brent C. ; Reinhold, Bruce ; Foster, John D. ; Rosenberry, Terrone L. / Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase : Analysis by mass spectrometry and by protein and DNA sequencing. In: Biochemical Journal. 1996 ; Vol. 314, No. 3. pp. 817-825.
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abstract = "Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-PO4-Hex-Hex-Hex(PO4-ethanolamine) (HexNAc)-HexN(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a...CTC... motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.",
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