牛肠碱性磷酸酶(biALP),一种膜结合的血浆金属酶,维持肠道稳态,调节十二指肠表面的pH值,并防止致病菌引起的感染。biALP的N-聚糖调节其酶活性,蛋白质折叠,和热稳定性,但是它们的结构没有完全报道。在这项研究中,通过液相色谱-电喷雾电离-高能碰撞解离-串联质谱法分析了biALP的N-聚糖的结构和数量。总的来说,对48个N-聚糖进行了鉴定和定量,包含高甘露糖[6个N-聚糖,33.1%(每种N-聚糖的相对数量之和)],混合(6,11.9%),和复杂(36,55.0%)结构[双(13,26.1%),三(16,21.5%),和四天线(7,7.4%)]。这些包括平分N-乙酰葡糖胺(33,56.6%),单-三岩藻糖基化(32,53.3%),单-到三-α-半乳糖基化(16,20.7%),和单-到四-β-半乳糖基化(36,58.5%)。没有确定唾液酸化。非平分GlcNAc的N-聚糖(9,10.3%),非岩藻糖基化(10,13.6%),非α-半乳糖基化(26,46.2%),和非β-半乳糖基化(6,8.4%)也被鉴定。biALP的活性(100%)降低至37.3±0.2%(通过脱岩藻糖基化),32.7±2.9%(通过去-α-半乳糖基化),和0.2±0.2%(通过去-β-半乳糖基化),与10-4至101mMEDTA的抑制作用相当,BiALP抑制剂。这些结果表明岩藻糖基化和半乳糖基化的N-聚糖,尤其是β-半乳糖基化,影响biALP的活性。这项研究首次鉴定了48种不同的N-聚糖结构和数量的牛以及人肠ALP,并证明了岩藻糖基化和半乳糖基化对维持biALP活性的重要性。
Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1 % (sum of relative quantities of each N-glycan)], hybrid (6, 11.9 %), and complex (36, 55.0 %) structures [bi- (13, 26.1 %), tri- (16, 21.5 %), and tetra-antennary (7, 7.4 %)]. These included bisecting N-acetylglucosamine (33, 56.6 %), mono-to tri-
fucosylation (32, 53.3 %), mono-to tri-α-galactosylation (16, 20.7 %), and mono-to tetra-β-galactosylation (36, 58.5 %). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3 %), non-
fucosylation (10, 13.6 %), non-α-galactosylation (26, 46.2 %), and non-β-galactosylation (6, 8.4 %) were also identified. The activity (100 %) of biALP was reduced to 37.3 ± 0.2 % (by de-
fucosylation), 32.7 ± 2.9 % (by de-α-galactosylation), and 0.2 ± 0.2 % (by de-β-galactosylation), comparable to inhibition by 10-4 to 101 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially β-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of
fucosylation and galactosylation for maintaining the activity of biALP.