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Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells.

ABSTRACT: To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.

SUBMITTER: Beaulieu JF

PROVIDER: S-EPMC1130497 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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Intracellular degradation and reduced cell-surface expression of sucrase-isomaltase in heat-shocked Caco-2 cells.

Project description:To investigate the role of post-translational events in intestinal cell differentiation we have studied the effects of heat shock on processing and cell surface delivery of sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (APN) in Caco-2 cells. In cells cultured at 42.5 degrees C there was a rapid decline in sucrase activity, while DPPIV and APN were unaffected over a 3-day period. Immunofluorescence staining confirmed the selective disappearance of SI from the surface membrane after only 1 day of culture at 42.5 degrees C. Cell-surface biotinylation of cells metabolically labelled with [35S]methionine 4 h after a switch from 37 degrees C to 42.5 degrees C demonstrated that newly synthesized APN and DPPIV were associated with the surface membrane, while SI was almost completely retained intracellularly. Pulse-chase experiments confirmed that, in these cells, DPPIV and APN were normally processed and vectorially delivered to the cell surface; in contrast, conversion between the two conformationally distinct high-mannose precursor forms of SI (hmP1 and hmP2) was markedly inhibited, a significant fraction of newly synthesized enzyme was degraded, probably in the ER, and an immature form of complex-glycosylated SI precursor (cP) was produced and mostly retained intracellularly. Double labelling of Caco-2 cells for SI and cathepsin D excluded an accumulation of SI in the lysosomes, suggesting that this organelle was not involved in the degradation of SI. These results indicate that the ER may play an important role in intestinal cell differentiation by regulating the conformational maturation, degradation and eventual cellular localization of some digestive enzymes.

| S-EPMC1134174 | biostudies-other

Structure-function analysis of human sucrase-isomaltase identifies key residues required for catalytic activity.

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

13C-Labeled-Starch Breath Test in Congenital Sucrase-isomaltase Deficiency.

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

Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-isomaltase N-terminal domains.

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

Congenital sucrase-isomaltase deficiency: identification of a common Inuit founder mutation.

Project description:BACKGROUND:Congenital 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. METHODS:We 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. RESULTS:In 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. INTERPRETATION:We 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

Topology and quaternary structure of pro-sucrase/isomaltase and final-form sucrase/isomaltase.

Project description:Pig sucrase/isomaltase (EC 3.2.1.48/10) was purified from intestinal microvillar vesicles prepared from animals with and without pancreatic-duct ligation to obtain the single-chain pro form and the proteolytically cleaved final form respectively. The purified enzymes were re-incorporated into phosphatidylcholine vesicles and analysed by electron microscopy after negative staining. The two forms of the enzyme were observed as identical series of characteristic projected views that could be unified in a single dimeric model, containing two sucrase and two isomaltase units. This shows a homodimeric functional organization similar to that of other microvillar hydrolases. The bulk of the dimer was separated from the membrane by a maximal gap of 3.5 nm, representing a junctional segment connecting the intramembrane section of the anchor to the catalytically active domain of sucrase/isomaltase. The enzyme complex protrudes from the membrane for a distance of up to 17 nm. From charge-shift immunoelectrophoresic studies of hydrophilic prosucrase/isomaltase and from electron microscopy of reconstituted pro-sucrase/isomaltase, there was no evidence to suggest the presence of anchoring sequences between the sucrase and isomaltase subunits.

| S-EPMC1147006 | biostudies-other

Congenital sucrase-isomaltase deficiency presenting with failure to thrive, hypercalcemia, and nephrocalcinosis.

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

Congenital Sucrase-isomaltase Deficiency: A Novel Compound Heterozygous Mutation Causing Aberrant Protein Localization.

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

Increased Prevalence of Rare Sucrase-isomaltase Pathogenic Variants in Irritable Bowel Syndrome Patients.

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

Lawsonia intracellularis infected enterocytes lack sucrase-isomaltase which contributes to reduced pig digestive capacity.

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

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