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

ABSTRACT: Sucrase-isomaltase (SI) is an intestinal membrane-associated ?-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.

SUBMITTER: Gericke B

PROVIDER: S-EPMC5491789 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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

Gericke Birthe B Schecker Natalie N Amiri Mahdi M Naim Hassan Y HY

The Journal of biological chemistry 20170518 26

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 anoth ...[more]

PMID: 28522605

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Project description:Staphylococcus aureus is an important nosocomial and community-acquired facultative intracellular pathogen. Many studies have reported that S. aureus infections are associated with intestinal symptoms, but little is known about the molecular mechanisms implicated in S. aureus-induced alterations of intestinal functions. In this study, we investigated the implication of lipid rafts in the interaction of S. aureus with Caco-2 cells. To assess potential alterations in the lipid raft structure and effects on the hydrolytic function, we utilized sucrase-isomaltase (SI) as the major intestinal α-glucosidase that is associated with and sorted to the apical membrane via lipid rafts. Seven days post-confluent, Caco-2 cells were infected with S. aureus Newman and further incubated for an additional 2 days. After 48 h, the levels of SI expression as well as the enzymatic function of this protein were assessed in the infected versus non-infected cells. Analysis of the sorting behavior of SI to the apical membrane constituted another crucial aspect in studying the effects of S. aureus on Caco-2 cells. For this purpose, the apical membranes or brush border membranes (BBMs; referred to as P2 fraction) were separated in both infected and non-infected cells from the basolateral and intracellular membranes (referred to as P1 fraction) by employing a cationic-based procedure using CaCl2. The data show that there is no significant change in the overall expression levels of SI in the infected versus non-infected cells as assessed by Western blotting analysis using monoclonal anti-SI antibodies. By contrast, a significant decrease in the localization as well as the specific hydrolytic activities of SI toward sucrose and isomaltose (Palatinose) was observed in the BBM (P2 fraction) in Caco-2 cells 48 h post-infection. Concomitantly, the specific SI activities increased in the basolateral membrane/intracellular fraction (P1). Noteworthy, the specific activity of SI in the BBM of infected cells was markedly reduced as compared with that of the non-infected counterparts. The data accumulated from this study strongly suggest that infections with S. aureus influence the final step in the lipid raft-associated trafficking of human SI and thereby may trigger secondary functional gastrointestinal disorders.

Dataset Information

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

Publications

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

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