Characterization of a new multigene family encoding isomaltases in the yeast Saccharomyces cerevisiae, the IMA family.

It has been known for a long time that the yeast Saccharomyces cerevisiae can assimilate alpha-methylglucopyranoside and isomaltose. We here report the identification of 5 genes (YGR287c, YIL172c, YJL216c, YJL221c and YOL157c), which, similar to the SUCx, MALx, or HXTx multigene families, are located in the subtelomeric regions of different chromosomes. They share high nucleotide sequence identities between themselves (66-100%) and with the MALx2 genes (63-74%). Comparison of their amino acid sequences underlined a substitution of threonine by valine in region II, one of the four highly conserved regions of the alpha-glucosidase family. This change was previously shown to be sufficient to discriminate alpha-1,4- to alpha-1,6-glucosidase activity in YGR287c (Yamamoto, K., Nakayama, A., Yamamoto, Y., and Tabata, S. (2004) Eur. J. Biochem. 271, 3414-3420). We showed that each of these five genes encodes a protein with alpha-glucosidase activity on isomaltose, and we therefore renamed these genes IMA1 to IMA5 for IsoMAltase. Our results also illustrated that sequence polymorphisms among this family led to interesting variability of gene expression patterns and of catalytic efficiencies on different substrates, which altogether should account for the absence of functional redundancy for growth on isomaltose. Indeed, deletion studies revealed that IMA1/YGR287c encodes the major isomaltase and that growth on isomaltose required the presence of AGT1, which encodes an alpha-glucoside transporter. Expressions of IMA1 and IMA5/YJL216c were strongly induced by maltose, isomaltose, and alpha-methylglucopyranoside, in accordance with their regulation by the Malx3p-transcription system. The physiological relevance of this IMAx multigene family in S. cerevisiae is discussed.

[Cloning and expression in Escherichia coli of oligo-1,6-glucosidase gene from Bacillus subtilis HB002].

The gene coding for oligo-1,6-glucosidase of Bacillus subtilis HB002 was cloned by the shotgun-cloning method and sequenced by the chain-termination method of Sanger et al. It consists of an open reading frame of 1683 bp. The amino acid sequence of oligo-1,6-glucosidase deduced from its nuecleotide sequence predicts a protein of 561 amino acid residues with a Mr of 65.985 kD, which is 81% and 67% identical to those of oligo-1,6-glucosidase from Bacillus sp. and Bacillus coagulans, respectively, 89% and 79% similar to those of oligo-1,6-glucosidase from Bacillus sp. and Bacillus coagulans, respectively. The oligo-1,6-glucosidase gene of Bacillus subtilis HB002 was cloned into Escherichia coli expression plasmid pBV220, the result of SDS-PAGE showed that the oligo-1,6-glucosidase gene had been expressed in Escherichia coli DH5 alpha, the expressed oligo-1, 6-glucosidase has enzymatic activity.

Effect of gut transposition on the expression of the endocrine gene neurotensin.

Expression of the gene encoding neurotensin (NT/N) is developmentally regulated in the adult small bowel with maximal expression noted in the distal ileum; the mechanisms responsible for this strict spatial-specific expression pattern are not known. The purpose of this study was to determine whether NT/N expression is altered by ileojejunal transposition. Rats underwent either sham operation or ileojejunal transposition and were killed 2 months after operation. The transposed (either jejunum or ileum) and sham-operated segments of gut were removed, a portion was processed for histologic examination, and the remainder was extracted for total RNA and analyzed by ribonuclease protection using a rat NT/N probe. For comparison, expression of another gut endocrine gene, peptide YY, and an enterocyte-specific gene, sucrase-isomaltase (SI), was also determined. Expression of the gut endocrine genes, NT/N and peptide YY, were minimally affected by transposition of either the jejunum or ileum. In contrast, SI expression was markedly altered in both the transposed jejunum and ileum compared with corresponding sham gut segments. Expression of the NT/N gene is minimally altered after jejunoileal transposition despite marked adaptive and morphologic changes in the transposed segments. These findings provide further support that the strict pattern of NT/N expression is "imprinted" in the gut and maintained regardless of location along the cephalocaudal gut axis.

Preservation of intestinal gene expression during hibernation.

The role of luminal nutrients in regulating enterocyte gene expression was studied in a natural model for long-term fasting, the hibernating ground squirrel. Squirrels were studied during the active season and during the hibernation season when they had not eaten for at least 12 wk. The specific activities of sucrase, isomaltase, and intestinal alkaline phosphatase in jejunal brush-border membranes were similar in hibernating and active squirrels, whereas amino-oligopeptidase was reduced in hibernators. Na(+)-K(+)-adenosinetriphosphatase activity in jejunal mucosa was unchanged by hibernation. Densitometric analysis of Western blots showed that abundance of sucrase-isomaltase (SI), amino-oligopeptidase, and the Na(+)-glucose cotransporter SGLT1 was similar in the two activity states. Preservation of SI abundance in hibernation was confirmed by immunocytochemistry. Slot-blot analysis revealed no differences in mRNA levels for these proteins between hibernating and active squirrels. Enterocyte proliferation and migration rates were greatly suppressed in torpid squirrels but increased immediately upon rewarming during arousals. These results demonstrate the striking constancy of enterocyte gene expression despite long-term fasting in a hibernating mammal.

Triiodothyronine stimulates the expression of sucrase-isomaltase in Caco-2 cells cultured in serum-free medium.

In a previous study we have shown that triiodothyronine (T3) added to a serum-free medium supplemented with insulin, transferrin, and selenous acid (ITS) can stimulate Caco-2 cell differentiation. In this study we have focused on the effects of T3 on sucrase activity. The results obtained demonstrate that T3 (50 nM) does not change Caco-2 cell proliferation but enhances sucrase activity from 50 to 80%. Similar increases were observed whether or not insulin was present in the culture medium, showing that there was no synergistic effect between T3 and insulin on sucrase activity. Moreover, T3 acts specifically during the differentiation period since addition of T3 to the defined TS medium before confluency is reached does not stimulate sucrase activity. Sucrase kinetic parameters were evaluated for the first time in Caco-2 cells under various culture conditions. The presence of a single enzyme was verified, with a Km of about 7 mM and a Vmax around 20 nmol of substrate hydrolyzed min-1 mg-1 of protein. Our results showed that T3 did not change the enzyme's affinity for sucrose but doubled the Vmax. Moreover, immunoblotting using anti-sucrase-isomaltase (SI) antibodies revealed an approximately twofold increase in the relative amount of SI immunoreactive protein in T3-stimulated cells compared to untreated cells. Results obtained by both Northern hybridization and RT-PCR amplification showed a significant increase in SI mRNA contents. These results suggest that T3 acts primarily on sucrase expression at the mRNA level.

Cellular fibronectin expression is down-regulated at the mRNA level in differentiating human intestinal epithelial cells.

The expression of human cellular fibronectin (HFN) was analyzed in relation to the differentiation process of the human enterocyte-like cell line Caco-2. A single approximately 290-kDa form of HFN, corresponding to a 10.0-kb mRNA species, was observed in Caco-2 cells. The expression levels of both protein and mRNA were found to decrease to undetectable levels concomitantly to the differentiation process of these cells. This decline in HFN expression was correlated with the expression of sucrase-isomaltase (a specific marker of intestinal cell differentiation). These results indicate that intestinal epithelial expression of HFN is down-regulated through its mRNA levels and is correlated with the acquisition of the differentiated enterocytic phenotype.

The constitutive exocytotic pathway in microvillous atrophy.

Microvillous atrophy is a disorder within the intractable diarrhea of infancy syndrome. The disease is believed to stem from a transport defect that prevents exocytosis of brush border-related material. We investigated this hypothesis by examining the direct constitutive exocytotic pathway using sucrase-isomaltase as a representative protein. We also studied various other brush border and lysosomal marker enzymes. The biosynthesis and localization of selected intestinal epithelial enzymes were studied in small-intestinal mucosal biopsy specimens from a total of nine children with microvillous atrophy by: (a) metabolic labeling in organ culture, (b) radioiodination and immunoprecipitation, (c) indirect immunoperoxidase immunocytochemistry, and (d) immunogold electron microscopy. The results demonstrated that brush border enzymes were synthesized normally and could be located in the apical brush border membrane and on microvillous membrane within microvillous inclusions. Brush border enzymes were not detected in the "secretory granules" that accumulated within the apical cytoplasm of epithelial cells. Lysosomal enzymes were only detected within lysosomal bodies. Thus, the direct constitutive pathway is not involved in microvillous atrophy, and a disturbance of endocytosis or the indirect constitutive pathway is unlikely. Any transport defect in the disease probably involves a different, unidentified exocytotic pathway.

Dietary CHO and stimulation of carbohydrases along villus column of fasted rat jejunum.

Adult rats when fed a high carbohydrate diet of 70% sucrose or glucose for 24 h following a 4-day fast showed increased concentrations of intestinal sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) but not lactase-phlorizin hydrolase (EC 3.2.1.23, EC 3.2.1.62). The concentration increases of these enzymes were accompanied by corresponding acceleration of their synthesis rates. Contrary to earlier studies by others, suggesting that upper villus cells in the fasted intestine are unresponsive to stimulation of sucrase activity by refeeding a high-sucrose diet, the concentration increases of both sucrase-isomaltase and maltase-glucoamylase were seen to occur in cells all along the length of the villus column. The earlier studies differed from the present study by basing enzyme assays relative to protein rather than the DNA content of villus cell fractions. We have shown that villus cells increase their protein content severalfold while migrating to villus tip, providing the basis for the difference between earlier and the present findings. Further evidence that stimulation of sucrase-isomaltase and maltase-glucoamylase by high carbohydrate is not restricted to the crypt and lower villus region was obtained by the finding that their synthesis rates appeared to be equally stimulated along the length of the villus column.

Synthesis and intracellular processing of sucrase-isomaltase in rat jejunum.

To examine directly the synthesis and intracellular processing of rat intestinal sucrase-isomaltase, [3H]leucine-labeled enzyme was solubilized from purified microsomal and microvillus fractions, immunoprecipitated, and analyzed. Immunologic identity between the microsomal and microvillus forms of the enzyme was demonstrated. Time-course studies using both intravenous and intraluminal labeling demonstrated an initial peak of microsomal sucrase-isomaltase labeling at 30 min followed by a sharp decline, and a subsequent rise in microvillus sucrase-isomaltase labeling, suggesting a precursor-product relationship. Fluorography and quantitative analysis of electrophoresed immunoprecipitates confirmed these results, and revealed the presence of two high molecular weight proteins in the microsomes. Susceptibility of the smaller high molecular weight precursor to cleavage with endo-beta-N-acetyl-glucosaminidase H indicated that this was the initially synthesized form of the glycoprotein. These data are consistent with the membrane flow hypothesis and the single polypeptide model suggested for sucrase-isomaltase synthesis.

Alcaptonuria and sucrase-isomaltase deficiency in three offspring of a consanguineous marriage.

Intestinal brush border membrane hydrolases and HLA lymphocyte antigens have been examined in three siblings with sucrose intolerance and alcaptonuria, and their consanguineous parents. Sucrase-isomaltase activity was absent in the three patients, and corresponded with the gel electrophoresis of SDS-solubilized brush border membranes, which failed to demonstrate the protein band normally associated with sucrase-isomaltase complex. The activities of all brush border membrane enzymes in the mother were normal, while those of the father were generally low. The use of hydrolytic capacity ratios, however, permitted the designation of both parents as heterozygotes. Significant homogentisic aciduria was found only in the three propositi, and no effect of homogentisic acid on the sucrase activities of two normal, unrelated children could be demonstrated in vitro. The HLA lymphocyte antigen profiles of all seven family members demonstrated remarkable histocompatibility in five of them.