Kyra Jones

Mapping the intestinal alpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase

Inhibition of intestinal α-glucosidases and pancreatic α-amylases is an approach to controlling blood glucose and serum insulin levels in individuals with Type II diabetes. The two human intestinal glucosidases are maltase-glucoamylase and sucrase-isomaltase. Each incorporates two family 31 glycoside hydrolases responsible for the final step of starch hydrolysis. Here we compare the inhibition profiles of the individual N- and C-terminal catalytic subunits of both glucosidases by clinical glucosidase inhibitors, acarbose and miglitol, and newly discovered glucosidase inhibitors from an Ayurvedic remedy used for the treatment of Type II diabetes. We show that features of the compounds introduce selectivity towards the subunits. Together with structural data, the results enhance the understanding of the role of each catalytic subunit in starch digestion, helping to guide the development of new compounds with subunit specific antidiabetic activity. The results may also have relevance to other metabolic diseases such as obesity and cardiovascular disease.

Enzyme-Synthesized Highly Branched Maltodextrins Have Slow Glucose Generation at the Mucosal α-Glucosidase Level and Are Slowly Digestible In Vivo.

For digestion of starch in humans, α-amylase first hydrolyzes starch molecules to produce α-limit dextrins, followed by complete hydrolysis to glucose by the mucosal α-glucosidases in the small intestine. It is known that α-1,6 linkages in starch are hydrolyzed at a lower rate than are α-1,4 linkages. Here, to create designed slowly digestible carbohydrates, the structure of waxy corn starch (WCS) was modified using a known branching enzyme alone (BE) and an in combination with β-amylase (BA) to increase further the α-1,6 branching ratio. The digestibility of the enzymatically synthesized products was investigated using α-amylase and four recombinant mammalian mucosal α-glucosidases. Enzyme-modified products (BE-WCS and BEBA-WCS) had increased percentage of α-1,6 linkages (WCS: 5.3%, BE-WCS: 7.1%, and BEBA-WCS: 12.9%), decreased weight-average molecular weight (WCS: 1.73×108 Da, BE-WCS: 2.76×105 Da, and BEBA-WCS 1.62×105 Da), and changes in linear chain distributions (WCS: 21.6, BE-WCS: 16.9, BEBA-WCS: 12.2 DPw). Hydrolysis by human pancreatic α-amylase resulted in an increase in the amount of branched α-limit dextrin from 26.8% (WCS) to 56.8% (BEBA-WCS). The α-amylolyzed samples were hydrolyzed by the individual α-glucosidases (100 U) and glucogenesis decreased with all as the branching ratio increased. This is the first report showing that hydrolysis rate of the mammalian mucosal α-glucosidases is limited by the amount of branched α-limit dextrin. When enzyme-treated materials were gavaged to rats, the level of postprandial blood glucose at 60 min from BEBA-WCS was significantly higher than for WCS or BE-WCS. Thus, highly branched glucan structures modified by BE and BA had a comparably slow digesting property both in vitro and in vivo. Such highly branched α-glucans show promise as a food ingredient to control postprandial glucose levels and to attain extended glucose release.

Modulation of Starch Digestion for Slow Glucose Release through “Toggling” of Activities of Mucosal α-Glucosidases

Background: Proper breakdown of starch by hydrolytic enzymes to yield glucose has profound implications for avoiding type 2 diabetes and obesity. Results: Starch digestion by the different human enzymes is controlled using a panel of compounds. Conclusion: Inhibitors can be used to switch off selectively the different enzyme activities. Significance: More refined control of starch hydrolysis with the aim of slow glucose delivery is possible. Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of “toggling”: differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC50 values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases.

Investigations of the structures and inhibitory properties of intestinal maltase glucoamylase and sucrase isomaltase.

53. Alfalah M, Keiser M, Leeb T, et al. Compound heterozygous mutations affect protein folding and function in patients with congenital sucraseisomaltase deficiency. Gastroenterology 2009;136:883–92. 54. Propsting MJ, Jacob R, Naim HY. A glutamine to proline exchange at amino acid residue 1098 in sucrase causes a temperature-sensitive arrest of sucrase-isomaltase in the endoplasmic reticulum and cis-Golgi. J Biol Chem 2003;278:16310–4. 55. Propsting MJ, Kanapin H, Jacob R, et al. A phenylalanine-based folding determinant in intestinal sucrase-isomaltase that functions in the context of a quality control mechanism beyond the endoplasmic reticulum. J Cell Sci 2005;118:2775–84. 56. Spodsberg N, Jacob R, Alfalah M, et al. Molecular basis of aberrant apical protein transport in an intestinal enzyme disorder. J Biol Chem 2001;276:23506–10. 57. Jacob R, Alfalah M, Grunberg J, et al. Structural determinants required for apical sorting of an intestinal brush-border membrane protein. J Biol Chem 2000;275:6566–72. 58. Jacob R, Zimmer KP, Schmitz J, et al. Congenital sucrase-isomaltase deficiency arising from cleavage and secretion of a mutant form of the enzyme. J Clin Invest 2000;106:281–7. 59. Keiser M, Alfalah M, Propsting MJ, et al. Altered folding, turnover, and polarized sorting act in concert to define a novel pathomechanism of congenital sucrase-isomaltase deficiency. J Biol Chem 2006;281: 14393–9. 60. Sander P, Alfalah M, Keiser M, et al. Novel mutations in the human sucrase-isomaltase gene (SI) that cause congenital carbohydrate malabsorption. Hum Mutat 2006;27:119. 61. Jacob R, Naim HY. Apical membrane proteins are transported in distinct vesicular carriers. Curr Biol 2001;11:1444–50. 62. Danielsen EM, van Deurs B. Galectin-4 and small intestinal brush border enzymes form clusters. Mol Biol Cell 1997;8:2241–51. 63. Delacour D, Cramm-Behrens CI, Drobecq H, et al. Requirement for galectin-3 in apical protein sorting. Curr Biol 2006;16:408–14. 64. Seelenmeyer C, Wegehingel S, Tews I, et al. Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1. J Cell Biol 2005;171:373–81. 65. Yeaman C, Le Gall AH, Baldwin AN, et al. The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells. J Cell Biol 1997;139:929–40. 66. Kitagawa Y, Sano Y, Ueda M, et al. N-glycosylation of erythropoietin is critical for apical secretion by Madin-Darby canine kidney cells. Exp Cell Res 1994;213:449–57. 67. Scheiffele P, Peranen J, Simons K. N-glycans as apical sorting signals in epithelial cells. Nature 1995;378:96–8. 68. Lin S, Naim HY, Rodriguez AC, et al. Mutations in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells. J Cell Biol 1998; 142:51–7. 69. Chuang JZ, Sung CH. The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells. J Cell Biol 1998;142: 1245–56. 70. Rodriguez-Boulan E, Gonzalez A. Glycans in post-Golgi apical targeting: sorting signals or structural props? Trends Cell Biol 1999;9:291–4. 71. Fiedler K, Simons K. The role of N-glycans in the secretory pathway. Cell 1995;81:309–12. 72. Sun AQ, Ananthanarayanan M, Soroka CJ, et al. Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells. Am J Physiol 1998;275:G1045–5. 73. Jacob R, Heine M, Alfalah M, et al. Distinct cytoskeletal tracks direct individual vesicle populations to the apical membrane of epithelial cells. Curr Biol 2003;13:607–12. 74. Jacob R, Heine M, Eikemeyer J, et al. Annexin II is required for apical transport in polarized epithelial cells. J Biol Chem 2004;279:3680–4. 75. Heine M, Cramm-Behrens CI, Ansari A, et al. Alpha-kinase 1, a new component in apical protein transport. J Biol Chem 2005;280:25637– 43. 76. Hein Z, Schmidt S, Zimmer KP, et al. The dual role of annexin II in targeting of brush border proteins and in intestinal cell polarity. Differentiation 2011;81:243–52. 77. Dork T, Wulbrand U, Richter T, et al. Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene. Hum Genet 1991;87:441–6. 78. Lamprecht G, Mau UA, Kortum C, et al. Relapsing pancreatitis due to a novel compound heterozygosity in the CFTR gene involving the second most common mutation in central and eastern Europe [CFTRdele2, 3(21 kb)]. Pancreatology 2005;5:92–6. 79. Jayandharan G, Viswabandya A, Baidya S, et al. Six novel mutations including triple heterozygosity for Phe31Ser, 514delT and 516T–>G factor X gene mutations are responsible for congenital factor X deficiency in patients of Nepali and Indian origin. J Thromb Haemost 2005;3:1482–7. 80. Nakamura A, Yazaki M, Tokuda T, et al. A Japanese patient with familial Mediterranean fever associated with compound heterozygosity for pyrin variant E148Q/M694I. Intern Med 2005;44:261–5. 81. Asp NG, Berg NO, Dahlqvist A, et al. Intestinal disaccharidases in Greenland Eskimos. Scand J Gastroenterol 1975;10:513–9. 82. Dahlqvist A. Assay of intestinal disaccharidases. Anal Biochem 1968;22:99–107. 83. Muldoon C, Maguire P, Gleeson F. Onset of sucrase-isomaltase deficiency in late adulthood. Am J Gastroenterol 1999;94:2298–9.

Substrate selectivity of C-terminal sucrase isomaltase and maltase glucoamylase

Carbohydrates make up a significant component of the human diet. One approach to controlling blood glucose and serum insulin levels in individuals with type II diabetes is inhibition of intestinal α-glucosidases and pancreatic α-amylases. Two intestinal αglucosidases, sucrase isomaltase (SI) and maltase glucoamylase (MGAM), are responsible for the final step of starch hydrolysis in mammals in the small intestine: the release of free glucose. Each enzyme consists of two catalytic subunits: N-terminal sucrase isomaltase (ntSI) and C-terminal sucrase isomaltose (ctSI); and N-terminal maltase glucoamylase (ntMGAM) and C-terminal maltase glucoamylase (ctMGAM). Here, residues hypothesized to impact substrate specificity of ctSI and ctMGAM will be presented, enhancing our understanding of the functionality of these enzymatic subunits as well as their overlapping substrate specificity.

Research progress reported at the 50th Anniversary of the Discovery of Congenital Sucrase-Isomaltase Deficiency Workshop.

C ongenital sucrase-isomaltase deficiency (CSID) is a rare autosomal intestinal disease caused by mutations of the sucrase-isomaltase gene. Patients with CSID have different phenotypes and enzymatic activities associated with sucrase-isomaltase ranging from mild reduction of sucrase activity to complete absence. Low sucrase activity leads to maldigestion of sucrose, resulting in dyspepsia-like symptoms such as chronic diarrhea, abdominal pain, and bloating. The severity of the symptom is related to the amount of sucrase activity and quantity of sucrose ingested. Reduced maltase activity is expected to occur in patients with CSID because both subunits of the SI complex contribute to the total mucosal maltose activity. Indeed, low maltase activity can also lead to maldigestion of starches contributing to the symptoms of dyspepsia. When children are assessed for this gene mutation, duodenal mucosal histology is invariably normal. Because there are no noninvasive methods for specific confirmation of sucrase-isomaltase deficiency, a novel noninvasive C-sucrose labeled substrate has been developed and validated as a sucrase activity breath test for screening and confirmation of CSID. It has been shown that primary sucrase deficiency can be confirmed using this new C breath test, as well as the effectiveness of sucrase replacement therapy. On December 21 and 22, 2011, the 8th Workshop of the Starch Digestion Consortium (SDC) was held at the Children’s Nutrition Research Center at the Baylor College of Medicine and Texas Children’s Hospital. The theme of the workshop was ‘‘50 Years of Progress Since Congenital Sucrase-Isomaltase Deficiency (CSID) Recognition.’’ This was a multidisciplinary workshop that blended clinical medicine with nutritional and food science. The purpose of this workshop was to review progress toward clinical diagnosis and management of sucrase-isomaltase enzyme deficiency during the past 50 years. The nutritional and food technological objectives of the conference were 2-fold: first, to define the role of sucrase-isomaltase enzyme complex in human starch digestion to glucose (a-glucogenesis), and second, by studying sucrase-isomaltase–deficient patients with CSID, envision approaches to botanical and technological alterations in food that will benefit all populations. More than 50 authors and attendees participated in this workshop from 12 different countries. Eighteen original communications were presented. Special thanks go to QOL Medical, the supplier of Sucraid oral enzyme supplement, for sponsoring this event. This supplement to the Journal of Pediatric Gastroenterology and Nutrition reports the proceedings of this workshop. E. Roseland Klein served as the technical editor of the workshop publication. Beth Mays was responsible for all travel arrangements and the workshop organization. Adam Gillum was responsible for graphic and audio arrangements. The group of participants standing among the lobby statuary at the SDC/CSID workshop held at the Children’s Nutrition Research Center, December 21–22, 2011 is shown in Figure 1. Statues are identified by italics; Borden is by Joseph Paderewski (1914–2000) and the others are copies of the work of Elizabet Ney (1833–1907). Research Progress Reported at the 50th Anniversary of the Discovery of Congenital Sucrase-Isomaltase Deficiency Workshop