Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase

• Published in: Journal of mass spectrometry. Vol. 36; no. 8; pp. 960 - 972
• Main Authors: Bublitz, Renate, Hoppe, Horst, Cumme, Gerhard A., Thiele, Mariana, Attey, Adrian, Horn, Anton
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01-08-2001; Wiley
• Subjects: Alkaline Phosphatase - chemistry; Analytical, structural and metabolic biochemistry; Animals; Asparagine; Biological and medical sciences; Carbohydrate Conformation; Carbohydrate Sequence; Carbohydrates; Cattle; DNA, Complementary; electrospray ionization mass spectrometry; exoglycosidase digestion; Fundamental and applied biological sciences. Psychology; glycan structures; Glycopeptides - chemistry; Glycoproteins - chemistry; Glycoside Hydrolases; Intestinal Mucosa - enzymology; matrix-assisted laser desorption/ionization mass spectrometry; Miscellaneous; Molecular Sequence Data; Oligosaccharides - chemistry; Other biological molecules; Polysaccharides - chemistry; Recombinant Proteins - chemistry; tryptic peptides; Alkaline phosphatase; Enzyme; Phosphoric monoester hydrolases; Esterases; Matrix assisted laser desorption ionization; Electrospray; Glycan; Branched chain; Glycopeptide; Aminoglycoside; Asparagine; Hydrolases; Mass spectrometry; Structural analysis
• ISSN: 1076-5174; 1096-9888
• DOI: 10.1002/jms.200

Surprisingly 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:
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:
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)