Structural study on the carbohydrate moiety of calf intestinal alkaline phosphatase

Abstract

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: [carbostructure: see text]. 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: [carbostructure: see text]. 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 alpha-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. (c) 2001 John Wiley & Sons, Ltd.

Identification of N-glycan of alpha-fetoprotein by lectin affinity microarray

“…5, 6). The high signal of AAL, ConA, DSA, PHA-L, RCA-I on AKP aYnity pattern suggested that the N-glycan structure of AKP are mainly non-fucosylated biantennary, triantennary with a bisecting GlcNAc, tetraantennary structures and GalNAc (Bublitz et al 2001). The high signal of ConA, DSA, WGA and RCA-I on CO aYnity pattern reXected that the N-glycan structure of CO consists mainly of high-mannose-type, hybrid-type oligosaccharide chains, GlcNAc, Sia and GalNAc (Mao et al 2003).…”

“…4 and Table 1). According to the literature, the N-glycan structure of AKP is mainly nonfucosylated, biantennary or triantennary structures with a bisecting N-acetylglucosamine (GlcNAC) and tetraantennary, fucosylated structures with a bisecting GlcNAc (Bublitz et al 2001). The N-glycan structure of CO consists of high-mannose (Man)-type, hybrid-type oligosaccharide chains and sialic acid (Sia) (Mao et al 2003).…”

Boronate affinity controllable oriented surface imprinting of glycans in organic phase using intact glycoproteins as semi-templates

“…印迹步骤如图1所 [41] . 取200 mg AMNPs, 结 构 的 糖 链 , 其 中 有 两 种 糖 链 的 含 量 可 以 达 到 75% [42] . 其结构如图3所示, 其尺寸用ChemBio 3D软 件估算, 得到两种糖链的长度分别为3.…”

Comparison of the single molecule activity distributions of recombinant and non-recombinant bovine intestinal alkaline phosphatase

“…Bovine intestinal alkaline phosphatase is a highly glycosylated enzyme [11]. Single molecule assays have shown this enzyme to be heterogeneous.…”