**1. Introduction**

α-Amylase has been found in many organisms, including bacteria, mainly Bacilli, seeds of cereals and legumes, and digestive glands of animals such as humans and pigs. These α-amylases can be obtained as homogeneous preparations and utilized in various situations. For example, mammalian α-amylase is used as a gastrointestinal medicine and digestive agent. Microbial α-amylase is used in sugar production, food processing, clothing production, and detergents because it can be produced on an industrial scale. The uses of these amylases all focus on the ability of the amylase to degrade polysaccharides. The α-amylase in pancreatic juice and saliva clearly functions as a digestive enzyme. It is an endo-type enzyme that randomly cleaves α-1,4 glycosidic bonds such as starch and glycogen to produce maltose and oligosaccharides [1]. Therefore, α-amylase in digestive juices is an essential enzyme for animals consuming a starch diet. α-Amylase has been found in liver [2], brain [3], blood, urine, and cancer cells [4] in humans, but its physiological significance has not been completely elucidated yet.

It has been reported that porcine pancreatic α-amylase binds to an *N*-linked glycan of glycoproteins [5]. It has been shown that the binding of porcine pancreatic α-amylase to *N*-linked glycans is different from that of the polysaccharide as a substrate. It shows that the carbohydrate-binding activities of pancreatic α-amylase are not common to all types of α-amylase. α-Amylases from barley, *Bacillus subtilis*, and mammalian saliva do not have this activity. Porcine pancreatic α-amylase binds mainly to transferrin having complex-type biantennary *N*-glycans, fetuin having complex-type triantennary *N*-glycans, and ribonuclease B having high-mannose *N*-glycans. However, it does not bind to bovine submandibular gland mucin having *O*-glycans or bovine serum albumin having no sugar chains. The carbohydratebinding activities of porcine pancreatic α-amylase are affected by pH. Porcine pancreatic α-amylase has a high affinity for both complex type and high-mannose *N*-glycans under pH 5.5. On the other hand, the binding activity is seen with only the high-mannose *N*-glycans, and the binding to the complex *N*-glycans is reduced under neutral pH. Thus, pancreatic α-amylase selectively recognizes the structure of *N*-glycans of glycoproteins depending on pH.

The biological significance of the *N*-glycan binding found in pancreatic α-amylase in vivo is unknown. Pancreatic α-amylase is synthesized in pancreatic acinar cells, packed into acidic zymogen granules (pH 5.5), and secreted into alkaline pancreatic juice (pH 8.0) [6]. Pancreatic juice flows into the duodenum and mixes with gastric acid where it is neutralized, and pancreatic α-amylase exerts its enzymatic activity. The pH in the middle of the duodenum falls from pH 6.5 to 4.5 in a 3-hour period after a meal [7]. There are glycoproteins with *N*-glycans in pancreatic zymogen granules, its membrane, and the lumen of the small intestine [8–11]. These suggest that the *N*-glycan-binding activity of pancreatic α-amylase may be play a role in the packing of pancreatic zymogen granules, its exocytosis in the pancreas, and digestion and absorption of carbohydrates in the small intestine.

In this study, we elucidated the roles of the *N*-glycan-binding activity of pancreatic α-amylase in the small intestine by identifying the glycoproteins to which the α-amylase binds on the small intestinal brush border membrane (BBM), the luminal surface of the small intestine where many glycoproteins are located. The functions and localizations of the identified α-amylase-binding-glycoproteins and analyses of interactions between α-amylase and the glycoproteins revealed that pancreatic α-amylase has the following regulatory functions other than as a digestive enzyme: (1) enhancement of α-glucosidase, sucrase-isomaltase, (2) regulation of sodium-dependent glucose uptake, and (3) localization of pancreatic α-amylase in the small intestine. Furthermore, the new discoveries show that duodenal epithelial cells express α-amylase, which is essential for proliferation and differentiation of small intestinal epithelial cells.
