**2.1 Binding of pancreatic α-amylase to small intestinal BBM**

Tissue sections of pig duodenum were immunostained with an anti-pancreatic α-amylase antibody. Entire duodenal tissue sections were stained with the pancreatic α-amylase antibody when there was a large amount of food in the stomach (during non-fasting), but hardly stained when there was little in the stomach (during fasting) [14]. The localization of exogenous pancreatic α-amylase was examined by staining a 1-cm section of the duodenum of a fasted pig with the pancreatic α-amylase antibody. Tissue sections fixed in formalin and embedded in paraffin were immunostained to detect pancreatic α-amylase. The duodenal sections were agitated in pancreatic α-amylase solution at 4°C. As a result, pancreatic α-amylase bound to the BBM, which is the upper end of the duodenal epithelium (**Figure 1**), and α-amylase staining increased in a time-dependent manner with agitation. Mannan inhibited the binding of pancreatic α-amylase to BBM duodenum sections incubated in α-amylase solution for 30 min. On the other hand, galactan and colominic acid inhibited binding of α-amylase to BBM after 10 min of agitation, but did not inhibit binding of α-amylase to BBM agitated for 30 min (data not shown) [14]. Pancreatic α-amylase bound to duodenum BBM, and its binding was inhibited by

**Figure 1.** *Binding of pancreatic α-amylase to pig duodenum BBM.*

*New Insights into Metabolic Syndrome*

has not been completely elucidated yet.

of *N*-glycans of glycoproteins depending on pH.

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

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

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

**2. Identification of glycoproteins that bind pancreatic α-amylase in the** 

In the small intestine, epithelial cells in the luminal villi have a brush border on the surface, and alkaline phosphatase and other membrane-type digestive enzymes

**200**

small intestinal epithelial cells.

**small intestinal BBM**

mannan, indicating that α-amylase binds to duodenal BBM in a mannose-specific manner, even at the tissue level.

One-centimeter duodenum sections from fasted pigs were incubated with pancreatic α-amylase (10 μM) in PBS (pH 7.2) including phenylmethylsulfonyl fluoride (final concentratation 1 mM) at 4°C for 30 min, then fixed and paraffinembedded. The paraffin sections were immunostained with rabbit anti-α-amylase IgGs-HRP. The color was developed with DAB/H2O2 and then counterstained with hematoxylin.
