Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase

Surprisingly alkaline phosphatase (AP) (EC 3.1.3.1) of calf intestine is fo und in large amounts, e.g. 80%, within chyme. Most of the enzyme is present as a mixture of four differently hydrophobic anchor-bearing forms and only the minor part is present as an anchorless enzyme. To investigate whether changes in the N-glycosylation pattern are signals responsible for large-sc ale liberation from mucosa into chyme, the glycans of the two potential gly cosylation sites predicted from cDNA were investigated by matrix-assisted l aser desorption/ionization and electrospray ionization mass spectrometry in combination with exoglycosidase treatment after Cryptic digestion and reve rsed-phase chromatography. The glycans linked to Asn249 are at least eight different, mainly non-fucosylated, biantennary or triantennary structures w ith a bisecting N-acetylglucosamine. For the most abundant glycopeptide (40 %) the following glycan structure is proposed: [GRAPHICS] The glycans linked to Asn410 are a mixture of at least nine, mainly tetraan tennary, fucosylated structures with a bisecting N-acetylglucosamine. For t he most abundant glycopeptide (35%) the following glycan structure is. prop osed: [GRAPHICS] For the structures the linkage data were deduced from the reported specific ities of the exoglycosidases used and the specificities of the transglycosi dases active in biosynthesis. The majority of glycans are capped by alpha - galactose residues at their non-reducing termini. In contrast to the glycan s linked to other AP isoenzymes, no sialylation was observed. Glycopeptide 'mass fingerprints' of both glycosylation sites and glycan contents do not differ between AP from mucosa and chyme. These results suggest that the obs erved large-scale liberation of vesicle-bound glycosylphosphatidylinositol (GPI)-anchored AP from mucosa into chyme is unlikely to be mediated by alte ration of glycan structures of the AP investigated. Rather, the exocytotic vesicle formation seems to be mediated by the controlled organization of th e raft structures embedding GPI-AP. Copyright (C) 2001 John Wiley & Sons, L td.