MICROHETEROGENEITY IN GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR STRUCTURES OF BOVINE LIVER 5'-NUCLEOTIDASE

In our study, 5'-nucleotidase was released from bovine liver by the tr eatment with Bacillus thuringiensis phosphatidylinositol-specific phos pholipase C and purified to a homogeneous state by concanavalin A-Seph arose and (diethylaminoethyl)-Toyopearl column chromatography and sodi um dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nuc leotidase were then cleaved by cyanogen bromide (CNBr), and then inosi tol phosphoglycan-containing C-terminal peptides (IPG peptides) were s eparated by C18 reverse-phase liquid chromatography and analyzed by pe ptide sequencer, amino acid analyzer, gas chromatography (GC), and GC- mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 6 07-613] is revealed to be the C-terminal amino acid to which a glycosy lphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Ei ght different molecular weight (MW) species of CNBr-cleaved IPG peptid es were detected. Three fractions of CNBr-cleaved IPG peptides were se parately treated by trypsin, and trypsinized IPG peptides were purifie d by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8 , and 1994.5) were detected by ESI-MS. Together with sequential exogly cosidase treatment and quantitative analysis of sugar moieties by GC a nd GC-MS, microheterogeneity in the structures of these five glycosylp hosphatidylinositol (GPI) anchor species was determined. The common co re structure was ethanolamine phosphate-mannose-mannose-mannose(-ethan olamine phosphate)-glucosamine-myo-inositol phosphate. Variations obse rved in additional mannose, N-acetylhexosamine, and ethanolamine phosp hate moieties form this heterogeneity. One additional ethanolamine pho sphate residue connected to the mannose next to glucosamine was common to these five GPI anchor structures.