Amy Hui-Mei Lin

Dynamics of Streptococcus mutans Transcriptome in Response to Starch and Sucrose during Biofilm Development

The combination of sucrose and starch in the presence of surface-adsorbed salivary α-amylase and bacterial glucosyltransferases increase the formation of a structurally and metabolically distinctive biofilm by Streptococcus mutans. This host-pathogen-diet interaction may modulate the formation of pathogenic biofilms related to dental caries disease. We conducted a comprehensive study to further investigate the influence of the dietary carbohydrates on S. mutans-transcriptome at distinct stages of biofilm development using whole genomic profiling with a new computational tool (MDV) for data mining. S. mutans UA159 biofilms were formed on amylase-active saliva coated hydroxyapatite discs in the presence of various concentrations of sucrose alone (ranging from 0.25 to 5% w/v) or in combination with starch (0.5 to 1% w/v). Overall, the presence of sucrose and starch (suc+st) influenced the dynamics of S. mutans transcriptome (vs. sucrose alone), which may be associated with gradual digestion of starch by surface-adsorbed amylase. At 21 h of biofilm formation, most of the differentially expressed genes were related to sugar metabolism, such as upregulation of genes involved in maltose/maltotriose uptake and glycogen synthesis. In addition, the groEL/groES chaperones were induced in the suc+st-biofilm, indicating that presence of starch hydrolysates may cause environmental stress. In contrast, at 30 h of biofilm development, multiple genes associated with sugar uptake/transport (e.g. maltose), two-component systems, fermentation/glycolysis and iron transport were differentially expressed in suc+st-biofilms (vs. sucrose-biofilms). Interestingly, lytT (bacteria autolysis) was upregulated, which was correlated with presence of extracellular DNA in the matrix of suc+st-biofilms. Specific genes related to carbohydrate uptake and glycogen metabolism were detected in suc+st-biofilms in more than one time point, indicating an association between presence of starch hydrolysates and intracellular polysaccharide storage. Our data show complex remodeling of S. mutans-transcriptome in response to changing environmental conditions in situ, which could modulate the dynamics of biofilm development and pathogenicity.

Proteolytic Cleavage of Ataxin-7 by Caspase-7 Modulates Cellular Toxicity and Transcriptional Dysregulation

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis.

Posttranslational Modification of Ataxin-7 at Lysine 257 Prevents Autophagy-Mediated Turnover of an N-Terminal Caspase-7 Cleavage Fragment

Polyglutamine (polyQ) expansion within the ataxin-7 protein, a member of the STAGA [SPT3-TAF(II)31-GCN5L acetylase] and TFTC (GCN5 and TRRAP) chromatin remodeling complexes, causes the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7). Proteolytic processing of ataxin-7 by caspase-7 generates N-terminal toxic polyQ-containing fragments that accumulate with disease progression and play an important role in SCA7 pathogenesis. To elucidate the basis for the toxicity of these fragments, we evaluated which posttranslational modifications of the N-terminal fragment of ataxin-7 modulate turnover and toxicity. Here, we show that mutating lysine 257 (K257), an amino acid adjacent to the caspase-7 cleavage site of ataxin-7 regulates turnover of the truncation product in a repeat-dependent manner. Modification of ataxin-7 K257 by acetylation promotes accumulation of the fragment, while unmodified ataxin-7 is degraded. The degradation of the caspase-7 cleavage product is mediated by macroautophagy in cell culture and primary neuron models of SCA7. Consistent with this, the fragment colocalizes with autophagic vesicle markers, and enhanced fragment accumulation increases in these lysosomal structures. We suggest that the levels of fragment accumulation within the cell is a key event in SCA7 neurodegeneration, and enhancing clearance of polyQ-containing fragments may be an effective target to reduce neurotoxicity in SCA7.

Importance of Location of Digestion and Colonic Fermentation of Starch Related to Its Quality

ABSTRACT For decades, quality of starch-based foods has been associated with the in vivo measured glycemic index or the in vitro digestion rate-based categories of rapidly digestible, slowly digestible, and resistant starch (RS). Glycemic index has been related to health-based endpoints mostly through correlative or observational studies, with mechanisms proposed but not well established. Here, we bring forth the concept of locational delivery of glucose from dietary starches to the distal small intestine to elicit an ileal brake effect, as well as short-chain fatty acid production from RS fermentation to cause a colonic brake. Both effects slow gastric emptying and, in turn, extend nutrient (i.e., energy) delivery to the body and may decrease appetite and promote weight management. Slowly digestible starches are currently a popular topic of research, although where they are digested and the released glucose is delivered in the small intestine is not known. A proposal is to further study and establish thi...

Unexpected High Digestion Rate of Cooked Starch by the Ct-Maltase-Glucoamylase Small Intestine Mucosal α-Glucosidase Subunit.

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.

Starch Source Influences Dietary Glucose Generation at the Mucosal α-Glucosidase Level

Background: α-Amylase was thought to be the sole enzyme that determines starch digestion rate. Mucosal α-glucosidases were considered to simply convert post-α-amylase dextrins to glucose. Results: The mucosal α-glucosidases can digest a wide range of α-limit dextrin molecules and digest starches from various sources differently. Conclusion: Starch chemical structure drives the digestion difference at the brush-border area. Significance: The findings provide new insight into controlling the glycemic response. The quality of starch digestion, related to the rate and extent of release of dietary glucose, is associated with glycemia-related problems such as diabetes and other metabolic syndrome conditions. Here, we found that the rate of glucose generation from starch is unexpectedly associated with mucosal α-glucosidases and not just α-amylase. This understanding could lead to a new approach to regulate the glycemic response and glucose-related physiologic responses in the human body. There are six digestive enzymes for starch: salivary and pancreatic α-amylases and four mucosal α-glucosidases, including N- and C-terminal subunits of both maltase-glucoamylase and sucrase-isomaltase. Only the mucosal α-glucosidases provide the final hydrolytic activities to produce substantial free glucose. We report here the unique and shared roles of the individual α-glucosidases for α-glucans persisting after starch is extensively hydrolyzed by α-amylase (to produce α-limit dextrins (α-LDx)). All four α-glucosidases share digestion of linear regions of α-LDx, and three can hydrolyze branched fractions. The α-LDx, which were derived from different maize cultivars, were not all equally digested, revealing that the starch source influences glucose generation at the mucosal α-glucosidase level. We further discovered a fraction of α-LDx that was resistant to the extensive digestion by the mucosal α-glucosidases. Our study further challenges the conventional view that α-amylase is the only rate-determining enzyme involved in starch digestion and better defines the roles of individual and collective mucosal α-glucosidases. Strategies to control the rate of glucogenesis at the mucosal level could lead to regulation of the glycemic response and improved glucose management in the human body.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function.

Direct starch digestion by sucrase-isomaltase and maltase-glucoamylase.

1. Jane J, Chen YY, Lee LF, et al. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chem 1999;76:629–37. 2. Quezada-Calvillo R, Robayo-Torres CC, Opekum AR, et al. Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal starch alpha-glucogenesis. J Nutr 2007;137:1725–33. 3. Nichols BL, Quezada-Calvillo R, Robayo-Torres CC, et al. Mucosal maltase-glucoamylase plays a crucial role in starch digestion and prandial glucose homeostasis of mice. J Nutr 2009;139:684–90. 4. Sim L, Quezada-Calvillo R, Sterchi EE, et al. Human intestinal maltaseglucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity. J Mol Biol 2008;375:782–92. 5. Ao Z, Quezada-Calvillo R, Sim L, et al. Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant). FEBS Lett 2007;581:2381–8.

Maltase-glucoamylase modulates gluconeogenesis and sucrase-isomaltase dominates starch digestion glucogenesis.

Objectives: Six enzyme activities are needed to digest starch to absorbable free glucose; 2 luminal &agr;-amylases (AMY) and 4 mucosal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) subunit activities are involved in the digestion. The AMY activities break down starch to soluble oligomeric dextrins; mucosal MGAM and SI can either directly digest starch to glucose or convert the post-&agr;-amylolytic dextrins to glucose. We hypothesized that MGAM, with higher maltase than SI, drives digestion on ad limitum intakes and SI, with lower activity but more abundant amount, constrains ad libitum starch digestion. Methods: Mgam null and wild-type (WT) mice were fed with starch diets ad libitum and ad limitum. Fractional glucogenesis (fGG) derived from starch was measured and fractional gluconeogenesis and glycogenolysis were calculated. Carbohydrates in small intestine were determined. Results: After ad libitum meals, null and WT had similar increases of blood glucose concentration. At low intakes, null mice had less fGG (P = 0.02) than WT mice, demonstrating the role of Mgam activity in ad limitum feeding; null mice did not reduce fGG responses to ad libitum intakes demonstrating the dominant role of SI activity during full feeding. Although fGG was rising after feeding, fractional gluconeogenesis fell, especially for null mice. Conclusions: The fGNG (endogenous glucogenesis) in null mice complemented the fGG (exogenous glucogenesis) to conserve prandial blood glucose concentrations. The hypotheses that Mgam contributes a high-efficiency activity on ad limitum intakes and SI dominates on ad libitum starch digestion were confirmed.

Contribution of the Individual Small Intestinal α-Glucosidases to Digestion of Unusual α-Linked Glycemic Disaccharides

The mammalian mucosal α-glucosidase complexes, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), have two catalytic subunits (N- and C-termini). Concurrent with the desire to modulate glycemic response, there has been a focus on di-/oligosaccharides with unusual α-linkages that are digested to glucose slowly by these enzymes. Here, we look at disaccharides with various possible α-linkages and their hydrolysis. Hydrolytic properties of the maltose and sucrose isomers were determined using rat intestinal and individual recombinant α-glucosidases. The individual α-glucosidases had moderate to low hydrolytic activities on all α-linked disaccharides, except trehalose. Maltase (N-terminal MGAM) showed a higher ability to digest α-1,2 and α-1,3 disaccharides, as well as α-1,4, making it the most versatile in α-hydrolytic activity. These findings apply to the development of new glycemic oligosaccharides based on unusual α-linkages for extended glycemic response. It also emphasizes that mammalian mucosal α-glucosidases must be used in in vitro assessment of digestion of such carbohydrates.