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Biochemical, Molecular, and Genetic Mechanisms

Luminal Starch Substrate "Brake" on Maltase-Glucoamylase Activity Is Located within the Glucoamylase Subunit^1–3,

⁴ CIEP-Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi, Zona Universitaria, San Luis Potosi, S.L.P., Mexico, 78360; ⁵ USDA, Agricultural Research Service, Children's Nutrition Research Center and Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030-2600; ⁶ Department of Medical Biophysics, University of Toronto and Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, M5G1L7 Canada; ⁷ Whistler Center for Carbohydrate Research and Department of Food Science, Purdue University, West Lafayette, IN 47907-2009; ⁸ Division of Biological Sciences, Section of Physiology, Cornell University, Ithaca, NY 14853; ⁹ Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 1Z3, Canada; and ¹⁰ Institute of Biochemistry and Molecular Medicine, University of Berne, Berne, CH-3012 Switzerland

^* To whom correspondence should be addressed. E-mail: bnichols{at}bcm.tmc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
The detailed mechanistic aspects for the final starch digestion process leading to effective -glucogenesis by the 2 mucosal -glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and -limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

	Introduction

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

Natural starches are formed by 2 main molecular structures: amylose, consisting of long linear chains of glucose associated by -1,4 glucosidic linkages and occasional branching with -1,6 glucosidic linkages; and amylopectin, consisting of relatively short -1,4, bound glucose chains of variable length with a relatively high content of -1,6 branching chains (9). The proportion of amylose to amylopectin, the average length of -1,4, chains, and frequency of -1,6 branching is variable between starches derived from different plant species and varieties (9) and has been recognized as a major determinant of their digestibility in the human small intestinal tract (10–12).

Intestinal digestion of starches to glucose in humans requires a consortium of 6 known enzyme activities. Two related -amylases (AMY),¹¹ salivary and pancreatic, have -1,4 endo-glucosidase activities that produce a range of linear and branched glucose oligomers called -limit dextrin (LDx). LDx consists primarily of maltose (G2), maltotriose (G3), and related -1,6 branched products (13,14) but very little free glucose (13). Efficient release of glucose from LDx requires the 4 small intestinal mucosal -1,4 exo-glucosidases associated with the sucrase and isomaltase activities of the membrane-bound intestinal enzyme human sucrase-isomaltase complex (SI) and with the membrane-bound enzyme human maltase-glucoamylase (MGAM) (15).

The studies on the effects of substrate chemical structure on human AMY have shown that, although the enzyme is active against G3, optimal activity is displayed on stretches of 5 or more linear -1,4 linked glucose molecules (16–18). The -1,6 branching of amylopectins interferes with this activity (14,16,18).

Little is known about the catalytic mechanisms of the mucosal -1,4 exo-glucosidases. Although MGAM and SI have classically been assayed as maltases (15,19), other glucose oligomers are substrates and some display a higher efficiency of hydrolysis than G2 (20,21). Two catalytic sites have been suggested to exist in human MGAM, each associated to a respective subunit: 1 appeared to contain 2 subsites for binding glucose residues and displays highest activity against G2 as substrate (20,22); the other appears to contain up to 4 subsites for binding of glucose residues and displays the highest catalytic efficiency against G3 and maltotetrose (G4). Interestingly, these 2 oligosaccharides also caused substrate inhibition (23). The substrate range and overlapping of the active sites has not been determined. Similar conclusions were obtained with the use of inhibitors such as acarbose and 1-deoxynojirimycin (24). The existence of 2 subunits in human MGAM was confirmed by cloning and sequencing of its coding mRNA (25,26) and revealed that both belonged to the glyco-hydrolase family 31 domain proteins (GH31) containing the signature sequence GXWIDMNE. The lack of information on the crystalline structure of GH31domains as well as of bound substrates has limited the definition of substrate specificities for each subunit.

Sucrase (S) and isomaltase (I) subunits of SI show, in addition to their -1,4 glucosidic activities, the only endogenous intestinal enzymes displaying specific activities against the -1,2 and -1,6 linkages of sucrose and isomaltose, respectively. However, due to the historical predominant role of starches in the human diet, glucose oligomers most probably constitute their main substrates. The only study addressing the catalytic properties of SI suggested that the sucrase subunit (S subunit) has maximal activity against G2, whereas the membrane-bound isomaltase subunit (I subunit) shows high activity against the -1,6 linkage of branched oligomers of up to 4 glucose residues (21).

The presence of the 2 N- and C-terminal subunits in all MGAM preparations obtained by biochemical methods has forced the simultaneous analysis of their catalytic properties. Recently, a soluble N-terminal subunit of MGAM (ntMGAM) lacking the transmembrane domain has been obtained in recombinant form by 1 of our laboratories (27). The availability of this recombinant protein and of recombinant human pancreatic AMY (rhpAMY) (28) opened the possibility to use a well-defined system for studying the process of starch digestion. This system would be more representative of the actual human starch digestion process than others using relatively crude preparations of fungal and animal enzymes. In vivo, the simultaneous presence of 3 intestinal glucosidases, AMY, SI, and MGAM, show important interactions of their activities leading to a strong synergism in the total -glucogenic activity (13). Similarly, we have demonstrated that mouse SI and MGAM show strong interactions with human pancreatic AMY, increasing by several-fold the -glucogenic activity from solubilized noncrystalline starch and derived products (29). Despite the strong synergic interactions observed, clear differences were also noted among the rates of glucose release from the different substrates, most probably caused by the different nature of the substrate chemical structures. These observations indicated that the efficiency of the intestinal -glucogenic machinery is sensitive to the chemical structure of starch and derived nutrients. The multiplicity of mucosal -glucosidases and their interactions with diverse substrates has lead us to the hypothesis that, as a group, they constitute a mechanism of adaptation to the wide range of food starches.

In this work, we compared the hydrolytic properties of ntMGAM to those of immunoisolated native human MGAM against maltoside substrates, including glucose oligomers G2 to maltopentose (G5), maltodextrins, and LDx. We show that the -glucogenic activity of ntMGAM against these different starch products allows a better understanding of the catalytic properties of native MGAM and its N and C subunits.

	Methods

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
    Recombinant ntMGAM. The production of recombinant ntMGAM was performed as described previously (27). Purified active ntMGAM was dialyzed against 10 mmol/L PBS containing 150 mmol/L NaCl, pH 6.8, and the protein concentration was adjusted to 1 g/L.

    Immunoprecipitation of native human MGAM, SI, and ntMGAM. Immunoprecipitated human MGAM and SI from an organ donor (Baylor College of Medicine Institutional Review Board approval H-1614) were obtained as described previously (13) using 1 single immunoprecipitating round and monoclonal antibodies LAMA 1/207/140/12, LAMA 1/77/6/2/1, and LAMA 1/127 specific against human MGAM, or HSI 3/42/1/2 and HSI 3/56/4/1 specific against human SI. In some experiments, normal mouse IgG fraction from BALB/c mouse serum was used in a similar way to monoclonal antibody. Efficiency of immunoprecipitation was measured by quantitation of maltase, sucrase, and isomaltase activities in each independent fraction and the original mucosa extract.

Immunoprecipitation of ntMGAM was performed with aliquots of 100 µL of the recombinant protein at a concentration of 25 mg/L mixed independently with 20 µL of 50% (v:v) slurries of the anti-MGAM, anti-SI, or normal IgG protein A-Sepharose (ProtA) beads prepared as described (13). The mixtures were incubated overnight at 4°C with rocking and the beads recovered by centrifugation and washed twice with washing buffer. The beads were finally resuspended in 100 µL of either PBS or PBS containing 2.5 mmol/L EDTA.

To discard the possibility that bound antibodies modify the catalytic properties of native human MGAM, SI-depleted supernatants of human intestinal mucosa were obtained after immunoprecipitation of SI in some experiments. These extract supernatants were used as a source of native MGAM free of SI activities.

    Kinetic properties of ntMGAM. The Michaelis constant (Km) and maximum rate of reaction (Vmax) of ntMGAM for G2 to G5 were determined with enzyme solutions containing 20 mg/L and substrate solutions in the range of 1.56–50 mmol/L for G2 and 0.39–12.5 mmol/L for G3 to G5 all in PBS-EDTA buffer. The reactions were performed by triplicate in 96-well plates with mixtures of 10 µL of the enzyme solution and 10 µL of each individual substrate solution during 30 min at 37°C. In assays to determine the inhibitory constant (Ki) of acarbose in the inhibition of the maltase activities, 10 µL of acarbose (Toronto Research Chemicals) solutions to attain final concentrations of 1, 10, 100, and 1000 µmol/L were added to the mixtures of substrate and jejunal mucosal homogenates before the incubation period. The reactions were developed with the Tris-glucose oxidase reagent during 60 min at 37°C and the optical density at 450 nm was measured in a SpectraMax190 microplate spectrophotometer (Molecular Devices).

    Real-time glucogenic assays. Real-time glucose release from oligosaccharides and starch products was measured as described previously (13,29) using 190 µL phosphate glucose-oxidase developing reagent and 10 µL of substrate solution at 5 g/L dissolved in PBS (240 mg/L final concentration). The mixtures were incubated for 10 min at 37°C for temperature equilibration and then 10 µL of enzyme preparations was added to the wells. The OD at 450 nm was measured in the SpectraMax190 microplate spectrophotometer at 37°C at 2-min intervals for a period of up to 60 min, with a 3-s shaking period before each reading. One unit of activity (U) was defined as that releasing 1 µmol glucose/min reaction.

    Effect of pH on relative activity. The effect of pH on the relative activity displayed by ntMGAM and immunoprecipitated MGAM (IP-MGAM) was investigated in assays using 50 mmol/L G2 solutions as substrate and ntMGAM or IP-MGAM resuspended in 1 of the following buffers: 50 mmol/L Gly, pH 3; 100 mmol/L acetate, pH 4; 100 mmol/L maleate, pH 5; 100 mmol/L citrate, pH 6; 100 mmol/L phosphates, pH 7; and 100 mmol/L HEPES, pH 8. Reaction conditions were similar to those described above for the analysis of the kinetic properties of ntMGAM.

    Statistics. Apparent Vmax, Km, and, when necessary, Ki values were calculated using nonlinear regression with the Marquardt-Levenberg algorithm and models adjusted to a classic single substrate Michaelis-Menten, competitive inhibition, or to substrate inhibition kinetics. Differences between calculated kinetic parameters of the respective strains were determined pairwise by the Z test. Rates of reaction for real-time glucogenesis assays were calculated under steady-state conditions by linear regression during intervals of 10–60 min of reaction. Indices of digestion for different starches and derived substrates were calculated as the ratio of the rates of reaction at steady state divided by the maltase activity observed after 30-min incubations for each enzyme mixture (29). Indices of digestion were analyzed by General Linear Model and 2-way-ANOVA with time as a covariate and enzyme mixture and/or substrate as classifying factors. Differences between indexes for each enzyme mixture and starch product were determined by 1-way ANOVA and Tukey's test (family error rate = 0.01). Correlation between the values of glucogenic index obtained with ntMGAM and IP-MGAM for different starch derived products was determined by Pearson's test and linear regression. In all cases, significance was considered at P < 0.05 unless otherwise noted.

	Results

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
    Immunoprecipitation of human native MGAM and ntMGAM. Using 1 single round of immunoprecipitation with monoclonal antibody bound to ProtA beads, recovery of maltase, isomaltase, and sucrase varied from 80 to 100% of the initial activities. A small amount of unspecific binding of SI molecules occurred even with ProtA beads coupled to normal mouse IgG (<5% of sucrase activity), indicating that some SI molecules may interact nonspecifically with the carbohydrate moiety of Sepharose. Therefore, a small amount of SI molecules may be contaminants in all IP preparations. Anti-SI ProtA beads were able to immunoprecipitate close to 50% of the initial maltase activity and >75% of the sucrase and isomaltase activities, whereas the supernatants after the immunoprecipitation with the anti-SI beads contained almost 50% of the maltase and 8% of sucrase and isomaltase activities (Fig. 1). Thus, more than 90% of the maltase activity in these supernatants was due to MGAM molecules. One single round of immunoprecipitation with ProtA beads bound to anti-MGAM antibodies was able to precipitate 30% of the original maltase activity (Fig. 1). In our previous work, at least 2 immunoprecipitation rounds of with anti-MGAM ProtA beads were required to quantitatively deplete the supernatants of MGAM (13). As expected, a small amount of sucrase and isomaltase activities (3%) were found associated with the anti-MGAM beads, also indicating that more than 95% of the maltase activity present in the IP-MGAM preparations was due to native MGAM (Fig. 1).

View larger version (19K): [in this window] [in a new window]   FIGURE 1  Recovery of enzymatic activities after immunoprecipitation with ProtA beads alone (PreClean), ProtA bound to normal mouse IgG (Normal IgG), ProtA bound to monoclonal antibodies against SI (anti-SI), or ProtA bound to monoclonal antibodies against MGAM (anti-MGAM). Values are means ± SD, n = 3 and are expressed relative to the activity in PreClean samples (100%).

    Catalytic properties of ntMGAM and native MGAM on glucose oligomers G2 to G5. ntMGAM showed moderate activity against G2 to G5, with Km values in ranging from 3 to 8 mmol/L (Table 1; Supplemental Fig. 1) with G4 showing the lowest Km. Values of Vmax were relatively close between G2 to G5 and varied from 7 to 10 U/mg. In this case, G3 displayed the highest Vmax. ntMGAM also displayed a slight substrate inhibitory brake effect when tested with G2, G3, and G4, with Ki in the range of 35–90 mmol/L, distinguishable only at high substrate concentrations.

To compare the kinetic patterns of hydrolytic activity, we also analyzed the kinetics of IP-SI against G2 to G5. The catalytic activity of IP-SI resembled that of ntMGAM, displaying values of Km in the range of 3.6 to 6.8 mmol/L, with G5 displaying the lowest value. However, in contrast to ntMGAM and IP-MGAM, no inhibitory brake effect existed for any of the 4 glucose oligomers used as substrate (Table 1; Supplemental Fig. 3).

    Activity of ntMGAM and native MGAM on starches, maltodextrins, and limit dextrins. Using a different set of monoclonal antibodies against human MGAM than those used in this work, we have shown previously that IP-MGAM experiences a strong substrate inhibitory brake effect by maltodextrins (13). In this work, we confirmed our previous finding that IP-MGAM experienced strong substrate inhibitory brake effect by maltodextrins evident at concentrations as low as 1.2 g/L and with an apparent Ki of 12.7 g/L. The apparent Km was 0.06 g/L, extremely low and within the limit of confident measurements of the method employed (Table 2; Supplemental Fig. 4). Because we speculated that the inhibitory brake behavior could be in part due to structural changes and relative immobility of MGAM caused by bound antibodies, we analyzed the behavior of the supernatants after immunoprecipitation of SI, whose -glucosidic activity is mostly due to solubilized MGAM. We observed a very similar substrate inhibitory brake effect to that of IP-MGAM, indicating that this effect is also exerted on the free MGAM enzyme molecule and is not caused by the immobilization and binding of antibodies. In contrast to IP-MGAM, ntMGAM showed robust -glucosidic activity with an apparent Km of 4.3 g/L and classic Michaelis-Menten kinetics. Vmax specific activity of ntMGAM was 7.5 U/mg (1U = 1 µmol/min of glucose released), very similar to that with glucose oligomers (Table 2; Supplemental Fig. 4).

We repeated these experiments using LDx obtained by extensive digestion of maltodextrins by porcine pancreatic amylase. These preparations of LDx contain a higher proportion of G2 to G5 that could modify the digestion process (13). We observed a behavior similar to that with corn maltodextrin (MDx) with high activities for IP-SI and ntMGAM and a relatively low activity and substrate inhibition for IP-MGAM and supernatant after immunoprecipitation of SI (Sup-SI). Apparent Km for LDx with IP-MGAM and Sup-SI enzyme preparations were increased 5–10 times, as well as the apparent substrate Ki. This suggested that this preparation of LDx could be largely composed of G2 or other substrates with low capacity for substrate inhibition (Table 2; Supplemental Fig. 5).

We wanted to measure and compare the properties of ntMGAM for the digestion of starches. Therefore, we defined an index of digestion based on maltase activity as denominator or correcting factor. Using these ratios of activities, we have shown that it is possible to distinguish structural differences of starches from different sources (29). In addition, we showed that at high enzyme concentrations, ntMGAM is able to partially hydrolyze native starch granules (30). Thus, we performed experiments to determine first, whether structural differences among starches from different sources could be distinguished based on the patterns or rates of -glucogenesis by human intestinal mucosa; second, whether ntMGAM could hydrolyze solubilized starch at concentrations of enzyme and substrate reasonably low for routine analytic work; and third, the degree in which the patterns of -glucogenesis by intestinal mucosa could be reproduced by ntMGAM.

Despite the belief that there is an absolute dependence on AMY for digestion of starch in the human tract, there was a substantial -glucogenesis by hydrolysis of solubilized normal corn starch [glucogenic index (GI): 0.0124) or its derivatives amylose (GI: 0.0088) and amylopectin (0.0131) by human intestinal mucosa homogenates (Table 3). Slightly higher -glucogenesis was observed with MDx as substrate (GI: 0.0191), and our synthetic LDx (O2–5), consisting of a equimolar mixture of G2 to G5 oligomers, displayed the highest rate of digestion (GI: 0.0337) (Table 3). In contrast with normal starch, the indexes for enzymatically modified slow digestion starches MG, barley β-amylase (BBA)/Aspergillus transglucosidase (TG), and MG/TA were substantially lower (GI: 0.008, 0.0069, and 0.0082, respectively). Thus, the GI in relation to G2 as defined and assayed here is also able to discriminate among starches and starch products with different chemical structures and digestibilities. Interestingly, ntMGAM at a final concentration in the assay wells of 120 µg/L and final substrate concentration of 240 mg/L displayed -glucogenic activity within or below the limit of detection against all the substrates but MDx and our synthetic LDx (O2–5). The last 2, MDx and 02-5, displayed GI of 0.0124 and 0.0300, respectively (Table 3), indicating a low activity of ntMGAM on complex starch molecules.

View this table: [in this window] [in a new window]   TABLE 3 Indexes of -glucogenesis from starch products based on maltase activity of ntMGAM and human small intestinal mucosa in presence and absence of rhpAMY1,2

A very important result was that despite the almost total absence of -glucogenic activity of ntMGAM against complex starch molecules, when assayed in presence of rhpAMY, there was a marked synergistic effect that caused increased GI values to levels slightly lower than those of human intestinal lysates in the presence of rhpAMY. In fact, statistical analysis showed that, in the presence of rhpAMY, there was an extremely good correlation (r = 0.91, P < 0.05; r² = 0.83, coefficient = 1.1, P < 0.05) between the values of GI by ntMGAM and human small intestinal mucosal lysate for any of the substrates. In addition, independent of the presence or absence of rhpAMY and of the -glucosidic enzymatic preparation, there were clear differences in the GI among substrates, maintaining the ability to differentiate them.

    Inhibition by acarbose. Acarbose is an aminoglucoside analogue of G4 that has been used in the control of glycemic levels in patients with type 2 diabetes. We were interested in testing the sensitivity of the ntMGAM and comparing it to that of native MGAM. Using substrate concentration in the vicinity of its Km, ntMGAM had a relatively low sensitivity to acarbose with Ki of 16 mmol/L (Supplemental Fig. 6). In contrast, IP-MGAM was highly sensitive to acarbose with a calculated Ki of 74 nmol/L. However, at acarbose concentrations as high as 10 µmol/L, 10–20% of the initial activity of IP-MGAM was still present, confirming the presence of N-terminal activities with lower affinity for acarbose (Supplemental Fig. S6).

To compare the relative effect of acarbose on ntMGAM and IP-MGAM against the effects on SI activities, we also analyzed the inhibition of sucrase and palatinase activity of human intestinal mucosa. Sucrase activity displayed a Ki for acarbose of 5.3 mmol/L, whereas this value was 205 mmol/L for palatinase activity (Supplemental Fig. 7), resembling the relationship of sensitivities between IP-MGAM and ntMGAM. Thus, S subunit was 1–2 orders of magnitude more sensitive than the I subunit to inhibition by acarbose (Supplemental Fig. 7). The order of sensitivity for all these activities was therefore: IP-MGAM > S > ntMGAM > I.

    Dependence of the activity on pH. We compared the dependence on the pH of ntMGAM with that of IP-MGAM and IP-SI to gain some information on the maltase catalytic mechanisms involved for each maltase enzyme. We found that IP-MGAM displayed peak maximal activity at pH 7 (Supplemental Fig. 8). In contrast, although ntMGAM displayed a maximal activity at the same pH 7 as IP-MGAM, this recombinant subunit showed substantial activity at a wide range of pH values from 4 to 7 (Supplemental Fig. 8). Different from the 2 MGAM preparations, IP-SI maltase activity showed an optimal pH of 6 but without observable evidence for the presence of the 2 maltase subunits.

	Discussion

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
The molecular structure of human MGAM has remained controversial for a number of years. In 1998 Nichols et al. (25) obtained a peptide structure derived from the cDNA coding for human MGAM. This structure consisted of an integral membrane protein extending extracellularly to form 2 subunits each with amino acid sequences that classified them as members of GH31. The closest related sequence was the human SI protein with overall homology of >60% and almost identical structural motives, such as the presence of a stalk region with high density of Thr residues, the presence of trefoil domains flanking the N extreme of each of the 2 GH31 domains, as well as highly conserved signature sequences GXWIDMNE characteristic of GH31 family domains. Homology in the regional arrangement of SI and MGAM was also evident because the N-terminal subunit of SI, the I subunit, displayed higher homology to ntMGAM, whereas the same relationship existed for the C-terminal subunit of MGAM and the S subunit of SI (25). Differences between the catalytic properties of each S and I subunits were recognized long ago and led to the description of the associated activities of sucrase and isomaltase, respectively. The original experiments of Dahlqvist and Telenius (15,19) by heat inactivation indicated the presence of 4 independent maltase activities in the human intestinal mucosa; 2 of them were associated with the sucrase and isomaltase activities. This association was later confirmed by purification and cloning of the SI molecule (15,31,32). The other 2 maltase activities were arbitrarily named maltase and glucoamylase. Additional experiments also indicated the existence of 2 active sites in isolated native human MGAM (15,20,22,24,32), but no experimental attempt was performed to differentiate and ascribe the substrate specificity and catalytic properties to the respective subunits due to the impossibility to obtain them in pure and independent form. Thus, the study of the properties of recombinant ntMGAM constitutes the first analysis, to our knowledge, of the catalytic properties of the independent subunits of MGAM.

Our analysis showed catalytic differences between native MGAM isolated by immunoprecipitation and the recombinant N subunit of human MGAM. In contrast to native MGAM, ntMGAM displayed relatively slow kinetics for the hydrolysis of oligomers with 2–5 glucose residues. In addition, different from the strong substrate inhibitory brake effect for G3 and G4 on native MGAM, ntMGAM had a low sensitivity to substrate inhibition by these glucans, suggesting that the binding site has fewer possible binding configurations than the native molecule. Similarly, the sensitivity to acarbose was >1 order of magnitude smaller for ntMAG than for the native molecule. Thus, ntMGAM appeared to have a lower affinity for these glucan ligands and the analogous acarbose than the native counterpart.

We have shown that IP-MGAM experiences a strong substrate inhibitory brake effect by MDx (13). In this work, using a different set of immunoprecipitating monoclonal antibodies against MGAM, we have confirmed the inhibitory brake effect of MDx and LDx on native MGAM, suggesting that this is a generalized inhibitory brake effect on MGAM and not a feature for a possible subpopulation of the enzyme molecules. In addition, mucosal extracts depleted of SI molecules by immunoprecipitation with anti-SI monoclonal antibodies displayed almost identical behavior to IP-MGAM against MDx and LDx substrates, indicating that the substrate inhibitory brake effect is not an artifact caused by the immunoprecipitating procedure. In view of the strong inhibitory brake effect with G3 and G4 as substrates, the presence of a high proportion of these 2, and probable other unidentified glucose oligomers, in MDx and LDx may explain the strong substrate inhibition with these 2 predigested substrates on native MGAM.

Despite our observation that ntMGAM can hydrolyze normal corn starch granules (30), the relatively low activity displayed by ntMGAM against different types of solubilized starches and starch-derived products was surprising when compared with that displayed by human mucosa. This apparently low activity against native starch can be explained by 3 facts: 1) the assays presented in this work were optimized for glucose oligomers G2 to G5 using enzyme concentrations 2 orders of magnitude lower (120 µg/L) than those of our previous work using native starch granules (30); 2), human mucosa contains the full complement of 4 -glucosidases displayed by SI and the whole MGAM molecule; and 3), at the low substrate concentrations used in the assay (240 mg/L), the transient concentration of intermediate products with inhibitory capacity against native MGAM do not reach inhibitory concentrations allowing the enzyme to display full activity against large glucan molecules. In contrast, when ntMGAM was assayed in the presence of rhpAMY, recombinant ntMGAM displayed very high -glucogenic activity, indicative of a high catalytic efficiency against short glucan molecules and a low efficiency against large ones. In addition, in the presence of rhpAMY, the -GI using ntMGAM were highly correlated with those of human mucosa, suggesting the combined use of the recombinant rhpAMY and ntMGAM molecules for the in vitro assay of digestibility of starches in a more representative manner for human physiology.

Because the sequence of the recombinant insert used for expression of the recombinant protein did not show any modification, its relative low catalytic efficiency against large glucan molecules could not be ascribed to artificial structural changes affecting the catalytic properties of the recombinant subunit. In addition, X-ray crystallographic data on the structure of ntMGAM (33) have shown a close resemblance of the putative catalytic site of ntMGAM with those of YicI and MalA bacterial proteins, the only GH31 proteins with known crystallographic structure, discarding a major misfolding of ntMGAM.

The presence of the C-terminal subunit in the native MGAM molecule is the most obvious structural difference with respect to the ntMGAM subunit studied in this work. This suggests that the catalytic differences are caused by the presence of this C-terminal subunit in the native molecule. These differences between native MGAM and ntMGAM allow some predictions on the catalytic properties of the C-terminal subunit of native MGAM: 1) displays fast catalytic behavior against short glucose oligomers; 2) shows important substrate inhibitory brake effects, probably due to a larger number of binding subsites for glucose residues; 3) has a broader spectrum of substrate specificities, including substantial glucoamylase activity against raw starches; and 4) has higher sensitivity to acarbose inhibition. These characteristics firmly establish that the N terminal contains predominantly maltase activities and the C terminal contains glucoamylase activities.

Our observations have extended the similarities between SI and MGAM from purely peptide features to functional aspects. Using Mgam KO Sv/129 mice and the naturally occurring Mgam-deficient CBA/CaJ mice with concurrent sucrase deficiency, we reached the conclusion that S subunit (C-terminal subunit) has a lower Km for G2 and higher acarbose sensitivity than the I subunit (N-terminal subunit) (29). Comparison of the kinetic data of human native MGAM with ntMGAM also supports the notion that the C-terminal subunit has higher activity than the N subunit. In this work, we showed the same relative sensitivity to inhibition by acarbose of the human S and I subunits of SI in mice. Importantly, this relationship is also present between human native MGAM and ntMGAM. Thus, from a functional point of view, SI and MGAM preserve similarities in their subunits.

In summary, we presented evidence that ntMGAM displays catalytic properties that correspond to the maltase subunit, whereas the C terminal is responsible for the glucoamylase activity contained in the whole native molecule. These catalytic features of MGAM resemble those of the intestinal SI complex, extending the structural homologies existing between these 2 intestinal -glucosidases to functional aspects. As a whole, our observations lend support to our hypothesis that the multiplicity of intestinal -glucosidic activities is an adaptation mechanism to handle the wide variety of chemical structures present in natural starches.

	FOOTNOTES

 
¹ Supported in part by federal funds from the USDA, Agricultural Research Service, under Cooperative Agreement no. 58-6250-1-003 (B.L.N.); the Swiss National Science Foundation, grant no. 3100A0-100772 (E.E.S.); and an operating grant from the Canadian Institutes of Health Research, grant no. MOP-13338 (G.D.B.). R.Q.C. was on sabbatical from Universidad Autonoma de San Luis Potosí (UASLP) as a Visiting Professor in the Nichols Laboratory 2004–2006 and received support from the "Fondo de Apoyo a la Investigación", UASLP (C06-FAI-11-16.53). The contents of this publication do not necessarily reflect the views or policies of the USDA, the Swiss National Science Foundation, or the Canadian Institutes of Health Research, nor does mention of trade names, commercial products, or organizations imply endorsement by the US, Swiss, or Canadian governments.

² Author disclosures: R. Quezada-Calvillo, L. Sim, D. R. Rose, Z. Ao, B. R. Hamaker, A. Quaroni, G. D. Brayer, E. E. Sterchi, C. C. Robayo-Torres, and B. L. Nichols, no conflicts of interest.

³ Supplemental Figures 1–8 are available with the online posting of this paper at jn.nutrition.org.

¹¹ Abbreviations used: AMY, -amylase; TG, Aspergillus transglucosidase; BBA, barley β-amylase; LDx, corn -limit-dextrin; MDx, corn maltodextrin; GI, glucogenic index; O2-5, glucosides from 2 to 5 glucose units long; GH31, glyco-hydrolase family 31 domain proteins; MGAM, human maltase-glucoamylase; IP-MGAM, immunoprecipitated maltase-glucoamylase; IP-SI, immunoprecipitated human sucrase-isomaltase complex; Ki, inhibitory constant; I, isomaltase subunit; G2, maltose; G3, maltotriose; G4, maltotetrose; G5, maltopentose; Vmax, maximum rate of reaction; Km, Michaelis constant; Mgam, mouse maltase-glucoamylase; ntMGAM, N-terminal subunit of human maltase-glucoamylase; ProtA, protein A-Sepharose beads; rhpAMY, recombinant human pancreatic amylase; S, sucrase subunit; SI, human sucrase-isomaltase complex; Sup-SI, supernatant after immunoprecipitation of human sucrase-isomaltase complex.

Manuscript received 16 August 2007. Initial review completed 20 November 2007. Revision accepted 17 January 2008.

	LITERATURE CITED

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