Microheterogeneity of the Hydrophobic and Hydrophilic Part of the Glycosylphosphatidylinositol Anchor of Alkaline Phosphatase from Calf Intestine

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Abstract
Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl-(Etn-P)2-glycosyl-PtdIns with HF generated 8 glycosyl-Ins species the largest of which (G1 and G2) have the following proposed structures: G3 and G5 are lower homologues of G1 and G2, respectively, being one αl-2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N -acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic ManxGlc-Ins species are either substituted with an N -acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular-mass determination by matrix-assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO3. Purification of the glycosylinositol species was achieved by a novel reverse-phase HPLC technique using fluorescent fluoren-9-yl-methoxy-carbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level. We observed recently that native alkaline phosphatase separates on octyl-Sepharose into four distinct fractions of increasing hydrophobicity (F1–F4). Here we show that all four fractions contain G1–G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residues/subunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10% which is in contrast to most glycosyl-PtdIns–anchored proteins of mammalian origin.