Topology and quaternary structure of pro-sucrase/isomaltase and final-form sucrase/isomaltase.
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ABSTRACT:
SUBMITTER: Cowell GM
PROVIDER: S-EPMC1147006 | biostudies-other | 1986 Jul
REPOSITORIES: biostudies-other
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Project description:Sucrase-isomaltase (SI) is an intestinal membrane-associated α-glucosidase that breaks down di- and oligosaccharides to absorbable monosaccharides. SI has two homologous functional subunits (sucrase and isomaltase) that both belong to the glycoside hydrolase family 31 (GH31) and differ in substrate specificity. All GH31 enzymes share a consensus sequence harboring an aspartic acid residue as a catalytic nucleophile. Moreover, crystallographic structural analysis of isomaltase predicts that another aspartic acid residue functions as a proton donor in hydrolysis. Here, we mutagenized the predicted proton donor residues and the nucleophilic catalyst residues in each SI subunit. We expressed these SI variants in COS-1 cells and analyzed their structural, transport, and functional characteristics. All of the mutants revealed expression levels and maturation rates comparable with those of the wild-type species and the corresponding nonmutated subunits were functionally active. Thereby we determined rate and substrate specificity for each single subunit without influence from the other subunit. This approach provides a model for functional analysis of the single subunits within a multidomain protein, achieved without the necessity to express the individual subunits separately. Of note, we also found that glucose product inhibition regulates the activities of both SI subunits. We experimentally confirmed the catalytic function of the predicted proton donor residues, and sequence analysis suggested that these residues are located in a consensus region in many GH31 family members. In summary, these findings reveal the kinetic features specific for each human SI subunit and demonstrate that the activities of these subunits are regulated via product inhibition.
| S-EPMC5491789 | biostudies-literature
Project description:BACKGROUND AND HYPOTHESES:Human starch digestion is a multienzyme process involving 6 different enzymes: salivary and pancreatic ?-amylase; sucrase and isomaltase (from sucrose-isomaltase [SI]), and maltase and glucoamylase (from maltase-glucoamylase [MGAM]). Together these enzymes cleave starch to smaller molecules ultimately resulting in the absorbable monosaccharide glucose. Approximately 80% of all mucosal maltase activity is accounted for by SI and the reminder by MGAM. Clinical studies suggest that starch may be poorly digested in those with congenital sucrase-isomaltase deficiency (CSID). Poor starch digestion occurs in individuals with CSID and can be documented using a noninvasive C-breath test (BT). METHODS:C-Labled starch was used as a test BT substrate in children with CSID. Sucrase deficiency was previously documented in study subjects by both duodenal biopsy enzyme assays and C-sucrose BT. Breath CO2 was quantitated at intervals before and after serial C-substrate loads (glucose followed 75?minutes later by starch). Variations in metabolism were normalized against C-glucose BT (coefficient of glucose absorption). Control subjects consisted of healthy family members and a group of children with functional abdominal pain with biopsy-proven sucrase sufficiency. RESULTS:Children with CSID had a significant reduction of C-starch digestion mirroring that of their duodenal sucrase and maltase activity and C-sucrase BT. CONCLUSIONS:In children with CSID, starch digestion may be impaired. In children with CSID, starch digestion correlates well with measures of sucrase activity.
| S-EPMC5957291 | biostudies-literature
Project description:BackgroundCongenital sucrase-isomaltase deficiency is a rare hereditary cause of chronic diarrhea in children. People with this condition lack the intestinal brush-border enzyme required for digestion of di- and oligosaccharides, including sucrose and isomaltose, leading to malabsorption. Although the condition is known to be highly prevalent (about 5%-10%) in several Inuit populations, the genetic basis for this has not been described. We sought to identify a common mutation for congenital sucrase-isomaltase deficiency in the Inuit population.MethodsWe sequenced the sucrase-isomaltase gene, SI, in a single Inuit proband with congenital sucrase-isomaltase deficiency who had severe fermentative diarrhea and failure to thrive. We then genotyped a further 128 anonymized Inuit controls from a variety of locales in the Canadian Arctic to assess for a possible founder effect.ResultsIn the proband, we identified a novel, homozygous frameshift mutation, c.273_274delAG (p.Gly92Leufs*8), predicted to result in complete absence of a functional protein product. This change was very common among the Inuit controls, with an observed allele frequency of 17.2% (95% confidence interval [CI] 12.6%-21.8%). The predicted Hardy-Weinberg prevalence of congenital sucrase-isomaltase deficiency in Inuit people, based on this single founder allele, is 3.0% (95% CI 1.4%-4.5%), which is comparable with previous estimates.InterpretationWe found a common mutation, SI c.273_274delAG, to be responsible for the high prevalence of congenital sucrase-isomaltase deficiency among Inuit people. Targeted mutation testing for this allele should afford a simple and minimally invasive means of diagnosing this condition in Inuit patients with chronic diarrhea.
| S-EPMC4312148 | biostudies-literature
Project description:BACKGROUND:Disaccharide Intolerance Type I (Mendelian Interance in Man database: *222900) is a rare inborn error of metabolism resulting from mutation in sucrase-isomaltase (Enzyme Catalyzed 3.2.1.48). Usually, infants with SI deficiency come to attention because of chronic diarrhea and nutritional evidence of malabsorption. CASE PRESENTATION:We describe an atypical presentation of this disorder in a 10-month-old infant. In addition to chronic diarrhea, the child displayed severe and chronic hypercalcemia, the evaluation of which was negative. An apparently coincidental right orbital hemangioma was detected. Following identification of the SI deficiency, an appropriately sucrose-restricted, but normal calcium diet regimen was instituted which led to cessation of diarrhea, substantial weight gain, and resolution of hypercalcemia. CONCLUSIONS:This case illustrates that, similar to congenital lactase deficiency (Mendelian Interance in Man database: *223000, Alactasia, Hereditary Disaccharide Intolerance Type II), hypercalcemia may complicate neonatal Sucrase-Isomaltase deficiency. Hypercalcemia in the presence of chronic diarrhea should suggest disaccharide intolerance in young infants.
| S-EPMC111192 | biostudies-literature
Project description:ObjectivesCongenital diarrheal disorders is a group of inherited enteropathies presenting in early life and requiring parenteral nutrition. In most cases, genetics may be the key for precise diagnosis. We present an infant girl with chronic congenital diarrhea that resolved after introduction of fructose-based formula but had no identified mutation in the SLC5A1 gene. Using whole exome sequencing (WES) we identified other mutations that better dictated dietary adjustments.MethodsWES of the patient and her parents was performed. The analysis focused on recessive model including compound heterozygous mutations. Sanger sequencing was used to validate identified mutations and to screen the patient's newborn sister and grandparents. Expression and localization analysis were performed in the patient's duodenal biopsies using immunohistochemistry.ResultsUsing WES we identified a new compound heterozygote mutation in sucrase-isomaltase (SI) gene; a maternal inherited known V577G mutation, and a novel paternal inherited C1531W mutation. Importantly, the newborn offspring carried similar compound heterozygous mutations. Computational predictions suggest that both mutations highly destabilize the protein. SI expression and localization studies determined that the mutated SI protein was not expressed on the brush border membrane in the patient's duodenal biopsies, verifying the diagnosis of congenital sucrase-isomaltase deficiency (CSID).ConclusionsThe novel compound heterozygote V577G/C1531W SI mutations lead to lack of SI expression in the duodenal brush border, confirming the diagnosis of CSID. These cases of CSID extend the molecular spectrum of this condition, further directing a more adequate dietary intervention for the patient and newborn sibling.
| S-EPMC8176889 | biostudies-literature
Project description:Patients with irritable bowel syndrome (IBS) often associate their symptoms to certain foods. In congenital sucrase-isomaltase deficiency (CSID), recessive mutations in the SI gene (coding for the disaccharidase digesting sucrose and 60% of dietary starch)1 cause clinical features of IBS through colonic accumulation of undigested carbohydrates, triggering bowel symptoms.2 Hence, in a previous study,3 we hypothesized that CSID variants reducing SI enzymatic activity may contribute to development of IBS symptoms. We detected association with increased risk of IBS for 4 rare loss-of-function variants typically found in (homozygous) CSID patients, because carriers (heterozygous) of these rare variants were more common in patients than in controls.1,4 Through a 2-step computational and experimental strategy, the present study aimed to determine whether other (dys-)functional SI variants are associated with risk of IBS in addition to known CSID mutations. We first aimed to identify all SI rare pathogenic variants (SI-RPVs) on the basis of integrated Mendelian Clinically Applicable Pathogenicity (M-CAP) and Combined Annotation Dependent Depletion (CADD) predictive (clinically relevant) scores; next, we inspected genotype data currently available for 2207 IBS patients from a large ongoing project to compare SI-RPV case frequencies with ethnically matched population frequencies from the Exome Aggregation Consortium (ExAC).
| S-EPMC6103908 | biostudies-literature
Project description:Lawsonia intracellularis is endemic to swine herds worldwide, however much is still unknown regarding its impact on intestinal function. Thus, this study aimed to characterize the impact of L. intracellularis on digestive function, and how vaccination mitigates these impacts. Thirty-six L. intracellularis negative barrows were assigned to treatment groups (n = 12/trt): (1) nonvaccinated, L. intracellularis negative (NC); (2) nonvaccinated, L intracellularis challenged (PC); and (3) L. intracellularis challenged, vaccinated (Enterisol® Ileitis, Boehringer Ingelheim) 7 weeks pre-challenge (VAC). On days post-inoculation (dpi) 0 PC and VAC pigs were inoculated with L. intracellularis. From dpi 19-21 fecal samples were collected for apparent total tract digestibility (ATTD) and at dpi 21, pigs were euthanized for sample collection. Post-inoculation, ADG was reduced in PC pigs compared with NC (41%, P < 0.001) and VAC (25%, P < 0.001) pigs. Ileal gross lesion severity was greater in PC pigs compared with NC (P = 0.003) and VAC (P = 0.018) pigs. Dry matter, organic matter, nitrogen, and energy ATTD were reduced in PC pigs compared with NC pigs (P ≤ 0.001 for all). RNAscope in situ hybridization revealed abolition of sucrase-isomaltase transcript in the ileum of PC pigs compared with NC and VAC pigs (P < 0.01). Conversely, abundance of stem cell signaling markers Wnt3, Hes1, and p27Kip1 were increased in PC pigs compared with NC pigs (P ≤ 0.085). Taken together, these data demonstrate that reduced digestibility during L. intracellularis challenge is partially driven by abolition of digestive machinery in lesioned tissue. Further, vaccination mitigated several of these effects, likely from lower bacterial burden and reduced disease severity.
| S-EPMC8214296 | biostudies-literature
Project description:Brush-border maltase-glucoamylase (MGA) activity serves as the final step of small intestinal digestion of linear regions of dietary starch to glucose. Brush-border sucrase-isomaltase (SI) activity is complementary, through digestion of branched starch linkages. Here we report the cloning and sequencing of human MGA gene and demonstrate its close evolutionary relationship to SI. The gene is approximately 82,000 bp long and located at chromosome 7q34. Forty-eight exons were identified. The 5' gene product, when expressed as the N-terminal protein sequence, hydrolyzes maltose and starch, but not sucrose, and is thus distinct from SI. The catalytic residue was identified by mutation of an aspartic acid and was found to be identical with that described for SI. The exon structures of MGA and SI were identical. This homology of genomic structure is even more impressive than the previously reported 59% amino acid sequence identity. The shared exon structures and peptide domains, including proton donors, suggest that MGA and SI evolved by duplication of an ancestral gene, which itself had already undergone tandem gene duplication. The complementary human enzyme activities allow digestion of the starches of plant origin that make up two-thirds of most diets.
| S-EPMC298790 | biostudies-literature
Project description:Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear alpha-1,4- and branched alpha-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear alpha-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both alpha-1,4- and alpha-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 A and in complex with the inhibitor kotalanol to 2.15 A resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic +1 subsite, which may account for its additional substrate specificity for alpha-1,6 substrates.
| S-EPMC2878540 | biostudies-literature
Project description:ObjectiveIBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase (SI) gene variants for their potential relevance in IBS.DesignWe sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population.ResultsCSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05).ConclusionsSI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.
| S-EPMC5563477 | biostudies-literature