Contribution of the Individual Small Intestinal α-Glucosidases to

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Contribution of the Individual Small Intestinal #-Glucosidases on Digestion of Unusual #-Linked Glycemic Disaccharides Byung-Hoo Lee, David R Rose, Amy Hui-Mei Lin, Roberto Quezada-Calvillo, Buford L. Nichols, and Bruce R. Hamaker J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01816 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Journal of Agricultural and Food Chemistry

Contribution of the Individual Small Intestinal α-Glucosidases on Digestion of Unusual αLinked Glycemic Disaccharides

Byung-Hoo Lee,†, § David R. Rose,‡ Amy Hui-Mei Lin,§,# Roberto Quezada-Calvillo,∆ Buford L. Nichols,⊥ Bruce R. Hamaker§,∥,*

†

Department of Food Science & Biotechnology, College of BioNano Technology, Gachon

University, Seongnam, Gyeonggi-do 13120, Republic of Korea §

Whistler Center for Carbohydrate Research, Department of Food Science, Purdue University,

West Lafayette, IN 47907, USA ‡

Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

#

Bi-State School of Food Science, University of Idaho and Washington State University,

Moscow, ID 83844, USA ∆

Department of Chemistry, Universidad Autonoma de San Luis Potosi, SLP 78360, Mexico

⊥

USDA, Agricultural Research Service, Children’s Nutrition Research Center and Department

of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA ∥

Department of Food Science & Technology, Sejong University, Gunja-Dong, Gwangjin-Gu,

Seoul 05006, Republic of Korea *

Corresponding Author:

Tel: +1-765- 494-5668; Fax: +1-765-494-7953 E-mail: [email protected]

1 ACS Paragon Plus Environment


1

2

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ABSTRACT The mammalian mucosal α-glucosidase complexes, maltase-glucoamylase (MGAM) and

3

sucrase-isomaltase (SI), have two catalytic subunits (N- and C-terminals). Concurrent with the

4

desire to modulated glycemic response, there has been a focus on di-/oligo-saccharides with

5

unusual α-linkages that are digested to glucose slowly by these enzymes. Here, we look at

6

disaccharides with various possible α-linkages and their hydrolysis. Hydrolytic properties of the

7

maltose and sucrose isomers were determined using rat intestinal and individual recombinant α-

8

glucosidases. The individual α-glucosidases had moderate to low hydrolytic activities on all α-

9

linked disaccharides, except trehalose. Maltase (N-terminal MGAM) showed a higher ability to

10

digest α-1,2 and -1,3 disaccharides, as well as α-1,4, making it the most versatile in α-hydrolytic

11

activity. These findings apply to the development of new glycemic oligosaccharides based on

12

unusual α-linkages for extended glycemic response. It also emphasizes that mammalian mucosal

13

α-glucosidases must be used in in vitro assessment of digestion of such carbohydrates.

14

Keywords: α-glucosidases, carbohydrate digestion, disaccharides, glycemic, slowly digestible carbohydrates

15 16

17


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INTRODUCTION Digestible carbohydrate-based diets such as those containing starch or sucrose depend

20

upon the activities of small intestinal mucosal α-glucosidases to produce glucose and fructose.

21

After hydrolysis, the released monosaccharides are absorbed into the blood stream via the

22

sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) for glucose

23

and glucose transporter 5 (GLUT 5) for fructose. The absorbed monosaccharides are utilized as

24

carbon and energy sources.1-3 The mucosal α-glucosidases consist of two complexes: maltase-

25

glucoamylase [MGAM; EC 3.2.1.20 for maltase (N-terminal MGAM, or ntMGAM) and EC

26

3.2.1.3 for glucoamylase (C-terminal MGAM, or ctMGAM)] and sucrase-isomaltase [SI; EC

27

3.2.1.48 for sucrase (ctSI) and EC 3.2.1.10 for isomaltase (ntSI)]. Both MGAM and SI consist of

28

two catalytic enzymes: the C-terminal luminal domains (ctMGAM and ctSI) and N-terminal

29

membrane-proximal domains (ntMGAM and ntSI). The N-terminal α-glucosidases are connected

30

to the brush-border membrane via an O-glycosylated linker in the small intestine (Figure 1).4, 5

31

Based on the carbohydrate-active enzymes (CAZY) classification,6 mucosal α-glucosidases are

32

classified as Glycoside Hydrolase Family 31 (GH31) enzymes due to structural and functional

33

similarities of their proteins. Furthermore, all four individual mucosal α-glucosidases include the

34

signature amino acid sequences “WIDMNE” in the catalytic site, and share 40-60% protein

35

sequence identity.7-9

36

While all four enzymes have α-1,4 hydrolytic activity, the individual α-glucosidases also

37

exhibit a variety of specific α-hydrolysis properties on different substrates.10, 11 MGAM has high

38

α-1,4 hydrolytic activity for digestion of maltose and larger starch-based oligomers and

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polymers.12-14 The SI complex accounts for the majority of mucosal maltase activity, because the

40

amount of SI protein is 40-50 times higher than MGAM in the human intestine.12, 13 The



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hydrolytic activity of ctMGAM, unlike the other subunits, is inhibited during α-1,4 linkage

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hydrolysis by concentration of its products, termed the “brake” effect.15-17 Both N-terminal α-

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glucosidases (ntMGAM and ntSI) have α-1,6 hydrolytic activity with ntSI having much higher

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hydrolytic activity than ntMGAM.18 Using powdered rat intestine (containing both MGAM and

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SI α-glucosidase complexes), it was previously shown that kojibiose (α-1,2) and nigerose (α-1,3)

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were digested at about 65 and 86% of maltose hydrolysis.19, 20 Prior to that study using pig

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intestinal MGAM complex showed hydrolysis of all types of α-glycosidic linkages between two

48

glucose molecules, except trehalose.10

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Low glycemic index foods are associated with reduced risk of common chronic health

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diseases,21 and this has led to interest in glycemic carbohydrates with slow digestion rate for

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modulated postprandial glycemic response. Apart from efforts to understand how to slow starch

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digestion, unusual α-linked carbohydrates, such as isomaltulose (α-1,6 sucrose isomer) and

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sucromalt (α-glucan containing α-1,3 and α-1,6 linkages) have been commercially developed as

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slowly digestible carbohydrates.22, 23 Even so, it is still difficult to devise precise strategies to

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produce specific slowly digestible carbohydrates based on linkage type and monosaccharide

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composition. In this research, digestion properties of a range of disaccharides of different linkage

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and monosaccharide composition were investigated using rat intestinal α-glucosidases and the

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four individual recombinant mucosal α-glucosidases with the aim of finding new ways to control

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the rate of glucogenesis from added dietary carbohydrates.

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MATERIALS AND METHODS

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Materials. Trehalose (1-O-(α-D-glucopyranosyl)-α-D-glucopyranose), kojibiose (2-O-(α-D-

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glucopyranosyl)-D-glucose), nigerose (3-O-(α-D-glucopyranosyl)-D-glucose), maltose (4-O-(α-D-


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glucopyranosyl)-D-glucose), and isomaltose (6-O-(α-D-glucopyranosyl)-D-glucose) were used as

65

substrates (Sigma-Aldrich Co., St. Louis, MO) to test the hydrolysis properties of α-glycosidic

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linkages between two glucose molecules. Sucrose (α-D-glucopyranosyl-(1,2)-β-D-

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fructofuranose), turanose (3-O-(α-D-glucopyranosyl)-D-fructose), maltulose (4-O-(α-D-

68

glucopyranosyl)-D-fructose), leucrose (5-O-(α-D-glucopyranosyl)-D-fructose), and isomaltulose

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(6-O-(α-D-glucopyranosyl)-D-fructose) were used to study the α-hydrolytic properties on sucrose

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isomers (Carbosynth, Berkshire, UK). The reagents for the glucose assay were purchased from

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Megazyme (Wicklow, Ireland). Other chemicals were purchased from Sigma-Aldrich Co. (St.

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Louis, MO).

73 74

Optimal pH range of rat intestinal α-glucosidases on different substrates. To test optimal pH

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range of mucosal α-glucosidases on different substrates, rat intestinal powder (Sigma-Aldrich

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Co., St. Louis, MO) was used as a source of mucosal α-glucosidases. Rat intestinal powder (2 g)

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was mixed with 20 mL of sodium phosphate buffer (100 mM, pH 6.9) and left at 4°C for 24 h.

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The mixture was centrifuged at 9000 × g for 15 min, and the supernatant was filtered through a

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0.8 µm Nylon membrane.24 The filtered extract was applied as a crude α-glucosidase solution.

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Maltodextrin DE 1 (dextrose equivalent), maltose, sucrose, and isomaltulose were applied as

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substrates (10 mg/mL, w/v) for the mucosal α-glucosidase assay. Optimum pH for individual α-

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glucosidase activities was determined using a phosphate-based universal buffer (pH 3-8).25 The

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mixture, containing each substrate solution (100 uL), 100 mM universal buffer (800 uL), and rat

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intestinal enzyme (100 uL), was incubated at 37˚C for 10 min for maltodextrin and maltose and

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60 min for sucrose and isomaltulose. The enzyme solution was inactivated by placing tubes in

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boiling water and the amount of released glucose was determined by the GOPOD method.26



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Hydrolysis properties of rat intestinal α-glucosidases on different α-linked disaccharides.

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Maltose isomers (trehalose, kojibiose, nigerose, and isomaltose) and sucrose isomers (turanose,

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maltulose, leucrose, and isomaltulose) were applied as substrates (100 mg/mL) for mucosal α-

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glucosidases. Maltose isomers were hydrolyzed in pH 6.5 and sucrose isomers were hydrolyzed

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in pH 6.0 sodium phosphate buffers (100 mM). The mixture, containing each substrate solution

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(100 uL), 100 mM sodium phosphate buffer (800 uL), and prepared-rat intestinal enzyme (100

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uL), was incubated at 37˚C for 120 min for maltose isomers and 360 min for sucrose isomers.

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The enzyme solution was inactivated by placing tubes in boiling water and the amount of

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released glucose was determined by the GOPOD method.26

97 98

Purification of the individual recombinant mucosal α-glucosidases. The individual

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recombinant mucosal α-glucosidases were expressed via the baculovirus system through

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different insect cell as a host as described by Jones, et al. 27 The recombinant baculovirus for ctSI

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and ctMGAM (both C-terminal genes from mouse mRNA) were transfected into the Sf9 insect

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cell, and the recombinant ntMGAM and ntSI (both N-terminal genes from human mRNA) were

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transfected to Drosophila S2 cells to express recombinant protein.28 The expressed individual

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protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography

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(Qiagen, Hilden, Germany), and then concentrated with Microcon YM-30 (MWCO 30,000;

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Millipore, CA, USA). Protein amount was measured by the Bradford method and compared with

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a bovine serum albumin (BSA) standard.29

108 109

Enzyme kinetics on different types of linkage and glucose composition. To study the enzyme


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kinetics of individual mucosal α-glucosidases on different substrates, 5 µg of each enzyme was

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reacted at 37°C in 10 mM PBS buffer (pH 6.9) with 1-200 mM of different concentrations of

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substrate. The amount (mM) of released glucose per 1 min (mM/min) from the different

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concentrations of the substrates (maltose and sucrose isomers) by the individual recombinant α-

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glucosidase reaction was determined by the GOPOD method.26 Kinetic values (Km and kcat)

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were calculated by the Michaelis-Menten equation using SigmaPlot 12 (Systat Software Inc., San

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Jose, CA, USA). Analyses were done in duplicate.

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Analysis of hydrolysis properties on different α-linkages on sucrose, isomaltose,

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isomaltulose. Different types of α-linked disaccharide solution [1.0%, w/v, (sucrose,

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isomaltulose and isomaltose)] as a substrate were reacted with 100 U (one unit enzyme activity

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was arbitrarily defined as the amount of enzyme that releases 1 µg of glucose from 1% maltose

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per 10 min at 37°C) of recombinant mucosal α-glucosidases at 37˚C with 10 mM PBS (pH 6.9)

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containing 0.02% of sodium azide. For testing α-hydrolytic activity, the mixture was reacted

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during 72 h. The resulting product was reacted with GOPOD solution to analyze the released

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glucose by enzyme reaction. Then, the amount of released glucose was divided by the number of

126

glucose units to calculate glucose recovery ratio. All reactions were performed in triplicate.

127 128

RESULTS AND DISCUSSION

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Mucosal α-glucosidases have different optimal pH range depending on substrates. In the

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small intestine, luminal pH increases from the acidic environment of the stomach to the neutral

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range, but still has a slightly increasing gradient from the proximal to distal parts.30, 31 Depending

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on the α-linkage type of the glycemic carbohydrate, optimal pHs of small intestinal α-



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glucosidase activities on different substrate were somewhat different (Figure 2). The optimal pH

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in vitro for maltose and maltodextrin digestion, which are connected by α-1,4 linkages, was pH

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6.5, while for sucrose (α-1,2) and isomaltulose (α-1,6) were pH 6.0, and all four substrates show

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relatively broad optimal pH ranges. The optimal pH data suggests that sucrose and α-1,6 linked

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digestible carbohydrates (e.g., isomaltose, isomaltulose, and branched α-limit dextrins from

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starch hydrolyzates by α-amylase) may be favorably hydrolyzed in the upper level of small

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intestine due to relatively low pH conditions.32, 33 Different optimal pH range is one of the factors

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that may affect the hydrolysis rate of digestible carbohydrates in the small intestine. The catalytic

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sites between the rat and human α-glucosidases are conserved,4 thus it is expected that the pH

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dependent-hydrolysis properties would be similar.

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Hydrolytic properties of rat intestinal α-glucosidases on maltose and sucrose isomers. A

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number of studies have focused on specific linkage-based small molecule slowly digestible

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carbohydrates to decrease postprandial blood glucose level.34-36 In the present study, various

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disaccharides bound by different linkages between α-D-glucosyl-D-glucose (Figure 3A) and α-D-

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glucosyl-D-fructose (Figure 3B) were tested using mammalian (rat) intestinal mucosal α-

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glucosidases to determine relative hydrolysis rates. Table 1 shows that all available types of two

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glucose α-linked disaccharides (maltose isomers) were hydrolyzed by crude rat intestinal α-

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glucosidases to glucose, but with different hydrolysis rates. Compared to maltose (α-1,4 linkage),

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the maltose isomers were slowly hydrolyzed (p < 0.05); and large differences were observed with

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relative hydrolysis rate of nigerose (α-1,3) at about 50%, followed by kojibiose (α-1,2),

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isomaltose (α-1,6), and trehalose (α-1,1). Thus, different types of α-linked maltose isomers might

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be applied, and in variations as larger oligomers, to make slowly digestible carbohydrates due to


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their slower glucose generation rates. The available sucrose isomers were also hydrolyzed to monosaccharides (glucose and

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fructose) by rat intestinal α-glucosidases at the optimal pH (6.0) of sucrose hydrolysis (Table 2).

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Maltulose (α-1,4) and leucrose (α-1,5) produced a similar amount of glucose as sucrose, while

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turanose (α-1,3) and isomaltulose (α-1,6) were more slowly hydrolyzed (p < 0.05). Glucose

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generation rates (µg/min) from the sucrose isomers were much lower than all of the maltose

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isomers except trehalose (Tables 1 and 2). Interestingly, sucrose is considered a rapidly digestible

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carbohydrate,37 though its glucose generation rate is lower than α-amylase products from starch

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digestion (e.g., maltose and maltotriose).38

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Enzyme kinetics on different α-glycosidic linkages between α-D-glucosyl-D-glucose.

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Glycemic substrates with different α-glycosidic-linked disaccharides [trehalose (α-1,1), kojibiose

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(α-1,2), nigerose (α-1,3), maltose (α-1,4), and isomaltose (α-1,6)] were investigated to

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understand their hydrolytic properties related to the individual recombinant α-glucosidases. As

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shown in the literature,17, 39, 40 all mucosal α-glucosidases have α-1,4 linkage (maltose)

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hydrolysis activity with different rates of glucogenesis; and ntSI is mainly responsible for α-1,6

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linkage hydrolysis (isomaltose).

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Trehalose (α-1,1), which does not have a reducing end, was not hydrolyzed to glucose by

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the individual recombinant mucosal α-glucosidases (Table 3), although it was hydrolyzed by rat

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intestinal α-glucosidases (Table 1). This is because the digestion of trehalose in the

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gastrointestinal tract is accomplished by trehalase,41 which has a different catalytic mechanism

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compared to mucosal MGAM and SI enzymes.

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For the maltose isomers, all four mucosal α-glucosidases hydrolyzed the α-1,2 (kojibiose)



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and α-1,3 (nigerose) glucosyl-glucose linkages, though with a significantly reduced rates of

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glucogenesis compared to maltase (α-1,4) activity (Table 3). Further, the α-1,2 linkage was more

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slowly hydrolyzed than the α-1,3 linkage, as it was with the rat intestinal enzymes. For α-1,2

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linkage hydrolysis, enzyme efficiency of glucose generation (kcat/Km) was approximately 8 to 56

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times lower than that of maltase activity for the four enzymes (kcat/Km of ctMGAM: 0.91;

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ntMGAM: 1.52; ctSI: 0.03; and ntSI: 0.06). Under the same conditions, glucose production rate

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(kcat/Km) from the α-1,3 linkage was 3 to 15 times lower than that of maltase activity (kcat/Km of

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ctMGAM: 2.73; ntMGAM: 4.45; ctSI: 0.17; and ntSI: 0.56). Notably, maltase (ntMGAM) had

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higher hydrolytic property on α-1,2 and α-1,3 linkages compared to the other α-glucosidases.

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This would be due to the comparatively open binding site of ntMGAM that can better

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accommodate glycosidic linkages to the C-2, -3 and -4 positions than the other enzymes tested.18,

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42

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Isomaltose was not digested well by the ctMGAM and ctSI which can be explained by

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the fact that the (α-1,6) glycosidic linkage of isomaltose is longer and more flexible than those of

193

kojibiose, nigerose and maltose (Figure 3A). This requires a narrower opening of the +1 sugar

194

binding site to accommodate isomaltose, as is observed in the ntSI (isomaltase) structure.18

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Because ntMGAM has minor hydrolytic activity towards the α-1,6 bond, it can be considered

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intermediate between ntSI and the C-terminal enzymes and thus is more promiscuous in

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specificity. The catalytic site is sufficiently close in structure to have low activity towards

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isomaltose, but sufficiently open to hydrolyze kojibiose and nigerose as well as its primary

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activity towards maltose.

200 201

Hydrolysis of sucrose, isomaltose, and isomaltulose by individual mucosal α-glucosidases.


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Sucrase activity was unexpectedly found in ctMGAM, though with 5 times lower activity than

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ctSI (Table 4). Considering the amount of SI in the small intestine is 40-50 times higher than

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MGAM,12, 13 it is reasonable that ctSI is considered as the only major α-glucosidase related to

205

sucrose hydrolysis. Contrary to our findings, previous investigations using pig and human

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intestine revealed no sucrase activity in MGAM.11 The clear, but low, sucrose hydrolysis activity

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of ctMGAM found here might be explained by the fact that the protein structure of ctSI is closer

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to that of ctMGAM (with 60% protein similarity) than to ntSI (around 40% sequence identity)

209

(Figure 1).42

210

Isomaltulose (the α-1,6 isomer of sucrose), which is recognized as a slowly digestible

211

sweetener,43 was hydrolysable by each recombinant α-glucosidase, but at markedly different

212

rates. ntSI and ntMGAM had major hydrolysis activities on isomaltulose with the ntSI showing

213

substantially higher activity (91.6 versus 29.4%, Table 4). The high hydrolysis rate of

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isomaltulose by ntSI confirms previous research.10 Both ctMGAM and ctSI had some, though

215

quite low, hydrolytic activity for isomaltulose (4.2 and 8.5%, respectively), while a previous

216

study by Lina, Jonker et al. (2002) showed no isomaltulose activity in the MGAM complex in rat

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and human small intestine.23

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Expectedly, isomaltose was almost fully hydrolyzed by the action of ntSI (Table 4).

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Notably, all mucosal α-glucosidases had some degree of isomaltase (α-1,6 linkage) activity,

220

particularly at longer reaction times. While 28.4 % of isomaltose was hydrolyzed to glucose by

221

the action of ntMGAM after 72 h reaction, ctMGAM and ctSI hydrolyzed only 8.8 and 18.1% of

222

the substrate (Table 4). Considering the amount of protein and hydrolysis activity in the small

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intestine, ntSI by far represents the major hydrolyzing enzyme for isomaltose and the branched

224

α-limit dextrins from α-amylase degraded starch.44



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It is interesting that isomaltulose has both structural similarity with sucrose (an α-

226

glycosyl-fructose disaccharide) and isomaltose (α-1,6 linkage), but is hydrolyzed mainly by ntSI.

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Considering that sucrose is digested only by ctSI and isomaltose is primarily digested by ntSI, it

228

appears that linkage type is the more important factor, rather than sugar composition, in

229

determining which of the individual α-glucosidases will digest isomaltulose. A further

230

investigation with changing carbohydrate structures by modifying the α-linkage could better

231

relate structures with binding properties on enzyme active sites.

232

In this study, there were clear differences in hydrolytic properties of the enzymes to

233

disaccharide substrates differing in linkages that appeared to parallel structural homologies of the

234

proteins. These investigations provide insight into the role of individual mucosal α-glucosidases

235

in the digestion of unusual linkage glycemic disaccharides for guidance in the development of

236

slowly digestible carbohydrate (i.e. oligosaccharide) ingredients based on α-linkage type. For the

237

first time, maltase (ntMGAM) was shown to have a particular capacity to digest α-1,2 and -1,3

238

disaccharides of glucose, ascribing a broader specificity to the catalytic site of this enzyme than

239

known before. Lastly, our findings imply that glycemic carbohydrates such as studied here must

240

be tested using mammalian mucosal α-glucosidases for evaluating digestion rate differences, and

241

not the glucoamylases from fungal or microbial sources commonly used in in vitro starch

242

digestion assays.

243 244


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Gray, G. M.; Lally, B. C.; Conklin, K. A., Action of intestinal sucrase-isomaltase and its

363 364 365

ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National

366

Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning

367

(NRF-2015R1C1A1A02036467) (BHL). We are thankful for support from the United States

368

Department of Agriculture (USDA) Agriculture and Food Research Initiative competitive grant

369

program, no. 08-555-03-18793 (BRH), Whistler Center for Carbohydrate Research at Purdue

370

University, Indiana, USA, and the Canadian Institutes for Health Research and Natural Science

371

and Engineering Research Council (DRR). Also, supported in part by federal funds from the

372

USDA, Agricultural Research Service, under Cooperative Agreement Number 58-6250-1-003

373

with Baylor College of Medicine (BLN, RQC).

374


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375


FIGURE CAPTIONS

376 377

Figure 1. Maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) protein complex

378

structures of the small intestinal lumen linked to the transmembrane domain via an O-

379

glycosylated linkage. Percentage comparisons between individual enzymes indicate sequence

380

identity. AA: amino acids

381 382

Figure 2. Optimal pH conditions for reaction of crude rat intestinal powder on different types of

383

α-linked substrates determined as the amount of glucose release at 37°C. The optimal pH in vitro

384

for maltose and maltodextrin digestion, which are α-1,4 linked, was pH 6.5; while for sucrose (α-

385

1,2) and isomaltulose (α-1,6) were pH 6.0. MD (DE1): maltodextrin dextrose equivalent 1

386 387

Figure 3. Various disaccharides based on different α-glycosidic linkages and monosaccharide

388

composition. A: α-linked disaccharides between two glucose molecules B: α-linked

389

disaccharides between glucose and fructose molecules

390



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Table 1. Glucose Production (µg/min) from Maltose Isomers (10 mg/mL, w/v) and Relative Hydrolysis Rate (%) Compared to Maltose (at 100%) by Crude Rat Intestinal αGlucosidases.a Trehalose (α-1,1)

Kojibiose (α-1,2)

Nigerose (α-1,3)

Maltose (α-1,4)

Isomaltose (α-1,6)

Glucose production rate (µg/min)

7.9 ± 1.3

21.4 ± 1.3

30.9 ± 0.7

59.3 ± 2.3

14.4 ± 1.4

Relative hydrolysis rate (%)

13 ± 2e

36 ± 2c

52 ± 1b

100 ± 4a

24 ± 2d

a

Enzymatic reactions were carried out in 100 mM sodium phosphate buffer (pH 6.5) at 37°C. Values represent the mean ± standard deviation and were performed in tripliicate. Values denoted by different letters (a – e) are significantly different from each other at p < 0.05 as determined using ANOVA and Tukey’s HSD multiple comparison tests.


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Table 2. Glucose Production (µg/min) from Sucrose Isomers (10 mg/mL, w/v) and Relative Hydrolysis rate (%) Compared to Sucrose (at 100%) by Crude Rat Intestinal αGlucosidases.a Sucrose (α-1,2)

Turanose (α-1,3)

Maltulose (α-1,4)

Leucrose (α-1,5)

Isomaltulose (α-1,6)

Glucose production rate (µg/min)

9.6 ± 0.1

6.6 ± 0.2

9.5 ± 0.3

9.0 ± 0.3

3.7 ± 0.1

Relative hydrolysis rate (%)

100 ± 1a

69 ± 3b

99 ± 3a

94 ± 4a

39 ± 1c

a

Enzymatic reactions were carried out in 100 mM sodium phosphate buffer (pH 6.5) at 37°C. Values represent the mean ± standard deviation and were performed in tripliicate. Values denoted by different letters (a – e) are significantly different from each other at p < 0.05 as determined using ANOVA and Tukey’s HSD multiple comparison tests.



Page 22 of 27

Table 3. Kinetic Parameters of Each Recombinant Mucosal α-Glucosidase on Differently αLinked Disaccharides with Two Glucoses. ctMGAM (glucoamylase)

ntMGAM (maltase)

ctSI (sucrase)

ntSI (isomaltase)

Km (mM)

nd*

nd

nd

nd

Kcat (s-1)

nd

nd

nd

nd

Kcat/Km

nd

nd

nd

nd

12.7 ± 2.5

11.6 ± 1.2

17.3 ± 2.2

53.7 ± 13.7

Kcat (s )

11.5 ± 0.6

17.6 ± 0.5

0.5 ± 0.0

3.2 ± 0.3

Kcat/Km

0.9 ± 0.2

1.5 ± 0.4

0.3 ± 0.0

0.1 ± 0.0

35.2 ± 3.6

27.1 ± 2.6

63.6 ± 13.0

44.4 ± 6.4

Kcat (s )

96.0 ± 3.3

120.9 ± 3.7

11.0 ± 1.0

24.8 ± 1.3

Kcat/Km

2.7 ± 0.9

4.4 ± 1.4

0.2 ± 0.1

0.6 ± 0.2

2.6 ± 0.6

8.7 ± 1.3

4.2 ± 1.4

11.1 ± 1.5

Kcat (s )

133.9 ± 4.3

110.2 ± 3.8

11.4 ± 0.8

18.3 ± 0.6

Kcat/Km

51.0 ± 7.0

12.7 ± 3.0

2.7 ± 0.5

1.7 ± 0.4

nda

128.0 ± 8.4

nd

15.2 ± 2.0

-1

Kcat (s )

nd

8.9 ± 0.3

nd

18.1 ± 0.7

Kcat/Km

nd

0.1 ± 0.0

nd

1.2 ± 0.3

Trehalose

Kojibiose Km (mM) -1

Nigerose Km (mM) -1

Maltose Km (mM) -1

Isomaltose Km (mM)

a

nd, not detected.


Page 23 of 27


Table 4. Hydrolysis (%) of Different Types of Substrates (sucrose, isomaltulose, and isomaltose; 10 mg/mL, w/v) to Glucose by Individual Recombinant Mucosal α-Glucosidases (100 U) after 72 h.a,b

ctMGAM (glucoamylase) ntMGAM (maltase) ctSI (sucrase) ntSI (isomaltase)

Sucrose (α-1,2; G+F) c

Isomaltulose (α-1,6; G+F)

Isomaltose (α-1,6; G+G)

14.8 ± 0.6

4.2 ± 0.6

8.8 ± 0.2

0.6 ± 0.1

29.4 ± 0.4

28.4 ± 1.0

73.1 ± 0.1

8.5 ± 0.4

18.1 ± 0.1

0.9 ± 0.0

91.6 ± 3.1

98.4 ± 3.6

a

One unit enzyme activity arbitrarily defined as the amount of enzyme that released 1 µg of glucose from 1% maltose per 10 min at 37°C b Mean value ± standard deviation of measurement of experiments performed in triplicate. c G: glucose and F: fructose



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Figure 1


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Figure 2



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Figure 3 26 ACS Paragon Plus Environment

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Table of Contents (TOC) 72x45mm (300 x 300 DPI)

ACS Paragon Plus Environment

Contribution of the Individual Small Intestinal α-Glucosidases to

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