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PubMed1989

Post-translational suppression of expression of intestinal brush border enzymes by fructose.

The two major dietary sugars, fructose and sucrose, were found to suppress effectively the biosynthetic renewal of brush border enzymes in the gut. When studied in cultured explants of pig small intestine mucosa, 10-50 mM concentrations of fructose completely prevented the expression of mature aminopeptidase N and severely reduced that of sucrase-isomaltase. The instantly occurring and reversible suppressive effect manifested itself as a leupeptin-sensitive degradation of newly synthesized brush border enzymes. The likely mechanism of action of the dietary sugar is by causing an abnormal cotranslational glycosylation that in turn triggers a rapid proteolytic breakdown. Our findings suggest that renewal of digestive brush border enzymes is transiently suppressed during intake of fructose- or sucrose-rich meals.

Aminopeptidases↗

PubMed1989

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.

Amino Acid Sequence↗

PubMed1989

Suramin inhibits cell growth and glycolytic activity and triggers differentiation of human colic adenocarcinoma cell clone HT29-D4.

Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis inhibits growth factor-induced mitogenesis. In the present report, we show that suramin inhibits the growth of human colic adenocarcinoma cells HT29-D4 and rapidly induces their differentiation into enterocyte-like cells. As soon as 6 days after the addition of suramin (100 micrograms/ml) in the culture medium, the cells form a polarized monolayer of regular columnar cells with occluding junctions delimiting two distinct membrane domains (apical and basolateral) and an apical brush-border expressing alkaline phosphatase and sucrase-isomaltase. The process of differentiation is fully reversible when the drug is removed from the culture medium. We also show that suramin inhibits both glucose consumption and lactate production so that the glycolytic activity of the treated cells is lowered by 42%. This observation would shed some light on the complex mechanisms involved during the induction of HT29 cell differentiation when glucose is removed from the culture medium.

Adenocarcinoma↗

PubMed1986

Inhibition of the post-translational processing of microvillar hydrolases is associated with a specific decreased expression of sucrase-isomaltase and an increased turnover of glucose in Caco-2 cells treated with monensin.

The biosynthesis and post-translational processing of sucrase-isomaltase and dipeptidylpeptidase IV were studied by L-[35S]methionine labeling, immunoisolation with monoclonal antibodies and SDS-PAGE in post-confluent Caco-2 cells treated with monensin (10 microM, 48 h). In addition to its classical effect on the post-translational processing of both hydrolases, i.e. an inhibition of the conversion of the high-mannose to the complex glycosylated form of the enzymes, monensin was found to have two other effects: a marked decrease of sucrase-isomaltase expression, but not of dipeptidylpeptidase IV; an increased turnover of glucose, as substantiated by increased rates of glucose consumption and lactic acid production and a decreased glycogen content. Whether these two effects are related to the particular differentiation and metabolic status of Caco-2 cells is discussed, as well as a possible role for the drug-induced modifications of glucose turnover on the decreased expression of sucrase-isomaltase.

Cell Line↗

PubMed1990

First purification and characterization of a sucrase-isomaltase from goose kidney microvillous membrane.

Goose (Anser anser) kidney microvillus sucrase-isomaltase (EC 3.2.1.48-EC3.2.1.10) was solubilized from isolated microvillus membranes using Emulphogen BC 720 or papain. Detergent-solubilized enzyme (D-SI) was purified 149 +/- 29 times with a yield of 15.7 +/- 2.6% by a two-step procedure which included chromatofocusing. The specific activity was 2.95 +/- 0.34 U/mg protein for sucrase, 1.02 +/- 0.13 for palatinase and 5.63 +/- 0.53 for maltase. D-SI was amphiphilic as indicated by its detergent-binding properties. These properties were not observed for sucrase-isomaltase released from the microvillus membrane by papain. The Mr of the enzyme purified after solubilization by Emulphogen and papain was 543,000 and 380,000, respectively, as determined by gel filtration. The difference in Mr indicates that an Emulphogen micelle is bound to the detergent-solubilized enzyme. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis, sucrase-isomaltase migrated as several polypeptide chains: a major band (Mr 280,000) and at least seven additional minor bands (Mr 220,000-100,000). It is suggested that the major band represents the precursor pro-sucrase-isomaltase and that the lower molecular weight bands are generated by PMSF or aprotinin-resistant proteinases during homogenisation and chromatography of the enzyme. Measured by chromatofocusing, the isoelectric point was found to be pH 4.6. Sucrase-isomaltase accounts for about 20% of total microvillus membrane proteins.

Animals↗

PubMed1989

Posttranslational regulation of sucrase-isomaltase expression in intestinal crypt and villus cells.

Expression and synthesis of sucrase-isomaltase (SI) were studied in human jejunum and in the colon tumor cell lines Caco-2 and HT-29. Twelve monoclonal antibodies produced against the adult human intestinal enzyme were shown to recognize specifically SI by immunoprecipitation of 14C-labeled membrane proteins, analysis of enzyme activities in the immunoprecipitates, and immunoblotting. These antibodies produced markedly different patterns of immunofluorescent staining of the intestinal mucosa. Three of them were specific for the absorptive villus cells, while the other nine also stained the luminal membrane of the proliferative crypt cells, with different intensities which paralleled their ability to recognize SI in immunoblots. Sequential immunoprecipitation of SI solubilized from purified brush borders or entire jejunum with four selected antibodies demonstrated the presence of different forms of the enzyme, expressed by either villus or crypt cells. Two immunologically distinct forms of high mannose precursor (hmP1 and hmP2) were also identified in both jejunal mucosa and colon tumor cells. They were present as monomers and their immunological differences were preserved under various ionic and pH conditions. Pulse-chase studies indicated that, in Caco-2 cells, hmP1 is converted into hmP2 within 30 min of chase, and hmP2 is then processed into the complex-glycosylated precursor destined for the brush border membrane. hmP1 was immunologically related to the mature SI present in crypt cells and lacked the epitopes specific for mature SI expressed by villus cells. These results demonstrated that sucrase-isomaltase is synthesized by both crypt and villus cells, but processing of the cotranslationally glycosylated high mannose precursor is dependent on the state of differentiation of the enterocytes. This may represent a general mechanism for the regulation of expression of differentiated cell products at the post-translational level.

Adult↗

PubMed1990

Detection and characterization of sucrase-isomaltase in adult human colon and in colonic polyps.

A panel of monoclonal antibodies specific for sucrase-isomaltase, but differing in their ability to stain the proliferative crypt cells in human jejunum, was used to investigate expression of this enzyme in adult human colon and colonic tumors. Immunofluorescence staining on cryostat sections demonstrated the presence of sucrase-isomaltase in the apical region of normal colonic crypt cells but not on surface epithelium. Colonic sucrase-isomaltase was purified by immunoprecipitation with selected monoclonal antibodies and identified predominantly as high-mannose and complex glycosylated single-chain precursors endowed with relatively low levels of enzyme activities. Most polyps examined (10/16) were also found to express significant amounts of sucrase-isomaltase. In contrast, only 3 of 45 adenocarcinomas were positive by immunofluorescence staining; no correlation was found between enzyme expression and tumor classification either by "Dukes" stage or degree of histological differentiation. These results demonstrate that colonic crypt cells and some benign tumor cells synthesize and express at their cell surface a form of sucrase-isomaltase immunologically distinct from that present in the brush borders of small intestinal villose cells.

Adenocarcinoma↗

PubMed1979

Developmental pattern of rat intestinal brush-border enzymic proteins along the villus--crypt axis.

At various postnatal stages, intestinal epithelial cells were isolated sequentially from villus tip to crypt base by successive EDTA treatments. According to the localization of marker enzymic activities, isolated cells were pooled into three cell compartments: villus (V), lower villus and upper crypt (VC) and crypt (C). Purified brush-border-membrane proteins were separated by 7.5%-polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Enzymic activities could be assigned to some protein bands: maltase/glucoamylase (protein band 3), sucrase-isomaltase (protein bands 3 and 6), lactase (protein band 5) and alkaline phosphatase (region of protein bands 8 and 9). The findings suggest the following. (1) Sucrase-isomaltase activities appeared in compartment C at 17 days with a simultaneous increase of the pre-existing protein band 3 and appearance of a well-defined protein band in position 6; the enzymic complex remained still present in the crypt cells until adulthood. From the day 21 onwards, sucrase-isomaltase was detected in compartments VC and V. (2) Lactase was only present in the three cell compartments until day 21; at this developmental stage its activity completely disappeared from compartment C, in spite of the persistence of a weak protein band. (3) Alkaline phosphatase activity could be detected as a single peak corresponding to protein band 9 in all three cell compartments until day 21; thereafter it was replaced by two peaks of activity showing a less precise correlation with the well-defined protein bands 8 and 9. In the crypt cells of the adult rat, however, the preweaning situation, which was regularly observed, is an unexpected phenomenon. (4) Maltase and glucoamylase did not display any marked qualitative or quantitative modifications either along the villus-crypt axis or during the period of postnatal development studied. Evidence is given from the present data that each brush-border enzyme investigated has a specific developmental pattern.

Age Factors↗

PubMed1990

Endocytosis, recycling, and lysosomal delivery of brush border hydrolases in cultured human intestinal epithelial cells (Caco-2).

Lysosomes of intestinal epithelial cells in vivo and in culture display strong immunoreactivity with monoclonal antibodies against various brush border enzymes as visualized by immunoelectron microscopy. Novel subcellular fractionation procedures were developed to study, by the pulse-chase technique and by internalization assays, the pathway along which two microvillar hydrolases, sucrase-isomaltase and dipeptidylpeptidase IV, are transported to lysosomes in the differentiated colon adenocarcinoma cell line Caco-2. 7-9% of metabolically labeled sucrase-isomaltase of dipeptidylpeptidase IV were present in lysosomes after 7-8 h of chase as intact complex-glycosylated molecules. Appearance of these enzymes in lysosomes was biphasic. Endocytosis studies with radioiodinated antienzyme monoclonal antibodies (monovalent antigen-binding fragments) and by means of cell surface iodination revealed only slow transport of the enzymes to lysosomes at a low level. However, both enzymes were internalized with different efficiencies and recycled to the cell surface via endosomes. These results suggest that in Caco-2 cells a significant amount of newly synthesized sucrase-isomaltase and dipeptidylpeptidase IV is directly imported into lysosomes bypassing the brush border membrane.

Adenocarcinoma↗

PubMed1991

Oligomerization and intracellular protein transport: dimerization of intestinal dipeptidylpeptidase IV occurs in the Golgi apparatus.

It was postulated that newly synthesized membrane proteins need to be assembled into oligomers in the endoplasmic reticulum in order to be transported to the Golgi apparatus. By use of the differentiated human adenocarcinoma cell line Caco-2, the general validity of this proposal was studied for small intestinal brush border enzymes which are dimers in most mammalian species. Chemical cross-linking experiments and sucrose gradient rate-zonal centrifugation revealed that dipeptidylpeptidase IV is present as a dimer in the brush border membrane of Caco-2 cells whereas the disaccharidase sucrase-isomaltase appears to be a monomer. Dipeptidylpeptidase IV was found to dimerize immediately after complex glycosylation, an event associated with the Golgi apparatus. Dimerization of this enzyme was inhibited by CCCP but did not depend on complex glycosylation of N-linked carbohydrates as assessed by the use of the trimming inhibitor 1-deoxymannojirimycin. It is concluded that dimerization of dipeptidylpeptidase IV occurs in a late Golgi compartment and therefore cannot be a prerequisite for its export from the endoplasmic reticulum.

Adenocarcinoma↗

PubMed1977

Carboxyl groups at the two active centers of sucrase-isomaltase from rabbit small intestine.

1. Seveal selective reagents were employed to identify the amino acid residues essential for the catalytic activity of sucrase-isomaltase. 2. Modification of histidine, lysine and carboxyl residues resulted in a partial inactivation of the enzyme. Substrates or competitive inhibitors provided protection against inactivation only in the reaction of carboxyl groups with carbodiimide (+lycine ethyl ester) or with diazoacetic ethyl ester. This indicated the occurrence of carboxyl groups at the two active centers of the enzyme complex. 3. Protection against inactivation of the enzyme by carbodiimide was provided also by the presence of alkali and alkaline earth metal ions, which are non-essential activators of sucrase-isomaltase. The presence of Na+ and Ba2+ protected approximately one carboxyl group per active center from reacting with carbodiimide plus glycine ethyl ester. 4. The carbodiimide-reactive groups were not identical with the two carboxylate groups recently found to react with conduritol-B-epoxide, an active-site-directed inhibitor of sucrase-isomaltase (Quaroni, A. and Semenza, G., 1976, J. Biol. Chem 251,3250--3253). A possible role for the carbodiimide-reactive carboxyl groups at the active centers of sucrase-isomaltase is discussed.

Animals↗

PubMed1989

Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry.

Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase-isomaltase. The labelling efficiency of RPGG was compared to that of protein A-gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12 nm particles, 10.3 and 6.2 particles/micron of length of microvillar membrane, 3.5 and 1.0 particles/micron2 of Golgi profile and 5.9 and 2.0 particles/micron2 of multivesicular body profile; and for the 6 nm particles, 49.6 and 15.7 particles/micron of length of microvillar membrane, 24.4 and 5.0 particles/micron2 of Golgi profile and 25.4 and 3.4 particles/micron2 of multivesicular body profile. Controls showed very little non-specific gold labelling (less than 0.02 gold particles/micron2 of section).

Animals↗

PubMed1989

Biosynthesis of intestinal microvillar proteins. Low temperature arrests both processing and intracellular transport.

The effect of culture at 20 degrees C on biosynthesis of microvillar enzymes was studied in pig small intestinal mucosal explants. At this temperature, aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48-10) both accumulated intracellularly, predominantly in their transient, high mannose-glycosylated form characteristic of the newly synthesized enzymes prior to the molecular processing taking place in the Golgi complex. The general morphology of the enterocyte was unaffected by culture at low temperature except for the Golgi complex where the cisternae appeared condensed and surrounded by numerous vesicles of 50 to 55 nm. Both molecular processing and microvillar expression could be restored by shifting the temperature to 37 degrees C. Culture at low temperature did not induce any missorting of newly synthesized aminopeptidase N, but both molecular processing and microvillar expression only resumed at a slow rate after increasing the temperature, suggesting that reorganization of the Golgi complex is a time-requiring process.

Animals↗

PubMed1986

Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression.

Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), sucrase-isomaltase (EC 3.2.1.48-10) and maltase-glucoamylase (EC 3.2.1.20), castanospermine caused the formation of novel transient forms of higher Mr than corresponding controls, indicating a blocked removal of glucose residues. For the first three enzymes, the 'mature' (Golgi-processed) forms were similar in size to or slightly smaller than corresponding controls and were, as shown for aminopeptidase N, endoglycosidase-H-sensitive, evidence of a blocked attachment of complex sugars. Maltase-glucoamylase did not undergo conversion into a 'mature' form, suggesting that, unlike other microvillar enzymes, it does not receive post-translational O-linked carbohydrate. Castanospermine suppressed the synthesis of the four enzymes, but did not block their transport to the microvillar membrane, showing that processing of N-linked carbohydrate is not required for microvillar expression. The proteinase inhibitor leupeptin partially restored the suppressed synthesis, indicating that the majority of the wrongly processed enzymes, probably because of conformational instability, become degraded soon after synthesis rather than being transported to the microvillar membrane.

Alkaloids↗

PubMed1992

Regulation of CFTR expression and function during differentiation of intestinal epithelial cells.

CFTR, the protein defective in cystic fibrosis is regulated during differentiation of intestinal epithelial cells. The undifferentiated cells (Caco-2 and HT-29) show a lower level of CFTR mRNA, while a 10-fold increase is seen in differentiated cells. These differences correlate well with those of other intestinal-specific genes, including sucrase-isomaltase, villin and alpha 1-antitrypsin, indicating that the regulation is cell specific. In Caco-2 cells the increase in CFTR mRNA cannot be accounted for by increased transcription of the gene. These data indicate that CFTR mRNA stabilizing factor(s) might be present in differentiated cells. The higher levels of CFTR mRNA in differentiated cells are accompanied by decreased protein levels, indicating, as well, involvement of translational control in the regulation of CFTR in these cells. Finally, fully differentiated cells show lowered levels of cyclic AMP-activated C1- transport, the characteristic function of CFTR. Thus, CFTR function in differentiated cells is modulated by a complex interaction of regulatory elements. Caco-2 and HT-29 cells provide a suitable in vitro system in which to study the mechanism of regulation of CFTR.

Blotting, Western↗

PubMed1992

The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells.

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5'-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.

Adenocarcinoma↗

PubMed1990

Basement membrane components are potent promoters of rat intestinal epithelial cell differentiation in vitro.

Basement membranes have been implicated in morphogenesis and cell differentiation. In this study, the effect of basement membrane components on intestinal epithelial cell maturation in a mesenchyme-free environment was investigated. Fetal rat small intestinal epithelial cells (from the 14th-17th day of gestation) were exposed to basement membrane-derived proteins (laminin, collagen type IV, and a complex basement membrane-enriched extract from the Engelbreth-Holm-Swarm sarcoma) and other extracellular matrix proteins (collagen type I and fibronectin) coated onto Petri dishes. The cells attached readily only to fibronectin and basement membrane proteins. For 5 days the developing epithelial colonies were monitored in vitro, assessing morphological and functional parameters of cell maturation. Colonies grown on laminin and the basement membrane extract were larger and of greater cell density. An increase in alkaline phosphatase and lactase activity was observed after 3-4 days in these colonies which could be enhanced to yield 90%-100% positive cells by the addition of dexamethasone to the medium while no sucrase-isomaltase activity was elicited. Electron microscopy confirmed a high degree of cellular polarization illustrated by tight junctions and apical microvilli in epithelial cells grown on a basement membrane-like support. In contrast, none of the other proteins stimulated the cells to mature in vitro. The authors conclude that certain basement membrane components actively promote fetal intestinal epithelial cell differentiation.

Animals↗

PubMed1991

Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases.

An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases.

Amino Acid Sequence↗