Graduate School of Humanities and Sciences and the Glycoscience Institute, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
J Biol Chem. 2012 Jun 29;287(27):23104-18. doi: 10.1074/jbc.M111.314658. Epub 2012 May 14.
Porcine pancreatic α-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680-4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na(+)/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycan-specific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic α-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic α-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other.
猪胰腺α-淀粉酶（PPA）与糖蛋白的 N 连接聚糖结合（Matsushita，H.，Takenaka，M.和 Ogawa，H.（2002）J. Biol.Chem.，277，4680-4686）。免疫染色显示 PPA 位于十二指肠肠上皮细胞的刷状缘膜（BBM）上，并且该结合被甘露聚糖但不是半乳糖聚糖抑制，表明 PPA 特异性地与 BBM 结合碳水化合物。BBM 中 PPA 的配体被鉴定为糖蛋白 N-聚糖，这些聚糖在葡萄糖同化中起重要作用，包括蔗糖酶-异麦芽糖酶（SI）和 Na（+）/Glc 共转运蛋白 1（SGLT1）。BBM 中 SI 和 SGLT1 与 PPA 的结合呈剂量依赖性，并被甘露聚糖抑制。使用 BBM 囊泡，我们发现由于 N-聚糖特异性相互作用，BBM 中的 PPA 和其配体的功能发生变化。PPA 的淀粉降解活性和 SI 的麦芽糖降解活性分别增强到 240%和 175%，而 SGLT1 的 Glc 摄取则由于 PPA 在高但生理上可能的浓度下被显著抑制，并且结合被甘露糖特异性凝集素的添加减弱，特别是来自雪花莲的凝集素。此外，在与糖生物素聚合物探针的结合测定中，在酵母中表达并通过单步亲和层析纯化的重组人胰腺α-淀粉酶表现出与 PPA 相同的碳水化合物结合特异性。结果表明，哺乳动物胰腺α-淀粉酶具有共同的碳水化合物结合活性，并且特异性地与肠 BBM 结合。与 BBM 中的 N-聚糖相互作用一方面激活了 PPA 和 SI，以产生更多的 Glc，另一方面通过 SGLT1 抑制肠上皮细胞对 Glc 的吸收，以防止血糖迅速升高。
