Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase
Authors: Bublitz Renate , Hoppe Horst , Cumme Gerhard A. , Thiele Mariana , Attey Adrian , Horn Anton 
Published: August 2002

AbstractSurprisingly alkaline phosphatase (AP) (EC 3.1.3.1) of calf intestine is found 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‐scale liberation from mucosa into chyme, the glycans of the two potential glycosylation sites predicted from cDNA were investigated by matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry in combination with exoglycosidase treatment after tryptic digestion and reversed‐phase chromatography. The glycans linked to Asn249 are at least eight different, mainly non‐fucosylated, biantennary or triantennary structures with a bisecting N‐acetylglucosamine. For the most abundant glycopeptide (40%) the following glycan structure is proposed:
magnified image The glycans linked to Asn410 are a mixture of at least nine, mainly tetraantennary, fucosylated structures with a bisecting N‐acetylglucosamine. For the most abundant glycopeptide (35%) the following glycan structure is proposed:
magnified image For the structures the linkage data were deduced from the reported specificities of the exoglycosidases used and the specificities of the transglycosidases active in biosynthesis. The majority of glycans are capped by α‐galactose residues at their non‐reducing termini. In contrast to the glycans 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 observed large‐scale liberation of vesicle‐bound glycosylphosphatidylinositol (GPI)‐anchored AP from mucosa into chyme is unlikely to be mediated by alteration of glycan structures of the AP investigated. Rather, the exocytotic vesicle formation seems to be mediated by the controlled organization of the raft structures embedding GPI–AP. Copyright © 2001 John Wiley & Sons, Ltd.
Abbreviations:

ESI‐MS

electrospray ionization mass spectrometry

MALDI‐MS

matrix‐assisted laser desorption/ionization mass spectrometry

Dionex HPLC

high‐pH anion‐exchange chromatography

Gal

galactose

Man

mannose

GlcNAc

β‐N‐acetylglucosamine

GlcNAcase

N‐acetylglucosaminidase

Fuc

fucose

PNGase F

peptide‐N‐glycosidase F

AP

alkaline phosphatase

GPI

glycosylphosphatidylinositol

PtdIns‐PLC

phosphatidylinositol‐specific phospholipase C

S. pn.

Streptococcus pneumoniae

J. b. m.

Jack bean meal

B. t.

bovine testes.

ENZYME

alkaline phosphatase

(EC 3.1.3.1)

β‐N‐acetylhexosaminidase

(EC 3.2.1.30)

endoglycosidase H

(EC 3.2.1.96)

α‐fucosidase

(EC 3.2.1.51)

α‐galactosidase

(EC 3.2.1.22)

β‐galactosidase

(EC 3.2.1.23)

α‐mannosidase

(EC 3.2.1.24)

peptide‐N‐glycosidase F

(EC 3.5.1.52)

phosphatidylinositol‐specific phospholipase C

(EC 3.1.4.10)

sialidase

(EC 3.2.1.18)