GLYCOINOSITOL PHOSPHOLIPID ANCHOR AND PROTEIN C-TERMINUS OF BOVINE ERYTHROCYTE ACETYLCHOLINESTERASE - ANALYSIS BY MASS-SPECTROMETRY AND BY PROTEIN AND DNA-SEQUENCING

Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethy lated on its amine groups and incubated with bacterial phosphatidylino sitol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fra gment that contained the residual GPI glycan was isolated by HPLC. Ana lysis by electrospray-ionization mass spectrometry revealed a parent i on of m/z 3798. The fragmentation patterns produced by collision-induc ed dissociation mass spectrometry of the +4 and +5 states of the paren t ion indicated a 23-amino acid peptide in amide linkage to ethanolami ne-PO4-Hex-Hex-Hex(PO4-ethanol (HexNAc)-HexN(Me)(2)-inositol phosphate , The glycan structure is completely consistent with that obtained pre viously for the GPI anchor of human erythrocyte AChE except for the ad dition of the HexNAc substituent. A nearly complete peptide sequence w as 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 establi shed the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTC-SGPAHG , 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 bo vine AChE contained two cysteine residues in a...CTC... motif, in cont rast with human AChE which contains only a single cysteine in this seg ment. Although bovine AChE contained no free thiol groups reactive wit h iodo[C-14]acetamide, partial reduction and alkylation with iodo[C-14 ]acetamide revealed that conversion into monomers occurred with an ove rall incorporation of only one alkyl group per monomer. An identical l evel 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 consider ed.