The gene for intestinal sucrase-isomaltase as member of a gene family

The synthetic oligonucleotide MF59, derived from the rabbit sucrase-isomaltase cDNA sequence, hybridizes to two classes of mRNA in the rat and pig intestinal epithelium as well as to various restriction fragments within the human genome. These results strongly support the hypothesis that the sucrase-isomaltase gene belongs to a gene family. The cloning of a human cosmid containing such a sequence similar to MF59 has allowed us to identify a new member of this gene family which detects a single 6.5 Kb intestinal mRNA in the adult pig.     

Reversible forskolin-induced impairment of sucrase-isomaltase mRNA levels, biosynthesis, and transport to the brush border membrane in Caco-2 cells

Hybridization analysis of mRNA with a cDNA probe for human sucrase-isomaltase, pulse-chase experiments with L-[35S]-methionine followed by SDS-PAGE, and immunofluorescence detection of sucrase-isomaltase were used to analyze the level(s) at which forskolin interferes with the expression of the enzyme in Caco-2 cells in culture. Three effects are observed in Caco-2 cells treated with forskolin: 1) a marked decrease in the level of sucrase-isomaltase mRNA, 2) a marked decrease in the biosynthesis of the enzyme without any alteration of its stability, and 3) an almost total inhibition of its transport to the brush border membrane. All three effects are reversible when the drug is removed from the culture medium, though this reversibility is asynchronous: transport to the brush border membrane resumes after 24 h, sucrase-isomaltase mRNA levels are back to the normal after 5 days, whereas the biosynthesis of the enzyme, although increasing progressively, remains lower than in control cells, even 10 days after removal of the drug. The possibility that some effects are directly dependent on cAMP and others a consequence of changes in glucose metabolism is discussed.     

mRNA localization in polarized intestinal epithelial cells

An important feature of enterocyte maturation is the asymmetrical distribution of cellular functions including protein localization. mRNA sorting is one mechanism for establishment and maintenance of this process in other systems, and we have previously demonstrated differential localization of mRNAs in human enterocytes. To study regulation of mRNA sorting, we established a model in polarized Caco-2 cells. Proxy cDNA constructs containing beta-galactosidase (beta-gal)/green fluorescence protein (GFP) and the 3'-untranslated region (3'-UTR) of either human sucrase-isomaltase or villin were transfected transiently or stably. A control construct contained poly-A sequence in place of 3'-UTR. Expression of GFP was observed by confocal microscopy; intracellular location of the construct mRNA was imaged by in situ hybridization. The sucrase-isomaltase mRNA proxy localized to an apical position in Caco-2 cells as in native enterocytes; the villin mRNA proxy did not show significant localization. The control construct was not localized and was found diffusely throughout the cell. Proxy GFP proteins tended to localize with their mRNA proxies, but with less precision. This study establishes a valuable model for the investigation of mRNA localization in intestinal epithelial cells. Mechanisms controlling asymmetrical distribution of intestinal mRNAs can be now be elucidated.     

Comparison of sucrase-free isomaltase with sucrase-isomaltase purified from the house musk shrew Suncus murinus

We purified sucrase-isomaltase and sucrase-free isomaltase from a normal and a sucrase-deficient line, respectively, of the house musk shrew Suncus murinus and examined the effects of mutation on enzyme structure and activities. Recent cDNA cloning studies have predicted that sucrase-free mutant isomaltase lacks the C-terminal 69 amino acids of normal isomaltase, as well as the entire sucrase. On SDS-polyacrylamide gel electrophoresis purified sucrase-free isomaltase gave a single protein band of 103 kDa, while sucrase-isomaltase gave two major protein bands of 106 and 115 kDa. The 115, but not 106, kDa band was quite similar to the 103 kDa band on Western blotting with Aleuria aurantia lectin and antibody against shrew sucrase-isomaltase, suggesting that the 115 and 103 kDa bands are due to normal and mutant isomaltases, respectively, in accordance with the above prediction. Purified isomaltase and sucrase-isomaltase were similar in Km and Vmax (based on isomaltase mass) values for isomaltose hydrolysis and in inhibition of isomaltase activity by antibody against rabbit sucrase-isomaltase, suggesting that the enzymatic properties of isomaltase are mostly unaffected by mutation.     

Isolation of a cDNA probe for a human jejunal brush-border hydrolase,sucrase-isomaltase,and assignment of the gene locus to chromosome 3

We report the nucleotide sequence and derived amino acid sequence of a cDNA clone encoding most of the N-terminal, isomaltase region of human sucrase-isomaltase (SI). A plasmid containing this cDNA, pS 12, identifies a 6-kb mRNA found in human jejunum and the human colon carcinoma cell Une Caco-2. This human SI cDNA shows extensive overall homology with recently published rabbit SI cDNA. Using pSI2 to probe DNA from a panel of somatic cell hybrids, we have assigned the gene encoding human SI to chromosome 3.     

Comparative expression of the mRNA for three intestinal hydrolases during postnatal development in the rat

The distribution of the mRNA for intestinal aminopeptidase-N, lactase-phlorizin hydrolase and sucrase-isomaltase was compared during rat postnatal development as well as along the longitudinal axis of the intestinal tract including small-intestine and colon. We found out that each mRNA exhibited a specific pattern of accumulation, suggesting proper regulation steps for the expression of the corresponding digestive enzymes.     

Evidence of degradation process of sucrase-isomaltase in jejunum of adult rats.

To evaluate degradation processes of sucrase-isomaltase in adult rat jejunum, we determined enzymic activity of sucrase and isomaltase and compared it with the amount of immunoreactive sucrase-isomaltase. In rats fed or starved for 18h, killed at 10:00 h or 22:00 h, sucrase activity (expressed on the basis of total protein or of immunoreactive sucrase-isomaltase) was significantly (P less than 0.02) lower in the lower jejunum than in the upper jejunum; isomaltase activity was similar in both segments. Crossed immunoelectrophoresis demonstrated the existence of a second sucrase-isomaltase antigen reacting with anti-(sucrase-isomaltase) serum. This antigen was present in larger amounts in the lower jejunum than in the upper jejunum, exhibited immunological partial identity with the intact sucrase-isomaltase, and had isomaltase activity but no sucrase activity. Results suggest that this antigen is a degradation product of sucrase-isomaltase in which the sucrase active site has been broken down. To examine the role of pancreatic enzymes in degradation of sucrase-isomaltase, common pancreatico-biliary ducts were ligated. Within 18 h after the operation, the difference of sucrase activity between the upper and the lower jejunum disappeared and the amount of the second sucrase-isomaltase antigen markedly decreased in the lower jejunum. Our results indicate that, during the degradation of intestinal sucrase-isomaltase by the pancreatic proteinases, degradation of the sucrase active site precedes that of the isomaltase active site.     

Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe.

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.     

Monensin inhibits the expression of sucrase-isomaltase in Caco-2 cells at the mRNA level

Using L-[35S]methionine labeling, SDS-PAGE and Northern blot analysis of sucrase-isomaltase mRNA, two different concentrations of monensin were used to delineate in Caco-2 cells the effect of the drug on the conversion of the high mannose to the complex form of sucrase-isomaltase from its dual effect on the biosynthesis of the enzyme and on the rate of glucose consumption. At 0.1 microM the drug has no effect on the rate of glucose consumption and, although it inhibits the conversion of the high mannose to the complex form of the enzyme, it has no effect on the level of sucrase-isomaltase mRNA and on the amount of neosynthesized enzyme. At 1 microM, in addition to its inhibiting effect on the maturation of the enzyme, monensin provokes concomitantly an increase in the rate of glucose consumption and a decrease in the level of sucrase-isomaltase mRNA and in the amount of neosynthesized enzyme. All these effects are reversible within 48 h after removal of the drug.     

Molecular cloning of silkworm paralytic peptide and its developmental regulation

The silkworm paralytic peptide (PP) is a member of the ENF peptide family that exerts multiple biological activities involved in defense reaction and growth regulation. We isolated its cDNA and examined mRNA expression profiles. cDNA encoded 131 amino acids from which the 23-residue PP sequence was found at the C-terminal portion. Immunoblot analysis and paralytic activity assay indicated that inactive pro-protein in larval hemolymph was processed into active peptide immediately after bleeding. In the last larval instar, 0.6-kb PP mRNA was expressed in various tissues, of which the fat body was predominant. Its expression in the fat body decreased during the feeding period and then increased during metamorphic process. Juvenile hormone and 20-hydroxyecdysone upregulated its expression. At the embryonic stage, 1.5-kb mRNA, in addition to 0.6-kb mRNA, was expressed from 1 day after oviposition to hatching. PP was thus expressed stage-specifically under hormonal control.     

Primary structure and processing of lysosomal alpha-glucosidase; homology with the intestinal sucrase-isomaltase complex.

Lysosomal alpha-glucosidase (acid maltase) is essential for degradation of glycogen in lysosomes. Enzyme deficiency results in glycogenosis type II. The amino acid sequence of the entire enzyme was derived from the nucleotide sequence of cloned cDNA. The cDNA comprises 3636 nt, and hybridizes with a messenger RNA of approximately 3.6 kb, which is absent in fibroblasts of two patients with glycogenosis type II. The encoded protein has a molecular mass of 104.645 kd and starts with a signal peptide. Sites of proteolytic processing are established by identification of N-terminal amino acid sequences of the 110-kd precursor, and the 76-kd and 70-kd mature forms of the enzyme encoded by the cDNA. Interestingly, both amino-terminal and carboxy-terminal processing occurs. Sites of sugar-chain attachment are proposed. A remarkable homology is observed between this soluble lysosomal alpha-glucosidase and the membrane-bound intestinal brush border sucrase-isomaltase enzyme complex. It is proposed that these enzymes are derived from the same ancestral gene. Around the putative active site of sucrase and isomaltase, 10 out of 13 amino acids are identical to the corresponding amino acids of lysosomal alpha-glucosidase. This strongly suggests that the aspartic acid residue at this position is essential for catalytic function of lysosomal alpha-glucosidase.